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

Transcription

1 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

2 INTRODUCTION

3 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

4 WHERE DID THE 3Rs START? Russell WMS and Burch RL (1959) The Principles of Humane Experimental Technique. Methuen, London Rs 3Rs 3Rs REPLACEMENT REDUCTION REFINEMENT 4 EVERY STEP OF THE WAY

5 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

6 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?

7 EURL-ECVAM JaCVAM ICCVAM NC3Rs NA3RsC SCIENTIFIC ORGANISATIONS This is NOT an exhaustive list European Union Reference Laboratory for Alternatives to Animal Testing Japanese Centre for the Validation of Alternative Methods Interagency Coordinating Committee on the Validation of Alternative Methods UK National Centre for 3Rs North American 3Rs Collaborative ( 7 EVERY STEP OF THE WAY

8 NORTH AMERICAN 3RS COLLABORATIVE 8 EVERY STEP OF THE WAY

9 WHAT IS INTEGRATED TOXICOLOGY?

10 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

11 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

12 A SIMPLIFIED MODEL OF INTEGRATED TOXICOLOGY Positive & negative feedback loops in silico +ve/ -ve feedback in chemico toxicity efficacy in vivo 12 EVERY STEP OF THE WAY in vitro

13 WHAT IS IN VITRO TOXICOLOGY?

14 WHAT IS 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

15 Is it new? No WHAT IS IN VITRO TOXICOLOGY? 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 In my opinion! 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 3Rs 15 EVERY STEP OF THE WAY

16 TOPICAL, DERMAL OR TRANSDERMAL?

17 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

18 A FOCUS ON DERMAL

19 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

20 DERMAL ABSORPTION AND DISTRIBUTION A brief introduction 20 EVERY STEP OF THE WAY

21 Cumulative Absorption (µg equiv./cm 2 ) 1.5 COMPARE SPECIES FOR TRANSLATION Human versus Rat Time (h) Skin from toxicology species are tested alongside human skin to derive estimates or to confirm safety and efficacy for translational toxicology

22 Flux (ng equiv./cm 2 /h) COMPARE FORMULATIONS Use flux for transdermal delivery Time (h) Select formulations with the chosen characteristics for the test item, remain on skin, target the skin or for transdermal drug delivery

23 Terminal Distribution of Radioactivity (ng equiv./cm 2 ) Absorption (ng equiv./cm 2 /h) ABSORPTION AND STRATUM CORNEUM TOGETHER Examine the entire data together for decision making Time (h) EVERY STEP OF THE WAY Tape Strip No.

24 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:

25 A CASE STUDY: BUTENAFINE HCl in LOTRIMIN ULTRA

26 GLP full mass balance studies can provide clinical trials justification or even to replace them 26 EVERY STEP OF THE WAY

27 REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 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

28 REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 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

29 REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 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 EVERY STEP OF THE WAY

30 REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 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/cm 2 to 6 skin samples from this same donor 30 EVERY STEP OF THE WAY

31 REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 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

32 REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 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 Stratum Corneum Unexposed Skin Total Unabsorbed Dose Total Absorbed Dose LLOQ LLOQ LLOQ Dermal Delivery Mass Balance By mass (µg/cm 2 ), very significant differences were observed between the matrices i.e. the method could determine formulation differences 32 EVERY STEP OF THE WAY

33 REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 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

34 REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 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) 34 EVERY STEP OF THE WAY Tested Cream DEA TEA Dislodgeable Dose Stratum Corneum Unexposed Skin Total Unabsorbed Dose Total Absorbed Dose Dermal Delivery Mass Balance Based on t-tests, no significant difference between old & new formulations observed for in vitro skin deposition & absorption of butenafine HCl

35 REGULATORY TOXICOLOGY AND PHARMACOLOGY 82; 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 Many forms of REPLACEMENTS! 3Rs REPLACEMENT 35 EVERY STEP OF THE WAY

36 REFOCUS ON DERMAL

37 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

38 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

39 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

40 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

41 AN EXAMPLE OF A DERMAL IND PROGRAMME A typical example for a dermal drug could be as follows

42 3Rs CANDIDATE SELECTION ELIMINATING KEY LIABILITIES A Dermal IND 3Rs REPLACEMENT In vitro & in vivo PK REFINEMENT herg Liability Formulation Development Candidate Drug Genotoxicity Cellular Toxicity CYP Inhibition 3Rs Bioanalysis REDUCTION 42 EVERY STEP OF THE WAY RGA Method Development

43 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

44 44 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)

45 Screening In vitro ion channels herg (NaV 1.5, CaV 2.5 etc) (CiPA) In vitro herg assay SAFETY PHARMACOLOGY Rodent respiratory function assay (IV/ SC) Modified rodent Irwin test (IV/ SC) A Dermal IND Non rodent cardiovascular telemetry (IV/ SC) 45 EVERY STEP OF THE WAY

46 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) A Dermal IND Rodent micronucleus assay with/without Comet 3Rs 3Rs REPLACEMENT 46 EVERY STEP OF THE WAY REFINEMENT

47 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 Ocular irritation BCOP and/or EpiOcular If positive for dermal irritation 3Rs REFINEMENT 47 EVERY STEP OF THE WAY REPLACEMENT

48 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

49 A THOUGHT TO THE FUTURE

50 3D Culture/ Microtissues WHERE WILL THESE TECHNOLOGIES GO? Content-based Principally cell and tissue biology based approaches Organoids On a Chip Technologies Engineering-based Principally relying on microfabrication based platforms 3D Bioprinting Organ-on-a Chip 50 EVERY STEP OF THE WAY

51 3D Culture/ Microtissues WHERE WILL THESE TECHNOLOGIES GO? Content-based Principally cell and tissue biology based approaches Organoids On a Chip Technologies Engineering-based Principally relying on microfabrication based platforms 3D Bioprinting Organ-on-a Chip 51 EVERY STEP OF THE WAY FOR ANOTHER PRESENTATION

52 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