Cytotoxicity assays: Principles and Applications

Transcription

1 Cytotoxicity assays: Principles and Applications Dr. Markus Kamber Xenometrix AG Gewerbestr. 25 CH-4123 Allschwil Switzerland

2 Overview 1. Major Applications 2. Why perform cytotoxicity assays? 3. What characteristics should a cytotoxicity assay have? 4. What tests does Xenometrix offer? 5. How do they work? 6. Advantages of Xenometrix kits 7. What problems/questions can arise?

3 Major Applications Testing of Medical Devices (e.g. Screws to fix broken bones; Dental Implants) Testing of materials that come in contact with injectables (e.g. rubber stoppers of infusion vials) Testing of new medicines (early preclinical phase)

4 A) Medical Devices Testing of devices by direct contact with test cells Testing after extraction of device ISO : Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity Suggested tests: NR, MTT, XTT

5 B) Testing of New Medicines Pharma, Early Phase Drug Development High Efficacy High Specificity Cost Efficient Minimal Unwanted Side Effects

6 Cell lines Primary Cells Organ Cultures Costs/Benefits of Tox Analyses High-Content Analysis Small Rodents Large Mammals Primates Man Relevance of Prediction Costs Number of Compounds Pre-clinical -----> Clinical

7 Why perform Cytotoxicity Assays? Generally: Identify compounds that are toxic for animal/human cells BUT:

8 Paracelsus (Theophrastus Bombast von Hohenheim ): "Alle Ding' sind Gift und nichts ohn' Gift; allein die Dosis macht, dass ein Ding kein Gift ist." (All things are poison and nothing is without poison; only the dose makes a thing a poison. ) NaCl LDlo (human) = 1000 mg/kg Identify the dose at which a compound becomes toxic

9 % Inhibition In vitro cytotoxicity test are one piece of information to decide if a compound has a favorable (in vitro) "therapeutic window" Activity vs. Cytotoxicity Concentration Activity cpd 1 Activity cpd 2 Activity cpd 3 Cytotoxicity 1 Cytotoxicity 3 Cytotoxicity 2

10 What characteristics should a cytotoxicity assay have? (1) Identify compounds with unfavourable activity/toxicity profile. - High specificity few false positives - High sensitivity few false negatives Advantage of multiple-endpoint tests - Little compound consumption - Reasonably fast, good throughput

11 What characteristics should a cytotoxicity assay have? (2) Robust, highly reproducible Especially for smaller labs: Easy handling, good protocol Reasonably priced No unusual or expensive hardware necessary Clear-cut results, no need for sophisticated analytical software Excellent customer support

12 What tests does Xenometrix Set of classic tests offer? Widely accepted methods Published data Single and combination kits Complete out-of-the-box kits Quality - tested reagents Validated protocols Pre- and post-sale dedicated free customer support

13 NEUTRAL RED - Lysosomal activity - Membrane permeability LDHe - Membrane permeability GLUCOSE - General physiological cell state - Glucose consumption XTT - Mitochondrial metabolism - Respiratory toxicity SULFORHODAMINE B - Cell proliferation - Total protein content

14 NEUTRAL RED - Lysosomal activity - Membrane permeability LDHe - Membrane permeability GLUCOSE - General physiological cell state - Glucose consumption XTT - Mitochondrial metabolism - Respiratory toxicity SULFORHODAMINE B - Cell proliferation - Total protein content

15 Test Principles Assay Parameter Principle XTT Mitochondrial activity Yellow tetrazolium salt is converted to water-soluble formazan derivative (MTT) Mitochondrial activity Yellow tetrazolium salt is converted to insoluble formazan derivative LDH Membrane permeability LDH reduces pyruvate to lactate NADH decrease by release of LDH from damaged cells is measured

16 Assay Parameter Principle SRB Protein synthesis Quantitative measurement of SRB which binds to cellular proteins, NR Lysosomal activity Quantitative measurement of NR which binds to viable cells and accumulates in the lysosomes Glu Glucose metabolism Glucose consumption by growing cells is measured

17 Multiple Parameter Kits NR XTT LDHe Glu SRB XTT SRB LDHe XTT NR - SRB LDHe XTT - NR LDHe XTT SRB XTT NR SRB LDHe Glu XTT SRB PAN I: LDHe XTT NR SRB

18 What tests does Xenometrix offer? (contd.) Advantages of multiple-assay kits Less cells needed (primary cells!) Less compound required (early development!) Improved sensitivity and specificity (Greater chance to catch toxicants if one looks at several different mechanisms; reduced incidence of false positive results) Possible indication of mechanism of action

19 IC50 (um) IC50 (µm) What characteristics should a cytotoxicity assay have? (2) False negatives under certain conditions: 5-FU IC50 with different assays Propranolol IC50 with different assays LDHe XTT NR SRB LDHe XTT NR SRB h Overnight 24 h 48 h 0 2 h Overnight 24 h 48 h Exposure time Exposure time

20 OD540-OD690 OD540-OD690 False negative: an actually toxic substance is not detected Example Propranolol (Beta blocker, Hypertension) SRB, Propranolol Blank SC TL Blank Propranolol (µm) 0.6 NR, Propranolol Blank SC TL Blank Propranolol (µm)

21 False positive: an actually harmless substance is classified as toxic. Example: assays whose read-out is based on an enzyme activity Inhibition of the enzyme can wrongly suggest a toxic effect. Example: Luminescence-based assay for the quantification of ATP

22 IC50 (µm) p-phenylenediamine (Hair dye) p-phenylenediamine IC50 with different assays LDHe XTT NR SRB h Overnight 24 h 48 h Exposure time It s dangerous to rely only on 1 parameter!

23 IC50 (µm) Chloroquine (anti-malaria, Lysosomotrop) Chloroquine LDHe XTT NR SRB 0 2 h Overnight 24 h 48 h Exposure time It s dangerous to rely only on 1 parameter!

24 IC50 (µm) Mechanism of action Chloroquine h 16 h 24 h 48 h Exposure time LDHe XTT NR SRB

25 What tests does Xenometrix offer? (contd.) Advantages of multiple-assay kits Possibility to avoid interfering conditions: - coloured compounds alternative wavelengths - enzymatic inhibitors alternative enzymes or a stain - known incompatibilities, e.g. lysosomotrophic compounds (chloroquine), redox-active compounds (ascorbic acid) - interfering medium components: FCS, pyruvate, phenol red

26 Advantages of Xenometrix kits Quality-controlled reagents and simple, detailed, and well-established protocols No need for sophisticated and expensive hardware Straightforward read-outs, no special evaluation software is required. Calculations can be done in Microsoft Excel Knowledgeable and dedicated free customer support before and after sale Unique multiple parameter kits

27 Questions? Xenometrix AG Gewerbestr. 25 CH-4123 Allschwil Switzerland

28 Rel. Sensitivities of InCytotox Assays (Cells per well, L929 cells) Lower Upper LDH XTT SRB NR

29 NR log IC50 (µm) NR log IC50 (µm) ATP vs. NR vs. XTT IC50 ATP vs. NR (48 hr incubation) R 2 = IC50 XTT vs. NR (48 hr incubation) ATP log IC50 (µm) 3.5 From Choi et al., R 2 = FU XTT log IC50 (µm)