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DMPK and Toxicity

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ADME

Absorption in Caco-2 Monolayer

Intestinal barrier permeability and toxicity in transwell.

Caco-2 barrier model, grown in a polarized culture system, is used

to evaluate the permeability of the test compounds. Compounds

loaded in the upper compartment are thereafter quantified in the

lower by LC-MS or LC-MS/MS analysis in order to calculate the

permeability coefficient (Papp value) .

Compounds are classified based on their solubility and

permeability according to the Biopharmaceutical Classification

System (BCS).

1 concentration (maximum non-toxic)

2 time points

Triplicates

A (Apical) to B (Basal) transport (B to A also available)

Transport buffer pH 7.4

Integrity marker: Lucifer Yellow (LY)

Permeability Controls: Atenolol/Metoprolol

Analytical method: LC_MS or LC-MS/MS

Endpoint: Coefficient of apparent permeability (Papp value) in cm/s

The application of a compound to a cell culture may induce in

some cases a cytotoxic effect depending on the concentration,

thus affecting the correct interpretation of observed effects. To

circumvent this problem, the cytotoxic effect of a concentration

range of the test compound, covering those used in the

permeability assay, is assessed.

3 concentrations

2 time points

Triplicates

A (Apical) to B (Basal) transport (B to A also available)


Integrity marker: Lucifer Yellow (LY)

Toxicity Control: 1% SDS

Analytical method: MTT/WST-1 (Absorbance)

Endpoint: % viability over control (vehicle)

ADME

Transporter Assays in MDCKII cell model

Efflux pumps (Pgp, BCRP and OATP2B1) play a key role in the

active transport mechanisms of drugs in the mammalian intestine

tissue.

Net efflux ratio calculated by performing a bidirectional study

(apical to basolateral (A-B) Papp data and basolateral to apical (B-

A) Papp data) either in Caco-2 cells or WT and Pgp, BCRP and

BCRP/OATP2B1-overexpressing MDCKII assays identifies which

compounds are substrates of these transporters (see decision

trees FDA Guidance 02/2012).

1 concentration (maximum non-toxic)

2 time points

Triplicates

A (Apical) to B (Basal) and B to A transport


Integry marker: Lucifer Yellow (LY)

Permeability Controls: Atenolol/Metoprolol

Transport substrates:

Digoxin (Pgp)

Estrone 3-sulphate (BCRP and OATP2B1)

Transport inhibitors:

Verapamil (Pgp),

KO134 (BCRP)

Erlotinib (OATP2B1)

Analytical method: LC-MS/MS

Endpoint: Efflux ratio = Papp (A-B)/ Papp (B-A)

ADME

Rapid biotransformation of compounds is not usually desired.

Therefore, drug metabolism is a key issue that needs to be

addressed at the early stages of drug development to improve

drug candidate selection. Hepatic Stability is assayed by

incubating test compounds with liver subcellular fractions

(microsomes, S9) or/and cultured isolated hepatocytes. Thereafter

generated metabolites are analyzed by LC-MS or LC-MS/MS. The

same protocol is followed to determine the Intrinsec Clearance

although samples are taken at 5 different times in order to

calculate compound’s in vitro half-life.

Hepatocytes and liver subcellular fractions

Species: Rodent and non-rodent

2 concentrations (maximum non-toxic)

3 time points for hepatic stability

5 time points for hepatic clearance

DuplicatesPositive control: Warfarin

Analytical method: LC-MS or LC-MS/MS

Endpoint: - % of compound disappearance over time 0 (hepatic

stability)- Half-life value (hepatic clearance)

Metabolic Stability & Intrinsic Clearance

ADME

Metabolic profiling

Metabolite detection/identification

Animals show significant metabolic differences when compared

with humans. Consequently, comparison of the metabolic profile of

a new drug across animal species, contributes to a more rationale

selection of the best species for preclinical toxicological studies.

Metabolites recovered after incubating test compounds with

hepatocytes or/and liver subcellular fractions are separated by

chromatography and analyzed by MS. At advanced stages of drug

development identification, generated metabolites and

determination of their chemical structures are also performed.

Hepatocytes and liver subcellular fractions

Species: Rodent and non-rodent


1 time point

Duplicates

Analytical method: LC-MS, LC-MS/MS, LC-Q-TOFMS

Endpoint: Major metabolite formation, Structure elucidation

CYP450 Phenotyping - Enzyme mapping

Knowledge of major enzymes involved in drug metabolite

formation can circumvent alterations in the metabolism of drugs.

Incubation of test compounds with microsomes individually

expressing human recombinant P450 isoenzymes contribute to

identify isoenzyme contributing to drug biotransformation.

Microsomes individually expressing human CYP isoforms

1 concentration

1 time point

Duplicates

Positive controls: Model substrates of most relevant human CYP

isoforms (FDA Guidance 02/2012)


Endpoint: Compound disappearance

ADME

Drug-Drug Interactions

CYP450 isoenzymes account for approximately 75% of total drug

metabolism and bioactivation. Many drugs can increase (enzyme

induction) or decrease (enzyme inhibition) the activity of these

isoenzymes, constituting a major source of adverse drug interactions.

CYP Induction

Test compounds are incubated with cultured human hepatocytes.

Thereafter a cocktail containing classical drug-metabolizing

enzyme substrates for inducible human isoforms CYP1A2, 2A6,

2B6, 2C9, 2C19 and 3A4 are added for an additional 2-h period.

Cultured human hepatocytes


1 time point

Triplicates

Positive controls: Methylcholanthrene, Rifampicin, Phenobarbital (FDA

Guidance 02/2012)


Endpoint: Fold-induction over control (vehicle)

CYP Inhibition

Microsomes expressing human recombinant P450 isoenzymes are

exposed to different concentrations of test compounds in the

presence of a specific substrate for each isoform.

Microsomes individually expressing human CYP isoforms

3 concentrations

1 time point

Triplicates

Substrates: Phenacetin, Coumarin, Bupropion, Diclofenac,

Mephenytoin, Midazolam (FDA Guideline 02/2012)

Inhibitors: Furafylline, Methoxalen, Tranylcypromine, Sulfaphenazole,

Ketoconazole (FDA Guidance 02/2012)


Endpoint: IC50

ADME

Protein Binding

Plasma protein binding can determine compound’s

pharmacokinetic behaviour ending in a profound effect on dosage

adjustments within the therapeutic range. Dialysis of compounds

in the absence/presence of plasma by the rapid equilibrium

dialysis technique allows quantifying free and plasma-bound

compounds.

3 concentrations

1 time point

Triplicates

Positive control: Warfarin

Analytical method: LC-MS or LC-MS/MS

Endpoint: % of bound compound to plasma

Toxicity and Safety

Acute systemic toxicity

OECD GD 129

BALB/c 3T3 Neutral Red Uptake Assay (3T3 NRU assay)Cytotoxicity test (MTT test) in mouse 3T3 fibroblast cells.

Normal Human Keratinocyte Neutral Red Uptake Assay (NHK

NRU assay)Cytotoxicity test in Normal Human Keratinocytes.

Acute systemic toxicity assessment through different assays,

on several species, in different cell types and systems.NRU, LDH release, MTT, WST-1, Resazurin test, ect. in human or

animal primary cells or cell lines (kidney, liver, pancreas, intestinal,

skin, etc.), among others, on demand.

ECVAM report on Acute systemic toxicity (2002) and

INVITTOX Protocol nº 51

LLC-RK1 Cell Test for NephrotoxicityCytotoxicity, Barrier integrity (Transepithelial resistance,TEER)

and paracellular permeability in LLC-PK1 (kidney proximal tubule

cell line).



MDCK test for acute toxicityCytotoxicity, Barrier integrity (Transepithelial resistance,TEER) and

paracellular permeability in MDCK (dog kidney epithelial cell line).



HepG2 Cell Test for HepatotoxicityCytotoxicity, Protein content and Cell growth. Morphology and

Cytoskeletal alterations, followed by Ph modifications in HepG2 liver

cell line (hepatoma).

Toxicity and Safety

Acute systemic toxicity


Chondrocyte functional toxicity testAlteration analysis on Proteoglycans production by chondrocytes

(Alcian Blue test) in Rabbit articular chondrocytes.

ECVAM report on Acute systemic toxicity (2002)

Haematotoxicity testAdenosine triphosphate (ATP) content, energy production and

metabolism y HL-60 human acute promyelocytic leukemia cell line.


Haematotoxicity test for acute neutropenia

Colony Forming Unit-Granulocyte/Macrophage (CFU-GM) Assay in

Human Cord Blood Mono Nuclear Cells (Hu-CBMNC) or Murine bone

marrow Mono Nuclear Cells (MNC)

Toxicity and Safety

Acute oral toxicity

OECD guideline nº 425

Up and Down procedureAnimal survival rate, LD50, periodically clinical observations, body

Weight and food/water consumption alterations, pathological analysis.

The assay could be performed in different rodent species (rat

preferred).


Repeat Dose 28-dayDaily clinical observations (health conditions, morbidity and mortality),

Functional test (sensory reactivity test, motor activity, ect.). Body

weight and food/water consumption alterations, Haematology,

biochemical analysis, gross necropsy and Histopathology. This assay is

performed in different rodent species.


Fixed Dose ProcedureAnimal survival rate, periodically clinical observations, body Weight and

food/water consumption alterations, pathological analysis. This assay

could be performed in different rodent species (rat preferred).


Acute Toxic Class MethodAnimal survival rate, periodically clinical observations, body Weight and

food/water consumption alterations, performed in different rodent

species (rat preferred).

Toxicity and Safety

Acute dermal toxicity

OECD GD 129

Basal cytoxicity test on skin cells

NRU, LDH release, MTT, WST-1, Resazurin test, ect. performed in

Human skin primary cells or cell lines (Human epidermal progenitor

cells, keratinocytes, dermal fibroblasts, melanocytes, HACAT, etc.),

among others on demand.

OECD guideline nº 402 (in vivo)

Acute dermal toxicityPeriodically clinical observations and pathological analysis in rat, rabbit

or guinea pig.


Repeated Dose Dermal Toxicity: 21/28-day StudyDaily clinical observations (health general conditions and toxicity

signs), haematology, biochemical analysis, gross necropsy and

histopathology in rat, rabbit or guinea pig.


Subchronic Dermal Toxicity: 90-day StudyDaily clinical observations (health general conditions and toxicity

signs), haematology, ophthalmological examination, biochemical

analysis, gross necropsy and histopathology in rat, rabbit or guinea

pig.

Toxicity and Safety

Skin corrosion


In Vitro Skin Corrosion: Transcutaneous Electrical Resistance

Test Method (TEER)TEER measurement and Sulforhodamine B dye permeation analysis in

rat skin discs.


In Vitro Skin Corrosion: Reconstructed Human Epidermis

(RHE) Test MethodCell Viability Measurements (MTT test) in Reconstructed Human

Epidermis (RHE).

Optional: Histological analysis.

Skin irritation

OECD guideline nº439

Reconstructed Human Epidermis (RHE) Test MethodCell Viability Measurements (MTT test) in Reconstructed Human

Epidermis (RHE).

Optional: Cytokine and inflammatory mediators release quantification

and histological analysis.

OECD guideline nº404 (in vivo)

Acute Dermal Irritation/CorrosionClinical observations and grading of the skin reaction (internal score) in

albino rabbit.

Toxicity and Safety

Ocular corrosives and severe irritants

identification


Bovine Corneal Opacity and Permeability Test Method for

Identifying Chemicals Inducing Serious Eye Damage and

Chemicals Not Requiring Classification for Eye Irritation or

Serious Eye DamageOpacity (light transmission through the cornea) quantification using an

Opacitometer and permeability of sodium fluorescein dye. Assay

performed in Bovine Eye (Following selection criteria detailed on the

OECD guideline).


Isolated Chicken Eye Test Method for Identifying Chemicals

Inducing Serious Eye Damage and Chemicals Not Requiring

Classification for Eye Irritation or Serious Eye DamageCorneal opacity, swelling, fluorescein retention, and morphological

effects performed in Chicken Eye (Following selection criteria detailed

on the OECD guideline).

Optional: Photographs acquisition.


Fluorescein Leakage Test Method for identifying Ocular

Corrosives and Severe IrritantsFluorescein permeability as an indicator of barrier function in MDCK

dog kidney epithelial cell line.

Toxicity and Safety

Eye irritation

INVITTOX protocol nº96

Hen’s Egg Test on the Chorio-allantoic Membrane (HET-CAM)Macroscopical observation of coagulation, haemorrhage and lysis of

blood vessels in the Chorio-allantoic Membrane in Hen's egg at day 10

after fertilisation.

Protocol Reference Pending (Under prevalidation phase by

ECVAM under a multicentric study)

Reconstructed Human Corneal Epithelium (RHCE) Test MethodCell Viability Measurements (MTT test) in Reconstructed Human

Corneal Epithelium (RHCE)

Optional: Cytokine and inflammatory mediators release quantification

and histological analysis.

Toxicity and Safety

Skin sensitization

OECD guideline nº442A (in vivo)

Local Lymph Node Assay (LLNA): DA

Proliferation of lymphocytes in the lymph nodes of the animals,

through the ATP content measurement by bioluminescence technique

(luciferase enzyme). Model: Mouse (CBA/J)

OECD guideline nº442B (in vivo)

Local Lymph Node Assay (LLNA): BrdU-ELISAProliferation of lymphocytes in the lymph nodes of the animals,

through the BrdU incorporation test. Model: Model: Mouse (CBA/J).

OECD guideline nº406 (in vivo)

Skin sensitizationClinical observations and grading of the skin reaction (internal score):

Erythema, swelling, etc. in Guinea pig.

Skin absorption

OECD Guideline nº 428

Skin absorption in vitro methodPermeation and skin absorption of the test item through skin by

chemical analytic techniques (UPLC/HPLC-UV, UPLC/HPLC-MS, HPLC-

MS/MS, HPLC-QTOF, etc) in Human and pig skin biopsies.

Toxicity and Safety

Phototoxicity


UV-VIS ABSORPTIONUV-VIS absorption spectrum analysis of the test item by

Spectrophotometric analysis.


In vitro 3T3 NRU phototoxicity testPhoto-Irritation-Factor (PIF) or Mean Photo-effect (MEF) calculation.

Classification of the product: No phototoxic, probable phototoxic or

phototoxic. Model: 3T3 murine fibroblast cell line.

Mutagenesis


Bacterial Reverse Mutation TestRevertant colonies cuantification (+/- S9). Data statistical analysis.

Strain Models: S. typhimurium (TA1535, TA1537, TA97, TA97a, TA98,

TA102 or TA100), E. coli (WP2 uvrA or WP2 uvrA (pKM101)).


In vitro Mammalian Cell Gene Mutation TestCytotoxicity and viability determination, colony quantification and

mutant frequencies calculation (+/- S9). Cellular models: L5178Y,

CHO, AS52, V79 or TK6 cells.

Toxicity and Safety

Genotoxicity


In vitro Micronucleous Test (Mnvit)Micronucleous quantification by fluorescence microscopy analysis (+/-

Cyt. B) in Cultured primary human peripheral blood lymphocytes and

cell lines (HL-60, CHO, L5178Y, etc.).

ASTM-E2186: Standard Guide for Determining DNA Single-

Strand Damage in Eukaryotic Cells Using the Comet Assay

COMET ASSAY in vitroDNA damage rate (Percentage DNA tail) in Cultured primary human

peripheral blood lymphocytes, hepatocytes, kupffer cells and cell lines

(HL-60, CHO, L5178Y, etc.).

Zebrafish toxicity models

Acute toxicity

Analysis of severe short-term effects of a new drug by incubation

of zebrafish embryos in 5 different concentrations in a logarithmic

progression of the molecule of interest.

Determination of the parameters at different time-points of

development. EC50, LC50, NOEC (No observed effect

concentration), LOEC (Lowest observed effect concentration) +

Teratogenicity:

Number of coagulated eggs.

Formation of somite.

Tail detachment.

Presence of heart-beat

Analysis of the long-term effect of the molecule of interest by

incubation of zebrafish embryos in NOEC concentration of the

molecule of interest for 21 days.

Observation of the following parameters:

Survival

No feeding inhibition

Normal behavior

Normal morphology and color

Chronic toxicity

At least 80 % of the cases long-term toxicity could be predicted by

results from studies with early life-stages, usage of zebrafish

embryo or larva in toxicology screenings is a potent tool to

streamline toxicity studies and reduce money expenditure.


Hepatotoxicity

Embryos of transgenic RFP liver from 5 days post fertilization (dpf)

to 7 dpf are incubated with the NOEC concentration of the

molecule of interest. Larvae are fixed and analyzed for the

following parameters:

Liver necrosis

Change in size/shape of the liver

Change in cell number and cell morphology

Yolk abnormality (yolk sac oedema)

Steatosis

Lethality

Cardiotoxicity

The majority of drugs that result in clinical problems interfere with

the human hERG.

Larvae of 48 hours post fertilization (hpf) of transgenic GFP heart

fishes are incubated with the NOEC concentration of the molecule

of interest and imaged during 3h. Monitoring of the following

dysfunctions:

Bradycardia

QT prolongation

Arrytmia

Fibrillation

Cardiac arrest


Ototoxicity

Different drugs show side effect to auditory system; hair cells

represent the sensory-transducing elements that transform the

mechanical force into electrical stimulus. Zebrafish has a

superficial organ composed by hair cells that is called neuromast

that enable its to detect vibration.

Larvae of transgenic GFP hair cells and neuromasts fishes are

exposed with the NOEC concentration from 3dpf of the molecule of

interest during 24hpf and then fixed and neuromasts counted.

Drug-induced neurological effects are remarkably common and

are among the most frequent reasons for poor drug compliance.

Larvae of transgenic fishes expressing GFP in a sub-population of

neurons exposed to the NOEC concentration of the molecule of

interest from 3 dfp to 4 dpf.

Larvae are fixed, and GFP neurons of defined region in the

prosencephalon are counted. Basal locomotor activity is also

measured.

Neurotoxicity

Citotoxic/Pro-Apoptotic molecules

Search for molecules that promote apoptosis through the

activation of p53 during development to find cytotoxic molecules

that could target highly proliferative cells, such as tumor cells.

Fluorescence TUNEL labeling of drug incubated embryos. Drugs

are added at 24 hpf and embryos are fixed at 48hpf for TUNEL

detection.

Zebrafish-based Cancer tests

Anti-angiogenic molecules

The growth and metastasis of tumors requires de novo

vascularization. Search for molecules that prevent angiogenesis

during development as a mean to find inhibitors or tumor

vascularization.

Lack of ISV (Intersegmental vessels) using transgenic fish

expressing GFP in the vessels, which labels by fluorescence all the

embryonic and adult vasculature.

Looking for null or affected vasculature pattern between control

and target individuals. Drugs are added at 24 hpf and embryos are

fixed at 48 hpf for imaging. Images are processed with Metamorph

for high-throughput analysis

Tubulin binding agents and other types of compounds are already

used by the industry as anti-cancer drugs: Taxol, Vinka alkaloids,

etc. This assays identify of molecules that prevent proliferation.

All type of compounds: Fluorescence pH3 labeling of drug

incubated embryos.

Only Tubulin Binding agents (Phenotypic assay): Tubulin inhibitors

affect epiboly. Therefore is possible to assess their phenotype

compared with control embryos by the relative position of the

embryo over the yolk.

Cell Cycle inhibitors


DNMTi (DNA Methyl Transferase Inhibitors)

Tumor suppressor genes experiment transcription block by hyper

methylation of their promoters in cancer cells. There are already

examples of DMNTi drugs in clinical trials (i.e.: Vidaza, Dacogen).

Search for molecules that prevent methylation as a mean to find

tumor suppressor activators during cancer progression using

following assays:

Phenotypic assay: Loss of Methylation during development cause a

no tail phenotype and somitic defects.

Fluorescence 5-methylcytosine labeling of drug incubated

embryos.

Oncogenes causing many types of leukemia function by

deregulating both proliferation and differentiation of hematopoietic

cells. Current treatments for leukemia focus primarily on

proliferation, but after aggressive cytotoxic treatment, 75% of

patients experience a recurrence within 2 years of remission.

Thus, targeting cell proliferation may be insufficient for eradicating

leukemia. Then, therapies that could reverse the effects of

oncogenes on LSC differentiation are promising alternatives or

complements to cytotoxic agents.

By using transgenic fishes overexpressing AML1-ETO,

erythropoiesis is convertedto granulopoiesis. That causes loss of

GATA1 and overexpression of Mpo in theblood lineage and

provides a model for searching molecules that prevent LSC

differentiation as a mean to find leukemia repressors.

Oncogene Hematopoietic Differentiation

Inhibitors


Neural Disorder Tests

ReadyCell is setting up different Neural Disorder tests based on

Zebrafish model. Do not hesitate in to contact us to learn more

about following assays:

Basal Activity

Learning and Memory

Fear and Anxiety

Social interaction

Cancer Signaling Pathways Disruption

When deregulated, developmental signaling pathways contribute

to cancerinitiation and progression. Thus, these pathways are

important cancer therapeutic targets.

Search for molecules that could modulate the activity of Cancer/

Developmental signaling pathways as a mean to find therapies to

fight cancer initiation or progression.

Assays using zebrafish fluorescence transgenic lines to find

repressor or activators of signaling pathways activity:

WNT Pathway reporter

BMP Pathway reporter

N pathway reporter

Shh pathway reporter

FGF pathway reporter

Observation of modulation of the fluorescent protein

expression in the reporter transgenic lines

Other Zebrafish-based tests

Bioinformatics

3D protein structure modeling: helps to analyze

interactions and to understand mechanisms of action.

Virtual screening: discover new active small molecules

for your target in a quick and easy way.

Protein-small molecule interaction analysis: to know

the key interaction points to guide the optimization of

your molecule.

Reprofiling/Side effects: to discover new uses for your

molecule, or to anticipate possible side effects.

Efficiency atlas: to navigate the chemico-biological

space and evolve your molecule to improve its

efficiency.

3D complex modeling: determine and

analyze macromolecular interactions that

helps to understand and modify their

behavior.

QSAR-3D: statistical analysis of 3D protein–small

molecule complexes to infer key points for

optimization and selectivity.

Transcriptomics

SAGE

SAGE (Serial Analysis of Gene Expression) is a powerful tool that

allows the analysis of overall gene expression patterns sequencing

between 10 and 14 nucleotides from the 5’ or 3’ end of

transcripts. SAGE enhances low abundance transcripts detection

and offers high reproducibility, being an effective and lower cost

alternative to whole-transcriptome sequencing.

RNA-Seq

The RNA-SEQ technique allows whole-transcriptome

characterization by Next-Generation Sequencing. RNA-SEQ

enhances low abundance transcripts detection, are highly

reproducible and identifies alternative isoforms from sequence

reads overlapping splice junctions and genomic variants as well.

miRNAs analysis

Identification and quantification of novel and known miRNAs, and

identification of thier targets as well. Study of their differential

expression and functional analysis.

Pharmacogenomics

Gene panels

Screening of specific gene regions, improving depth and reliability

offers a cost effective solution for current clinical needs. High

depth (>100x) and reliability (specificity>0.97) make Gene Panels

an ideal tool for patient characterization in clinical trials, and in

clinical diagnosis.

Whole exome

Drug response profiles and pathogenic variants associated with

genetic diseases could be monitored by Whole Exome analysis.

This tool allows the screening of the vast majority of known coding

regions in a unique sequencing process, identifying potential

genes linked with disease or drug effectiveness.

Consulting

Data Mining and Biostatistical Analysis

Technological advances allow the generation of larger volumes of

data, and demand new storage and processing solutions for

dealing with such massive datasets.

Sequence Analysis

Custom in silico projects

From experienced professionals to proprietary technology

and development, ReadyCell offers custom solutions for

in silico studies

Nucleotide and protein sequence analyses have become critical for

the interpretation of a broad spectrum of biologicalquestions.

Sequence Analysis includes a wide range of services as genome

annotation, functional prediction, phylogenetic studies or 3D

protein structure prediction.

Contact

ReadyCell S.L.Barcelona Science Park

Baldiri Reixac 10

08028 Barcelona

SpainTel +34 93 403 70 77

Fax +34 93 789 19 06

www.readycell.com

[email protected]

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