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(µg equiv./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 (ng equiv./cm 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).