INTEGRATED TOXICOLOGY (3Rs IN ACTION) AND HOW IN ......REGULATORY TOXICOLOGY AND PHARMACOLOGY 82;...

INTEGRATED TOXICOLOGY (3RsIN ACTION) AND HOW IN VITROTOXICOLOGY IS CRITICAL FOR SUCCESS FOR A DERMAL INDClive Roper BSc PhD CBiol CSci MRSB

28 September 2017

NorCal SOT, South San Francisco, CA, USA

EVERY STEP OF THE WAY

EVERY STEP OF THE WAY

INTRODUCTION

A LITTLE HISTORY

3 EVERY STEP OF THE WAY

In vitro testing has been mainstream in toxicology since the 1970’s• In discovery, on and off target efficacy screens are performed in cellular, and

increasingly, computational (in silico) models

• The hERG channel test and computational models are well integrated into the safety pharmacology testing paradigm

• A genetic toxicology programme is initiated with screening in vitro and in silico assays then progresses to GLP in vitro bacterial and mammalian cellular and finally rodent in vivo

This presentation focuses on utilizing 3Rs approaches to integratedin silico, in chemico, in vitro and in vivo for dermal IND

WHERE DID THE 3Rs START?Russell WMS and Burch RL (1959) The Principles of Humane Experimental Technique. Methuen, London.


3Rs

REPLACEMENT

3Rs

REDUCTION

3Rs

REFINEMENT

1959

1959• Politics

– Dwight D Eisenhower was US President

– Harold McMillan was UK Prime Minister

• Music

– Mack the Knife – Bobby Darin

– A fool such as I – Elvis Presley

– Pillow talk – Doris Day

• Sport

– Los Angeles Dodgers won the World Series Baseball

– Boston Celtics won the NBA Basketball

• Politics

• Donald J Trump is the US President

• Theresa May is the UK Prime Minister

• Music

• Too Good at Goodbyes - Sam Smith

• Sport

• Who will be in the World Series Baseball?

• Will the Cubbies repeat 2016?

• Who will be in the NBA Basketball?

2017

SCIENTIFIC ORGANISATIONSThis is NOT an exhaustive list


EURL-ECVAM

European Union Reference Laboratory for Alternatives to Animal Testing

JaCVAM

Japanese Centre for the Validation of Alternative Methods

ICCVAM

Interagency Coordinating Committee on the Validation of Alternative Methods

NC3Rs

UK National Centre for 3Rs

NA3RsC

North American 3Rs Collaborative (www.NA3RsC.org)

NORTH AMERICAN 3RS COLLABORATIVEhttp://www.na3rsc.org/home.html


http://www.na3rsc.org/home.html

WHAT IS INTEGRATED TOXICOLOGY?

WHAT IS INTEGRATED TOXICOLOGY?There are many different definitions!


We define this as

A testing strategy or regime that utilises the best tests available (in silico, in chemico, in vitro or in vivo) in order to confirm the test article safety

Use the right tools in your toolbox for each test article!!!

REGULATORY ACCEPTANCE OF AN INTEGRATED TOXICOLOGYExample: Genetic Toxicology


Testing strategies include:• In silico (e.g. LHASA, Leadscope, Simulations Plus)• Bacterial (bacterial reverse mutation assay aka Ames)• Mammalian (chrom abs, MLA, in vitro micronucleus)• In vivo mammalian (rodent micronucleus, comet)

Regulators accept these strategies• Pharma ICH S2(R1) & M7• Reach, consumer, agrochemicals• industrial chemicals …

A SIMPLIFIED MODEL OF INTEGRATED TOXICOLOGYPositive & negative feedback loops


in silico

in vivo

in vitro

in chemico

+ve/ -ve feedback

toxicity

efficacy

WHAT IS IN VITRO TOXICOLOGY?

WHAT IS IN VITRO TOXICOLOGY?https://en.wikipedia.org/wiki/In_vitro_toxicology


“In vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and food additives”

https://en.wikipedia.org/wiki/In_vitro_toxicology

WHAT IS IN VITRO TOXICOLOGY?


Is it new? No

What does it do? Replaces in vivo, works alongside in vivo, answers different questions (e.g. AOPs)

Does it replace in vivo models? Sometimes

Is it validated? Sometimes

Do regulatory authorities accept it? Sometimes

Can strategic decisions be made? Yes

Do they only involve human samples? No

It’s not simple to answer these and other questions

In my opinion!

3Rs

TOPICAL, DERMAL ORTRANSDERMAL?

TOPICAL, DERMAL OR TRANSDERMAL DRUGDefinitions


Topical drug• Active on the skin e.g. treatment for hospital MRSA

Dermal drug• Active in the skin e.g. treatment for basal cell carcinoma or actinic keratosis

Transdermal drug• Active elsewhere, i.e. requires to be delivered via the systemic circulation

e.g. hormonal drug therapy and nicotine replacement

A FOCUS ON DERMAL

APPLICATIONS FOR DERMAL PRODUCTSIn vitro skin penetration/ distribution


In vitro skin penetration/ distribution with human skin are used• to screen in new actives at early discovery

• to support development & aid selection of formulations at lead optimisation

• in support for choosing whether the drug should be used for

• topical, dermal or transdermal

• to repurpose drug candidates

• that have been deselected due to, for example,

• poor oral bioavailability

• high first pass metabolism

DERMAL ABSORPTION AND DISTRIBUTIONA brief introduction


COMPARE SPECIES FOR TRANSLATIONHuman versus Rat

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Skin from toxicology species are tested alongside human skin to deriveestimates or to confirm safety and efficacy for translational toxicology

COMPARE FORMULATIONSUse flux for transdermal delivery

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Select formulations with the chosen characteristics for the test item, remain on skin, target the skin or for transdermal drug delivery

ABSORPTION AND STRATUM CORNEUM TOGETHERExamine the entire data together for decision making


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Tape Strip No.

TIME COURSE DISTRIBUTIONPendlington et al. (2008). Development of a Modified In Vitro Skin Absorption Method to Study the

Epidermal/Dermal Disposition of a Contact Allergen in Human Skin. Cutaneous and Ocular Toxicology, 27: 283–294

24

A CASE STUDY: BUTENAFINE HCl in LOTRIMIN ULTRA®


GLP full mass balance studies can provide clinical trials justification or even to replace them


Lotrimin Ultra® contains• butenafine hydrochloride (1%, w/w ) – active ingredient

• diethanolamine (DEA) at 0.3% (w/w) as pH adjuster

BUT, DEA became a listed substance on California's Proposition 65 (June 13)

SO, reformulate Lotrimin Ultra®• by replacing DEA with triethanolamine (TEA) at 0.43% (w/w) – molar

equivalent

BUT, there was a need to confirm bioequivalence!!!

An in vitro skin penetration & distribution study was designed, following discussions with the US FDA, as a surrogate for a clinical bioequivalence test

REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 14-19Mitra A, Kim N, Spark D, Toner F, Craig S, Roper C and Meyer T (2016). Use of an in vitro human skin permeationassay to assess bioequivalence of two topical cream formulations containing butenafine hydrochloride (1%, w/w)


FDA asked us to consider and answer 3 components• Full FDA bioanalysis method

• Demonstrate differences in a single formulation in a single donor for

• 50%, 100% and 150% of required butenafine HCl concentration

• Main test for equivalence between

• New and old formulations



The LC-MS/MS method was validated for • Selectivity, sensitivity, linearity of the calibration curve,

• precision & accuracy, recovery, stability and dilution integrity

According to the• US FDA guidance document for bioanalytical method validation (FDA, 2001)

• EMA guidelines on bioanalytical validation (EMA, 2012a,b) and

• VICH GL1 and VICH GL2 guidelines for validation of analytical procedures

• VICH GL1 (Validation Definition) and

• VICH GL2 (Validation Methodology)

• October 1998; effective October 1999



Concentration differences detectable in the method were tested by• Preparing the formulation containing DEA with either

• Butenafine HCl at 0.5, 1.0 and 1.5%, w/w

• Obtaining full thickness human abdomen skin sample from a

• 34 years old female patient

• Dermatomed

• 18 samples of skin placed into Franz cells

• Barrier test

• Each formulation was applied at 2 mg/cm2 to

• 6 skin samples from this same donor



Samples collected• Receptor fluid (represents systemically available)

• 0 h (predose), 1, 2, 4, 8 and 24 h post dose

• Skin washed & dried

• 24 h post dose

• Stratum corneum removed with 20 tape strips

• 24 h post dose

• Exposed epidermis and dermis separated

• 24 h post dose

Samples analysed by LC-MS/MS



Distribution (% Applied Dose)


Tested Cream 0.5% (w/w) 1.0% (w/w) 1.5% (w/w)

Dislodgeable Dose 83.58 86.39 90.75

Stratum Corneum 5.01 4.45 3.56

Unexposed Skin 0.11 0.06 0.01

Total Unabsorbed Dose 88.70 90.91 94.31

Total Absorbed Dose LLOQ LLOQ LLOQ

Dermal Delivery 2.51 2.46 1.44

Mass Balance 91.21 93.37 95.75

By mass (µg/cm2), very significant differences were observed betweenthe matrices i.e. the method could determine formulation differences


Main test for equivalence between New and Old formulations• Only differences

• 24 samples of skin obtained from 6 different donors

• Each of the 2 formulations were applied to

• 12 samples of skin obtained from 6 donors in duplicate

• Same donors used for both formulations




Tested Cream DEA TEA

Dislodgeable Dose 85.95 85.66

Stratum Corneum 4.40 4.15

Unexposed Skin 0.15 0.01

Total Unabsorbed Dose 90.49 89.82

Total Absorbed Dose 0.04 0.04

Dermal Delivery 1.71 2.16

Mass Balance 92.20 91.99

Distribution (% Applied Dose)

Based on t-tests, no significant difference between old & new formulations observed for in vitro skin deposition & absorption of butenafine HCl



The results of the study demonstrating similarity of the two formulations was accepted by the FDA and resulted in authorization to market the new Lotrimin Ultra® cream

WITHOUT a clinical trial

Many forms of REPLACEMENTS!

3Rs

REPLACEMENT

REFOCUS ON DERMAL

LOCAL EFFECTSRisks must be quantified or, ideally, ruled out


In silico QSAR models are predicting these, and other, toxic outcomes• Lhasa, Leadscope etc

In vitro human tissue tests are increasingly replacing the in vivo animal tests• Ocular irritation and severe damage

• BCOP and EpiOcular versus ocular Draize

• Skin irritation and corrosion

• EpiSkin versus dermal Draize

• Skin sensitization

• DPRA, KeratinoSens, U-Sens/ hCLAT versus LLNA or Buehler

• Skin 3T3-NRU is Tier 1 in phototoxicology

OCULAR IRRITATION AND DAMAGEIn vitro screen and replacement


No single replacement test can distinguish NC, Class 1 and Class 2

BCOP (OECD 437)• Most Class 1 & NC

• Not enough information

EpiOcular (OECD 492)• NC & positive (cannot distinguish between Class 1 and Class 2)

3Rs

REPLACEMENT

3Rs

REDUCTION

DERMAL IRRITATION AND CORROSIONEpiSkin


EpiSkin corrosion (OECD 431) and EpiSkin irritation (OECD 439) distinguish between

• Not Classified, Irritation and Corrosion

• Cytotoxicity assay using MTT assessment by colorimetric change

3Rs

REPLACEMENT

DERMAL IRRITATION AND CORROSIONCorrositex


In vitro membrane barrier test method for skin corrosion aka Corrositex(OECD 435) is an in chemico model for corrosion testing

• Can be used in conjunction or replacement for EpiSkin Corrosion

• Simple kit test

• Time to color change

3Rs

REPLACEMENT

AN EXAMPLE OF ADERMAL IND PROGRAMME

A typical example for a dermal drug could be as follows

CANDIDATE SELECTION – ELIMINATING KEY LIABILITIES


Candidate Drug

In vitro &

in vivo PK

Genotoxicity

Cellular Toxicity

CYP Inhibition

Bioanalysis

Formulation Development

hERGLiability

RGA Method Development

A Dermal IND

3Rs

REPLACEMENT

3Rs

REDUCTION

3Rs

REFINEMENT

DMPK


Screening• Early PK to determine systemic exposure

Species selection• In vitro metabolic profiling in hepatocyte/microsome• Rats & minipig are often chosen

Screening skin penetration & distribution• Franz cell in vitro skin penetration & distribution assay

• Dermal drug candidate• Formulation selection• Translation; human versus pig versus rat

A Dermal IND

LOCAL TOXICITY


Phototoxicity• In vitro 3T3 neutral red uptake (first tier assessment)• In vivo phototoxicology programme

Dermal Sensitization• In silico LHASA Derek• In chemico DPRA• In vitro KeratinoSens and hCLAT or U-Sens• Buehler assay (guinea pig) or LLNA (mouse)

Skin Irritation and Corrosion• In vitro EpiSkin• In chemico Corrositex• In vivo in rabbits – when negative in vitro• or where regulator specifically requests confirmation in vivo)

A Dermal IND

SAFETY PHARMACOLOGY


Screening• In vitro ion channels

• hERG• (NaV1.5, CaV2.5 etc)

• (CiPA)

In vitro hERG assay

Rodent respiratory function assay (IV/ SC)

Modified rodent Irwin test (IV/ SC)

Non rodent cardiovascular telemetry (IV/ SC)

A Dermal IND

GENETIC TOXICOLOGY


Screening• Ames MPF• Screening versions of GLP assays• BlueScreen/ GreenScreen• 3D skin (EpiDerm) comet or micronucleus

In silico (e.g. LHASA Derek, Leadscope)

In vitro (bacterial reverse mutation assay; Ames)

In vitro (chromosome aberration assay)

In vitro (micronucleus)

Rodent micronucleus assay with/without Comet

A Dermal IND

3Rs

REFINEMENT

3Rs

REPLACEMENT

GENERAL TOXICOLOGYA Dermal IND


Rodent DRF (IV/ SC) including TK

Rodent 28 day toxicity (IV/ SC) including TK

Non rodent MTD (dermal) including TK

Non-rodent 28 day toxicity (dermal) including TK

Consider add-on pigA for genetic toxicology

Ocular irritation• BCOP and/or EpiOcular

• If positive for dermal irritation

3Rs

REFINEMENT

3Rs

REPLACEMENT

NOTEA Dermal IND


A testing programme should be planned on a case-by-case basis and, if possible, reviewed by a regulator

A THOUGHT TOTHE FUTURE

WHERE WILL THESE TECHNOLOGIES GO?


On a Chip Technologies

Organoids3D Culture/ Microtissues

Content-basedPrincipally cell and tissue biology based approaches

3D Bioprinting Organ-on-a Chip

Engineering-basedPrincipally relying on microfabrication based platforms

WHERE WILL THESE TECHNOLOGIES GO?


On a Chip Technologies

Organoids3D Culture/ Microtissues

Content-basedPrincipally cell and tissue biology based approaches

3D Bioprinting Organ-on-a Chip

Engineering-basedPrincipally relying on microfabrication based platforms

FOR ANOTHER PRESENTATION

IN CONCLUSION


In conclusion, a well planned and executed integrated toxicology testing programme will

• apply many aspects of the 3Rs

• whilst delivering reduced cost,

• improved regulatory compliance and

• the best chance of success for efficacy and safety

This is already a reality in modern toxicology!!!

3Rs

REPLACEMENT

3Rs

REDUCTION

3Rs

REFINEMENT

INTEGRATED TOXICOLOGY (3Rs IN ACTION) AND HOW IN ......REGULATORY TOXICOLOGY AND PHARMACOLOGY 82;...

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