State of Science for Respiratory Sensitization

State of Science for Respiratory Sensitization Dose-Response Models for the OEL-Derivation of Chemicals Jürgen Pauluhn Global Drug Discovery, Bayer HealthCare, Bayer Pharma AG, Wuppertal, Germany Hannover Medical School, Hannover, Germany ILSI/HESI-Workshop on Assessment of Respiratory Sensitization 8-9 May 1, Alexandria, VA 1

Outline Hypotheses, mechanisms & etiopathological relationship of induction and elicitation Test approaches & protocols Respiratory irritation vs. sensitization vs. allergy Metrics & dose Derivation of OELs for irritant asthmagens such as TDI-vapor & MDI-aerosol

Hypothesis: What do we intend to model and how do we define Sensitization? Skin and/or inhalation exposure C x t Inhalation exposure Lymph node priming & Th-cell polarization Systemic sensitization and induction of status asthmaticus are interconcatenated but independent processes! Occupational Asthma 3

What can be quantified and controlled? Skin induction quantifiable: NO Lung induction quantifiable-twa: YES Excursions: NO C x t Inhalation exposure quantifiable: YES aggravation Lymph node priming & Th-cell polarization Expression of cytokines irritation vs. allergy: NO Systemic sensitization Compartmentalized expression of immunoglobulines, e.g. IgE/G specific or total: NO Occupational Asthma Integrating phenotypes of OA: YES

Skin/Lung Induction: Dose-Metric-Regimen Biomarker of susceptibility vs. adversity Compartmentalization Inflammation response to irritant and/or allergic stimulus Neurogenic modulation 1 Reciprocal Relationship of Induction and Elicitation Dose metrics and induction sitespecific constraints Dose of Induction 1 8 Induction Elicitation Interrelationship of irritation, sensitization, and elicitation of sensitization Human relevance 8 1 Dose of Elicitation 5

Compartmentalization of PMNs in the BNR-Ova-Model Duplicated from Schuster et al. ()

Serum total IgE an Index of Inflammation or Allergy? 1 Topical induction x mg MDI/m³ x 3 min 8 Eosinophils 5 Counts [# 1 /Lung] IgE in Serum [ng/ml] 3 C-/- C-/+ Topical-low Topical-high 1 3 5 PMN C-/- C-/+ Top-low Top-high Low/high: /15 l MDI on both days and 7 Counts [# 1 /Lung] 15 1 x1 5 The most discriminatory endpoint of respiratory allergy is PMN in BAL (in BN rats). C-/- C-/+ Topical-low Topical-high 7

Situation more complex for reactive Vapors: C vs. C x t - Relationships Reactive vapor Neurogenic inflammation Reactive aerosol Nasal Reflexes Bronchial Airways the site of asthma manifestation Reactive aerosols: Site of deposition is aerosol size-dependent, the lung dose and POD is Cxt-dependent. Reactive vapors: The degree of penetration into lower airways is C-dependent. C determines the depth of penetration into the lung & Cxt the site-specific dose. 8

Respiratory Tract Irritation: Similarities to Skin? Definitely NOT! MDI Aerosol - Rats Threshold for acute alveolar irritation Normal range: 1±% 5 Regulatory stumbling blocks: RT = ET + T + P [US: separated, EU: combined] Aerosols & vapors dosimetrically different. Also the response to injury is entirely different ET-T-P irritation sites have to be identified Relative to Control [%] 3 15 1 9 8 Protein in BALF 1xh (postexposure day 1) 1xh (postexposure day 3) Cxt-dependent adaptation 1 1 Concentration [mg/m³] 9

Respiratory Tract Irritation: Selection of Challenge Cxt for Aerosols Protocol: Single h exposure of naïve BNor Wistar rats to MDI-aerosol C-dependence of BAL-protein after and 1-day post-exposure Acute LRT-threshold:.5 mg/m³ x h (18 mg MDI/m³ x min) Minimal LRT-threshold 3 mg/m³ x h (18 mg MDI/m³ x min) Converted to challenge duration: 3 mg/m³ x 3 min Experimental validation of Cxt needed Relative to Control [%] 5 3 15 Protein in BALF day day 1 y =.7+.31x; r =.99 1 9 8 1 1 Concentration [mg/m³] 1

Searching for the POD of Airway Irritation for Aerosols Sensitization (skin/lung) C x t 1 mg MDI/m³ x min day day 35 day 5 day 5 BAL, lung function day Naive BN Rats Change Relative to Control [%] 5 3 1 BAL LDH - 1 x h BAL Protein - 1 x h BAL LDH - x 3 min BAL Protein - x 3 min POD- Alveolar irritation LDH Protein 9 1 1 988 7 Concentration x Time [mg/m³ x min] 11

The Induction of Phenotypes of Asthma require a repeated Challenge Protocol Before Challenge After Challenge PMN in BALF [%] 5 3 C x t Challenge: x 15 mg MDI/m³ C-/+ topical inhalation (5 x 8 mg/m³ - 3 hrs/day) C-/---+ aggravation Penh 1 8 1 8 1 8 Chamber 1 / before MDI Challenge Chamber Chamber 3 topical - day 1 topical - day 35 topical - day 9 control - day 5 topical - day 5 Penh 1 8 1 8 1 8 Chamber 1 / after MDI Challenge topical - day 1 topical - day 35 topical - day 9 control - day 5 topical - day 5 Chamber Chamber 3 1 1 35 5 1 8 Chamber 1 1 1 1 8 Chamber Day 8 1 1 Time [min] 8 1 1 Time [min] MDI aerosol: optimally inhalation challenges are required to demonstrate the asthma response 1

Sensitization Efficacy (MDI): Topical vs. Inhalation C x t 1 mg MDI/m³ x min day day 35 day 5 day 5 or escalation BAL, lung function day Dose Induction Priming / Chall. 18 1 PMN C I-IV : mg MDI/m³ C I-III : mg MDI/m³ C V : 5-15- mg MDI/m³ C x t day (mg/m³ x min) 5x1-min (mg/m³) 5x3-min (mg/m³) x3-min 1 (L) 1 3 --- 5 (M) 5 15 --- Cytodifferentiation [%] 1 1 1 8 1 (H) 1 3 --- 1 (H) 1 -- --- 5-15- C- C+ MDI_1-L MDI_3-L MDI_1-M MDI_3-M MDI_1-H MDI_3-H C+_1-C5 MDI_1-C5 C+_1-C15 MDI_1-C15 C+_1-C MDI_1-C Topical (x) vs. Inhalation (5x): Topical (SD.±3%) > inhalation (SD.±5%); high-concentration x short-time protocol > low-concentration x long-time protocol; elicitation Cxt-dependent 13

Compartmentalization and Impact of Vehicle: BNR-MDI- Model Relative to Control (%) 5 3 1 IL- BALF BALC LALN Th -Priming Topical induction x mg MDI/m³ x 3 min ## # Control SEBA AOO MDI SEBA: di-n-octyl sebacic acid ester (%) AOO: acetone:olive oil (%) MDI: neat (same dose in all groups) Pro-inflammatory cytokines show different vehicle-effect relationships from one compartment to another. Relative to Control (%) Relative to Control (%) 5 15 1 5 18 1 1 1 1 8 IFN- BALF BALC LALN ## ## # ## ## Control SEBA AOO MDI IL-1 BALF BALC LALN Th 1 -Priming ## ## ## ## Control SEBA AOO MDI 1

Summary: Rationalization of Protocol Topical induction x mg MDI/m³ x 3 min 5 Absolute Cytodifferentiation Each compartment of the lung gives a different read-out. Cytodifferentials [x1 cells/lung] 15 1 5 Macrophages NC PMN Lymphocytes Eosinophils Foamy cells The response in LALNs using different vehicles for sensitization shows variable outcomes. BAL-PMN most appropriate effect-based discriminator for both irritation and sensitization. Control SEBA AOO MDI Concentration [mg/m³] SEBA: di-n-octyl sebacic acid ester (%) AOO: acetone:olive oil (%) MDI: neat (same dose in all groups) Lung priming & elicitation threshold: repeated encounters above irritation threshold Cxt required. 15

Searching for the POD of Airway Irritation of Vapors: Acute Exposure (TDI) Change relative to Preexposure Period [%] Change relative to Preexposure Period [%] 18 1 1 1 1 1 1 1 1 8 8 Minute Volume (BN) 1 3 5 air control 5 mg/m³ 1 mg/m³ 81 mg/m³ 113 mg/m³ 1% pre-exposure Time Time air control 5 mg/m³ 1 mg/m³ 81 mg/m³ 11 mg/m³ 113 mg/m³ Tidal Volume (BN) 1 3 5 Relative Depression of Ventilation [%] 8 Respiratory Minute Volume Brown Norway rat Wistar rat y BN =13.9+5.1(1-exp(-.7x)) y Wis =-1.1+9.5(1-exp(-.11x)) 8 1 1 Concentration [mg TDI/m³] C MUST be high enough to reach the alveoli The depression of ventilation MUST converge against stable breathing 1

Searching for the POD of Airway Irritation of Vapors: Recurrent Exposure (TDI) Volume [ml/min] 5 3 1 ppm.15 ppm. ppm.3 ppm 1 3 5 Time [min] Day Minute Volume-Day Day Minute Volume Minute Volume [ml/min] 5 3 1 day day day - last 3 min day - last 3 min Volume [ml/min] 5 3 1 Minute Volume - Day 1 3 5 Time [min] ppm.15 ppm. ppm.3 ppm 5 1 15 Concentration [mg/m³] Single exposure: POD =.3 x RD 5 ~.1 ppm. Repeated exposure: POD based on BMDL(95%) =.35 ppm (1 st day) and.8 ppm ( th day). Reason: apparent expression of TRPA receptors on pulmonary C-fibers. 17

Searching for the POD of Airway Irritation of Vapors: Recurrent Exposure (TDI) Cumulative Score 7 5 3 Nasal Cavity I Focal cell infiltration Inflammation Epithelial atrophy/degeneration Epithelial necroses Cumulative Score 3 1 Lung Mucus in ariways BALT activation Increased alveolar macrophages Focal cell infiltration 1 C TDI-L TDI-I TDI-H Group C TDI-L TDI-I TDI-H Group 7 Nasal Cavity IV Cumulative Score 5 3 Focal cell infiltration Inflammation Epithelial atrophy/degeneration Epithelial necroses POD-URTI =.8 ppm ( th day). Most TDI scrubbed between location 1-1 C TDI-L TDI-I TDI-H What we see in the lung is response to injury but not irritant-inflammation Group 18

Conceptual Approach for a Respiratory Sensitization / Elicitation Protocol for TDI-Vapor Prevailing experimental evidence suggests that Respiratory Sensitization is a multi-step process depending on two independent processes: - Induction of a state of increased susceptibility to future encounters. This process is potentially reversible (apart from memory effect ). - The induction of this process requires irritant (inflammatory) encounters at high doses. This can most suitably be achieved by skin exposure(s). - This state is evidenced by the determination of pro-inflammatory factors which do not necessarily distinguish the irritant and allergic etiopathologies. 19

Conceptual Approach for a Respiratory Sensitization / Elicitation Protocol for TDI-Vapor - Continuation Hence any Respiratory Sensitization can only be revealed and quantified by Respiratory Elicitation in animals predisposed to asthma - Repeated inhalation elicitation encounters above the lung irritant threshold dose (Cxt) are needed for progression & aggravation. - The induction of this process requires multiple highly rationalized irritant (mildly inflammatory) encounters at defined Cxt s to produce an asthmatic rat. - The elicitation-threshold in asthmatic rats is irritation (Cxt)- dependent.

Comparison of Respiratory Sensitization/Elicitation Protocols for TDI-Vapor Pilot Inhalation Escalation Challenge - C var x t const BAL-PMN [%] 3 %-TDI - 3x8 mg/m³ - escalation challenge 3-min 1%-TDI - 3x8 mg/m³ - escalation challenge 3-min %-TDI - 3x8 mg/m³ - escalation challenge 3-min 5%-TDI - 3x8 mg/m³ - escalation challenge 3-min linear Cxt no irritation-no response 1 5 1 15 5 5 Inhalation Escalation Challenge-IV C const x t var Concentration x Time [mg/m³ x min] Aerosols: Dosimetrically & physiologically reproducible (MDI) Vapors: Dosimetrically & physiologically very complex (TDI) Hence, C const x t var protocols are deemed to be most robust for both aerosols & vapors PMN [%] 3 1 Control / TDI TDI / TDI progressive Cxt no irritation-no response 1 3 5 Concentration x Time [mg/m³ x min] 1

Conceptual Approach for a Respiratory Sensitization / Elicitation Protocol for TDI-Vapor Pre-step: Irritation inhalation assays as typically used in inhalation toxicology as ancillary studies for the dose-selection of repeated inhalation exposure studies. Step I: Elicitation threshold after first inhalation challenge Induction Control Skin day day 7 1 x 85 mg/m³ day Elicitation challenge: C const x t var t = 1 3 min post-challenge day 1 Endpoints: 1. NO exhaled (pc &1). BAL-PMN (pc 1) 3. Delayed response (pc -1) Step II: Elicitation threshold after repeated inhalation challenges Induction Control Skin day day 7 Priming Challenge day 5 day 1 x 85 mg/m³ day 5 Elicitation challenge: C const x t var t = 1 3 min post-challenge day 1 Endpoints: 1. NO exhaled (pc &1). BAL-PMN (pc 1) 3. Delayed response (pc -1) 3 x 85 mg/m³ x 3 min t 15 days Human translation (HEC): 3 min x 85 mg/m³= 55 mg/m³ x min. HEC=55x1/7x1/(1x3x3)=. ppmv x 9 min;. ppmv x 9 min resulted in 33% responders in workers with TDI-asthma

Translational patho(physio)logical Hallmarks of Human and BNR-Asthma 8 eno-post-challenge days -1 combined 5 Escalation Challenge I NO adj [ppb] Control / TDI TDI / TDI NO in exhaled breath Cytodifferentials [x1 cells/lung] 15 1 5 Macrophages PMN Lymphocytes Eosinophils Foamy cells Non-classifiable cells Eos/PMN in BAL I-1 I-3 I- IV-1 IV-3 IV- C-1 TDI-1 C-3 TDI-3 C- TDI- Challenge Group Penh 1 1 8 3c/c control / TDI TDI / TDI Nocturnal asthma Cytodifferentials [x1 cells/lung] 5 15 1 5 Escalation Challenge IV Macrophages PMN Lymphocytes Eosinophils Foamy cells Non-classifiable cells PMN in BAL 8 1 1 Time [min] C-1 TDI-1 C-3 TDI-3 C- TDI- Group 3

Dose-Response Analysis: TDI-Vapor & MDI-Aerosol 5 TDI MDI Inhalation Escalation Challenge-IV C const x t var 5 BAL-PMN - Topical vs. Inhalation skin Control / TDI TDI / TDI MDI topical (x mg MDI/15 g bw) MDI inhalation 5x9 mg MDI/m³-1 min MDI inhalation 5x93 mg MDI/m³-1 min Naive challenged control (step I) PMN [%] 3 PMN [%] 3 1 NOAEL 1 NOAEL skin NOAEL lung Inhalation (5 x 1 mg/m³ x min) 1 3 5 Concentration x Time [mg/m³ x min].1 1 1 1 Concentration (mg/m³) TDI: The acute irritant threshold, which is.35 ppm x h for TDI, corresponds to ~1-times the elicitation threshold of 1/(7x3) mg/m³ x min =. ppm x h. MDI: The acute irritant threshold, which is.5 mg/m³ x h for MDI (18 mg/m³ x min), corresponds to ~-times an elicitation threshold of 9 mg/m³ x min. These apparent differences in potency are related to dosimetric differences (vapor vs. aerosol)

MDI: Dosimetric Adjustment & Species Extrapolation.5mg / m³ 3 min 8 min Irritation threshold rat-bal-protein.38 8hrs ( LRI) mg m³ Irritation threshold human.5mg / m³ 8 min 1 Irritation 3mg / m³ 3min 8 min 8-h workday adjustment ( rat).19 8hrs( RTS) rat-elicitation.19mg / m³ 3 Dosimetric adjustment (nasal:oronasal). 8hrs( RTS) Susceptibility adjustment 1 human OEL irritation-based OEL respiratory sensitization-based mg m³ mg m³.15mg / m³ 3min mg.1 8hrs 8 min m³.5mg (5 ppb) m³.1mg (1 ppb) m³ RTS: Respiratory Tract Sensitization; 1) Leroyer et al. (1998) Specific bronchoprovocation test 1 5 1 1 1 1 1 Allergy 5

TDI: Dosimetric Adjustment & Species Extrapolation Irritation threshold Irritation threshold 1 human mg mg ( RD5).3. ( URTI, mice) m³ m³ mg PODrepeated.1 3min ( URTI, rat) m³ mg.35mg / m³ 3 min.35 ( URTI) m³ 1 Irritation 8-h workday adjustment sensi 1mg / m³ min mg.1 8hrs( RTS) 8 min m³ Dosimetric adjustment.1mg / m³ 3 3 MV retention.1 mg (.3 ppmv ) m³ Susceptibility adjustment human OEL irritation-based.13mg / m³ 9min mg. 8hrs (.3 ppmv ) 8 min m³.1 73 3 MV ret mg m³ 8hrs URT.3 ppmv 1 1 Allergy OEL respiratory sensitization-based.3 ppmv RTS: Respiratory Tract Sensitization; 1) Henschler et al., 19; ) Vandenplas et al. (199), Sastre et al. 3); RD : Barrow et al. (1978)

Summary The major prejudice of Respiratory Sensitization is believed to depend on inhalation sensitization. It is likely more an inhalation priming of already predisposed/sensitized subjects. The protocols devised duplicate the key hallmarks of human asthma, including the structure & dosing protocols used in human inhalation bronchial challenges. All endpoints measured are quantifiable in terms of dose and integrated effect. For MDI-aerosol and TDI-vapor the dosimetrically-adjusted irritant dose was remarkably close to the effective human challenge dose. To be effective, elicitation doses must be above the irritant threshold Cxt to prime the respiratory tract for Respiratory Sensitization. 7

Conclusion Respiratory sensitization requires recurrent irritant inhalation exposures to induce asthma. Both irritation and elicitation of respiratory sensitization are clearly threshold dose- (and NOT concentration-) dependent. Protection from irritation (conventional basis of NOAEL) protects also from sensitizing the respiratory tract. OELs and DNELs can be derived using standard inhalation bioassays. Proof-of-principle studies may require a slightly higher degree of sophistication. 8