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41	Conception et synthèse de molécules à visée anti-infectieuse selon deux stratégies : le criblage à haut débit et l’approche par fragments / Design and synthesis of anti-infectious molecules using two different strategies : high throughput screening and fragment-based drug discovery approaches Prevet, Hugues 30 September 2016 (has links) La découverte d’un candidat médicament repose sur l’identification de hits, présentant des propriétés physico-chimiques adéquates pour leur optimisation. Le criblage à haut débit et l’approche par fragments sont deux techniques couramment utilisées lors de cette étape d’identification et elles ont été mises en œuvre au cours de ma thèse dans le but de découvrir de nouveaux composés ciblant d’une part le complexe CD81/CLDN-1 pour empêcher l’entrée du virus de l’hépatite C (VHC) dans les hépatocytes et d’autre part EthR2, un régulateur transcriptionnel mycobactérien, afin de potentialiser l’activité d’un antituberculeux sur les souches résistantes de M. tuberculosis.Dans une première partie, un criblage à haut débit sur le complexe CD81/CLDN-1 a permis d’identifier des modulateurs en série thiéno[2,3-c]pyrazole. Ces composés ont été pharmacomodulés et un composé spécifique de l’étape d’entrée du VHC, non toxique et présentant une activité submicromolaire a pu être ainsi identifié. Cette sonde pharmacologique permettra de mieux comprendre les mécanismes impliqués dans le processus d’entrée virale.Dans une deuxième partie, nous nous sommes intéressés à la conception de nouveaux fragments dits privilégiés. Ainsi, le développement des voies de synthèse, sous irradiation micro-onde, de deux entités moléculaires, le noyau 1,4-benzodiazepine-2,5-dione et le noyau spirohydantoïne, nous a permis d’obtenir 34 composés originaux. Afin d’évaluer le potentiel de cette stratégie, une librairie virtuelle de fragments a été générée et son criblage in silico sur la protéine MDM2 a été effectué. La mesure in vitro de l’activité des hits identifiés permettra de valider l’intérêt de cette approche pour la découverte de nouveaux ligands ciblant les interactions protéine-protéine.Dans une troisième partie, des inhibiteurs d’un répresseur transcriptionnel mycobactérien impliqué dans la potentialisation de l’activité de l’éthionamide ont été développés. A l’issue d’un criblage de 960 fragments, l’identification d’un hit en série tropinone, et sa cocristallisation avec la protéine EthR2, a permis d’entamer une optimisation rationnelle qui a conduit à l’obtention rapide de composés présentant de meilleures activités. / The discovery of drug candidates is based on the identification of hits with appropriated physico-chemical properties for further development. High throughput screening and fragment-based drug discovery approaches are two strategies commonly used for this identification. These strategies were applied during my PhD research work for identifying not only new modulators of the CD81/CLDN-1 complex to prevent entry of the Hepatitis C virus (HCV) into hepatocytes but also inhibitors of the mycobacterial transcriptional repressor, called EthR2, to boost ethionamide antibacterial activity against resistant strains of M. tuberculosis.Firstly, a high throughput screening assay was developed to identify molecules bearing a thieno[2,3-c]pyrazole scaffold that modulate the CD81/CLDN-1 complex. The structure-activity relationships allowed us to design and synthesize one non-toxic compound that inhibits viral entry with an IC50 in the submicromolar range. This best analog will be used as pharmacological tool to understand the molecular mechanism involving the CD81/CLDN-1 interaction during virus entry.Secondary, we worked on the design and synthesis of a new generation of fragments called privileged fragments. We focused our interest on the 1,4-benzodiazepine-2,5-dione and spirohydantoin scaffolds and using microwave-assisted conditions 44 original privileged fragments have been synthesized. To further illustrate the potential of our privileged fragments, a virtual focused library has been generated and screened in silico on MDM2 protein. The in vitro evaluation of the identified hits will allow us to validate our approach and to show the potential of our privileged fragments for the discovery of new hits against protein-protein interactions.Finally, inhibitors of a new mycobacterial transcriptional repressor involved in the boosting of ethionamide activity have been developed. Screening of 960 fragments allowed us to identify a hit bearing a tropinone scaffold which was cocrystallized with EthR2. A rational design from this cocrystal structure led rapidly to more potent ligands. Virus de l'hépatite C CD81 Claudine-1 Inhibiteurs Hépatocytes HTS Frangments privilégiés 3D FBDD Chimiothèque Tuberculose Éthionamide EthR2 Potentialisation Hepatitis C virus CD81 Claudin-1 Inhibitors Hepatocytes HTS Priviliged fragments 3D FBDD Chemical library Tuberculosis Ethionamide EthR2 Boosting
42	Oncoleaking gene therapy: a new suicide approach for treatment of pancreatic cancer Pahle, Jessica 17 July 2018 (has links) Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien zeigten eine selektive optCPE vermittelte Verminderung der Tumorvitalität in Verbindung mit Nekrose, die in fast allen Fällen mit einer Reduktion des Tumorvolumens einher ging. Die tierexperimentellen Studien belegen damit die Effektivität der CPE-basierten Gentherapie im Pankreaskarzinom. Mit diesen neu gewonnenen Erkenntnissen zum „Onkoleaking“ Konzept der CPE Suizid-Gentherapie und deren Wirkungsmechanismen sind Kombinationen mit konventionellen Therapien möglich. / Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3  /  4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated the selective CPE mediated tumor growth inhibition, associated with reduced tumor viability and effective induction of tumor necrosis. This further corroborated the advantages of this novel oncoleaking strategy. With this gain of knowledge about our new oncoleaking concept of suicidal gene therapy and its mechanism of action, novel combinations with conventional therapies are possible to further improve therapeutic efficacy and to overcome resistance in pancreas carcinoma. Gentherapy Pankreaskarzinom Bakterientoxin Clostridium perfringens enterotoxin CPE Claudin pancreatic cancer bacterial toxins gene therapy Clostridium perfringens enterotoxin CPE suicide gene therapy oncoleaking claudins cldn 500 Naturwissenschaften und Mathematik 610 Medizin und Gesundheit 570 Biowissenschaften; Biologie WG 7400 XH 7515 XH 7512 XH 4104 ddc:500 ddc:610 ddc:570
43	Biomarkers in esophageal cancer Takala, H. (Heikki) 05 June 2012 (has links) Abstract Mediators of epithelial permeability, angiogenesis and invasion may serve as prognostic indicators and targets for therapies in esophageal cancer (EC). The expressions of claudins, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor-A (VEGF), nitric oxide synthases (iNOS, eNOS, nNOS) and toll-like receptor 9 (TLR9) were evaluated by immunohistochemistry in EC. The results were compared with clinicopathological variables, tumor proliferation and apoptosis. All of the claudins were expressed in most of the cancers. Esophageal adenocarcinomas (EAC) displayed more often increased claudin 3 and 5 expression than esophageal squamous cell carcinomas (ESCC). Loss of claudin 3 expression associated with distant metastases in EC and a tendency in this direction was also observed for claudin 4. Cancers with stronger claudin 4 expression showed increased apoptosis in both EAC and ESCC. HIF-1α was present in most of the ECs and like iNOS more often in ESCC than in EAC. Strong HIF-1α expression tended to associate with positive VEGF immunostaining. In ESCC, both strong HIF-1α expression and VEGF positivity tended to associate with increased microvessel density (MVD). In EAC, tumors showing VEGF positivity associated with increased MVD outside the tumor. Patients with strong HIF-1α expression had more often distant metastases than other patients in EC. There was no VEGF expression in normal esophageal mucosa and T3-4 tumors tended to be more often VEGF positive than T1-2 tumors. The expression of TLR9 was more intensive in dysplasia than in normal epithelium and ESCC and abundant TLR9 expression could serve as a marker of squamous cell high grade dysplasia. Intensive TLR9 expression was associated with higher grade tumors and the presence of nodal and distant metastases in ESCC. EC and its progression may be related to increased angiogenesis regulated by VEGF and HIF-1α. In EC, claudin expression varies along with the histology of the tumor. Claudin expression may be associated with apoptosis or proliferation and contribute to tumor behavior. An association was detected between moderate to strong expression of claudin 3 and a high TLR9 histoscore in ESCC. Altered expression of claudin 3 may result to upregulation of endosomal TLR9. TLR9 may serve as a marker for squamous cell dysplasia and ESCC progression. / Tiivistelmä Solukerrosten läpäisevyyttä, verisuonten uudismuodostusta ja kasvainsolujen liikkuvuutta säätelevät tekijät voivat toimia ruokatorvisyövän ennustetekijöinä ja hoidon kohteina. Tässä tutkimuksessa selvitettiin klaudiinien, hypoksia-indusoituvan tekijän 1α (HIF-1α), verisuonen endoteelin kasvutekijän A (VEGF), kolmen typpioksidisyntaasin (iNOS, eNOS ja nNOS) sekä tollin kaltaisen reseptorin 9 (TLR9) ilmentymistä ja merkitystä ruokatorvisyövässä immunohistokemiallisin menetelmin. Tuloksia arvioitiin suhteessa proliferaatioon ja apoptoosiin. Useimmat syöpäkasvaimet ilmensivät jokaista tutkittua klaudiinia. Ruokatorven rauhassyövässä ilmeni levyepiteelisyöpää useammin klaudiineja 3 ja 5. Klaudiinin 3 vähäinen ilmentyminen oli yhteydessä etäpesäkkeiseen tautiin. Sama suuntaus näkyi klaudiinin 4 kohdalla. Apoptoosia todettiin enemmän kasvaimissa, jotka ilmensivät muita enemmän klaudiini 4:ä. Useimmat syöpäkasvaimet ilmensivät HIF-1α:a. Sekä iNOS että HIF-1α ilmentyivät runsaammin levyepiteeli- kuin rauhassyövässä. Tutkimus viittasi yhteyteen voimakkaan HIF-1α:n ilmentymisen ja VEGF:n ilmentymisen välillä. Voimakas HIF-1α:n ilmentyminen ja VEGF:n ilmentyminen vaikuttivat liittyvän ruokatorven levyepiteelisyövän uudissuonituksen lisääntymiseen. Rauhassyövän lisääntynyt uudissuonitus kasvaimen ulkopuolella saattaa liittyä VEGF:n ilmentymiseen. Potilailla, joiden kasvaimissa HIF-1α:n ilmentyminen oli voimakasta, todettiin etäpesäkkeitä muita useammin. VEGF:a ei todettu normaalissa limakalvossa, ja sen ilmentyminen vaikutti olevan yleisempää syvälle kasvavissa kuin pinnallisissa syövissä. TLR9 ilmentyi voimakkaammin levyepiteelin dysplasiassa kuin normaalissa tai kasvainepiteelissä. Huonosti erilaistuvissa ja levinneissä kasvaimissa TLR9 ilmentyi voimakkaammin kuin muissa kasvaimissa. Ruokatorvisyövän synty ja eteneminen voivat liittyä HIF:n ja VEGF:n säätelemään verisuonten uudismuodostukseen. Klaudiinit saattavat vaikuttaa syövän käyttäytymiseen myös apoptoosin ja proliferaation kautta. Tutkimuksessa todettiin yhteys lisääntyneen klaudiinin 3 ilmentymisen ja voimakkaasti ilmentyvän TLR9:n välillä. Muutos klaudiinin 3 ilmentymisessä saattaa lisätä levyepiteelin läpäisevyyttä johtaen TLR9:n aktivoitumiseen. TLR9 voi vaikuttaa ruokatorven levyepiteelin dysplasian ja syövän syntyyn sekä toimia vaikean dysplasian ja aggressiivisen levyepiteelisyövän merkkiaineena. angiogenesis claudin esophageal cancer hypoxia inducible factor-1α (HIF-1α) nitric oxide synthase (NOS) toll-like receptor 9 (TLR9) klaudiini ruokatorvisyöpä tollin kaltainen reseptori 9 (TLR9) typpioksidisyntaasi (NOS) verisuonten uudismuodostus
44	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology
45	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology

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