INTEGRATED TOXICOLOGY (3Rs IN ACTION) AND HOW IN VITRO TOXICOLOGY IS CRITICAL FOR SUCCESS FOR A DERMAL IND

Clive 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

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 integrated in silico, in chemico, in vitro and in vivo for dermal IND

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

3Rs 3Rs 3Rs

REPLACEMENT REDUCTION REFINEMENT

4 EVERY STEP OF THE WAY 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 2017 • 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? SCIENTIFIC ORGANISATIONS This 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)

7 EVERY STEP OF THE WAY NORTH AMERICAN 3RS COLLABORATIVE http://www.na3rsc.org/home.html

8 EVERY STEP OF THE WAY 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!!!

10 EVERY STEP OF THE WAY REGULATORY ACCEPTANCE OF AN INTEGRATED TOXICOLOGY Example: 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 …

11 EVERY STEP OF THE WAY A SIMPLIFIED MODEL OF INTEGRATED TOXICOLOGY Positive & negative feedback loops in silico

+ve/ -ve feedback in chemico

toxicity

efficacy in vivo in vitro 12 EVERY STEP OF THE WAY 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”

14 EVERY STEP OF THE WAY WHAT IS IN VITRO TOXICOLOGY? In my opinion! 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 3Rs Can strategic decisions be made? Yes Do they only involve human samples? No

It’s not simple to answer these and other questions

15 EVERY STEP OF THE WAY TOPICAL, DERMAL OR TRANSDERMAL? TOPICAL, DERMAL OR TRANSDERMAL DRUG Definitions

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

17 EVERY STEP OF THE WAY A FOCUS ON DERMAL APPLICATIONS FOR DERMAL PRODUCTS In 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

19 EVERY STEP OF THE WAY DERMAL ABSORPTION AND DISTRIBUTION A brief introduction

20 EVERY STEP OF THE WAY COMPARE SPECIES FOR TRANSLATION Human versus Rat 1.5

) 1.2 2

0.9

0.6

0.3 Cumulative Absorption (µg equiv./cm Absorption(µgequiv./cm Cumulative 0.0 0 4 8 12 16 20 24 Time (h) Skin from toxicology species are tested alongside human skin to derive estimates or to confirm safety and efficacy for translational toxicology COMPARE FORMULATIONS Use flux for transdermal delivery

40

35

30

/h) 2 25

20

15

Flux (ng equiv./cm equiv./cm (ng Flux 10

5

0 0 4 8 12 16 20 24 Time (h) 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 TOGETHER Examine the entire data together for decision making

60

50

/h) 2 40

60

) 2

30 50

20

40 Absorption (ng (ng equiv./cm Absorption

10 30

0 20 0 4 8 12 16 20 24 Time (h) 10

Terminal Distribution of Radioactivity (ng equiv./cm (ng Radioactivity of Distribution Terminal 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

23 EVERY STEP OF THE WAY Tape Strip No. TIME COURSE DISTRIBUTION Pendlington 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

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

27 EVERY STEP OF THE WAY REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 14-19 Mitra A, Kim N, Spark D, Toner F, Craig S, Roper C and Meyer T (2016). Use of an in vitro human skin permeation assay 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

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

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

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

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

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

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

31 EVERY STEP OF THE WAY REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 14-19 Mitra A, Kim N, Spark D, Toner F, Craig S, Roper C and Meyer T (2016). Use of an in vitro human skin permeation assay to assess bioequivalence of two topical cream formulations containing butenafine hydrochloride (1%, w/w) 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 between the matrices i.e. the method could determine formulation differences

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

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

33 EVERY STEP OF THE WAY REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 14-19 Mitra A, Kim N, Spark D, Toner F, Craig S, Roper C and Meyer T (2016). Use of an in vitro human skin permeation assay to assess bioequivalence of two topical cream formulations containing butenafine hydrochloride (1%, w/w) Distribution (% Applied Dose) 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 Based on t-tests, no significant difference between old & new formulations observed for in vitro skin deposition & absorption of butenafine HCl

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

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 3Rs Many forms of REPLACEMENTS!

REPLACEMENT

35 EVERY STEP OF THE WAY REFOCUS ON DERMAL LOCAL EFFECTS Risks 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

37 EVERY STEP OF THE WAY OCULAR IRRITATION AND DAMAGE In 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 3Rs

REPLACEMENT 38 EVERY STEP OF THE WAY REDUCTION DERMAL IRRITATION AND CORROSION EpiSkin 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

39 EVERY STEP OF THE WAY REPLACEMENT DERMAL IRRITATION AND CORROSION Corrositex 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

40 EVERY STEP OF THE WAY REPLACEMENT AN EXAMPLE OF A DERMAL IND PROGRAMME

A typical example for a dermal drug could be as follows CANDIDATE SELECTION – ELIMINATING KEY LIABILITIES A Dermal IND 3Rs 3Rs

In vitro & REFINEMENT REPLACEMENT in vivo PK

Genotoxicity hERG Liability Cellular Toxicity Candidate Drug 3Rs Formulation CYP Development Inhibition

REDUCTION Bioanalysis

42 EVERY STEP OF THE WAY RGA Method Development DMPK A Dermal IND 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

43 EVERY STEP OF THE WAY LOCAL TOXICITY A Dermal IND 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)

44 EVERY STEP OF THE WAY SAFETY PHARMACOLOGY A Dermal IND 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)

45 EVERY STEP OF THE WAY GENETIC TOXICOLOGY A Dermal IND Screening • Ames MPF 3Rs • Screening versions of GLP assays • BlueScreen/ GreenScreen • 3D skin (EpiDerm) comet or micronucleus REPLACEMENT In silico (e.g. LHASA Derek, Leadscope) In vitro (bacterial reverse mutation assay; Ames) In vitro (chromosome aberration assay) In vitro (micronucleus) 3Rs Rodent micronucleus assay with/without Comet

46 EVERY STEP OF THE WAY REFINEMENT GENERAL TOXICOLOGY A Dermal IND

Rodent DRF (IV/ SC) including TK Rodent 28 day toxicity (IV/ SC) including TK Non rodent MTD (dermal) including TK 3Rs Non-rodent 28 day toxicity (dermal) including TK

Consider add-on pigA for genetic toxicology REFINEMENT Ocular irritation • BCOP and/or EpiOcular • If positive for dermal irritation 3Rs

47 EVERY STEP OF THE WAY REPLACEMENT NOTE A Dermal IND

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

48 EVERY STEP OF THE WAY A THOUGHT TO THE FUTURE WHERE WILL THESE TECHNOLOGIES GO? On a Chip Technologies

Content-based Engineering-based Principally cell and tissue biology based approaches Principally relying on microfabrication based platforms 3D Culture/ Microtissues Organoids 3D Bioprinting Organ-on-a Chip

50 EVERY STEP OF THE WAY WHERE WILL THESE TECHNOLOGIES GO? On a Chip Technologies

Content-based Engineering-based Principally cell and tissue biology based approaches Principally relying on microfabrication based platforms 3D Culture/ Microtissues Organoids 3D Bioprinting Organ-on-a Chip

51 EVERY STEP OF THE WAY 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, 3Rs • improved regulatory compliance and • the best chance of success for efficacy and safety REFINEMENT

This is already a reality in modern toxicology!!! 3Rs

3Rs REDUCTION

52 EVERY STEP OF THE WAY REPLACEMENT