Bioavailability of protein therapeutics in rats following inhalation exposure: Relevance to occupational exposure limit calculations

Accepted Manuscript Bioavailability of protein therapeutics in rats following inhalation exposure: Relevance to occupational exposure limit calculations Janet C. Gould, Irvith Carvajal, Todd Davidson, Jessica Graham, Jedd Hillegass, Susan Julien, Alex Kozhich, Bonnie Wang, Hui Wei, Aaron P. Yamniuk, Neil Mathias, Helen G. Haggerty, Michael Graziano PII:

S0273-2300(18)30254-X

DOI:

10.1016/j.yrtph.2018.10.003

Reference:

YRTPH 4233

To appear in:

Regulatory Toxicology and Pharmacology

Received Date: 16 April 2018 Revised Date:

27 September 2018

Accepted Date: 2 October 2018

Please cite this article as: Gould, J.C., Carvajal, I., Davidson, T., Graham, J., Hillegass, J., Julien, S., Kozhich, A., Wang, B., Wei, H., Yamniuk, A.P., Mathias, N., Haggerty, H.G., Graziano, M., Bioavailability of protein therapeutics in rats following inhalation exposure: Relevance to occupational exposure limit calculations, Regulatory Toxicology and Pharmacology (2018), doi: https://doi.org/10.1016/ j.yrtph.2018.10.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Bioavailability of protein therapeutics in rats following inhalation exposure: relevance to

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occupational exposure limit calculations

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3 Janet C. Goulda, Irvith Carvajalb, Todd Davidsonb, Jessica Grahamb, Jedd Hillegassb*, Susan Julienb, Alex

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Kozhichb, Bonnie Wangb, Hui Weib, Aaron P. Yamniukb, Neil Mathiasb, Helen G. Haggertyb, Michael

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Grazianob

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SafeBridge Consultants, Inc., 330 Seventh Avenue – Suite 1101, New York, NY 10001, USA

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Bristol-Myers Squibb, 1 Squibb Drive, New Brunswick, NJ 08903, USA

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10 *Corresponding author:

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Jedd Hillegass, Bristol-Myers Squibb, 1 Squibb Drive, New Brunswick, NJ 08903, USA

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[email protected]; 732-227-6093

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Word count: Abstract (200); Text (7712); References (978)

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Abstract

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Protein therapeutics represent a rapidly growing proportion of new medicines being developed by the

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pharmaceutical industry. As with any new drug, an Occupational Exposure Limit (OEL) should be

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developed to ensure worker safety. Part of the OEL determination addresses bioavailability (BA) after

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inhalation, which is poorly understood for protein therapeutics. To explore this, male Sprague-Dawley

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rats were exposed intravenously or by nose-only inhalation to one of five test proteins of varying

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molecular size (10-150 kDa), including a polyethylene glycol-conjugated protein. Blood, lung tissue and

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bronchoalveolar lavage (BAL) fluid were collected over various time-points depending on the expected

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test protein clearance (8 minutes-56 days), and analyzed to determine the pharmacokinetic profiles. Since

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the BAL half-life of the test proteins was observed to be >4.5 hours after an inhalation exposure,

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accumulation and direct lung effects should be considered in the hazard assessment for protein

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therapeutics with lung-specific targets.

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inhalation exposure for all test proteins (~≤1%) which did not appear molecular weight-dependent. Given

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that this study examined the inhalation of typical protein therapeutics in a manner mimicking worker

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exposure, a default 1% BA assumption is reasonable to utilize when calculating OELs for protein

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therapeutics.

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The key finding was the low systemic bioavailability after

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Key words: Inhalation, Protein Therapeutic, Antibody, Pharmacokinetics, Occupational Exposure Limit,

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Bioavailability

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Abbreviations:

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AUC – area under the curve

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BA – bioavailability

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BAL – bronchoalveolar lavage

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BSA – bovine serum albumin

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BTLA mAb – B and T lymphocyte attenuator human monoclonal IgG1antibody 2

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Cmax – maximum serum concentration

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Da – dalton

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DNA – deoxyribonucleic acid

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EDC – ethyl(dimethylaminopropyl) carbodiimide

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Fc – crystallization fragment

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FcRn – neonatal constant region fragment receptor

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FDA – Food and Drug Administration

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GSD – Geometric standard deviation

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IgG – immunoglobulin G

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IL23 – interleukin 23

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IL23 scFv – single chain fragment variable against IL-23

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HPLC – high performance liquid chromatography

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HRP – horseradish peroxidase

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IV – intravenous

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KD – dissociation constant

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kDa – kilodalton

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λz – elimination rate constant

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LLOQ – lower limit of quantitation

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LOAEL – lowest observed adverse effect level

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µg – microgram

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µm – micrometer

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mg – milligram

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MMAD – mass median aerodynamic diameter

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MSD – meso scale discovery

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MW – molecular weight

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NBCA– non-binding control adnectin

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NBCA-PEG – pegylated non-binding control adnectin

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nm – nanometer

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NIOSH – National Institute of Occupational Safety and Health

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NOAEL – no observed adverse effect level

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OEL – occupational exposure limit

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PBS – phosphate buffered saline

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PoD – point of departure

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PEG – polyethylene glycol

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pI – isoelectric point

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PK – pharmacokinetic

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PNOS – particles not otherwise specified

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RNA – ribonucleic acid

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SPR – surface plasmon resonance

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T1/2 – terminal half-life

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Tmax – time to maximum concentration

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UPLC/MS – ultra performance liquid chromatography/mass spectrometry

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100 1. Introduction

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Biologics are an increasing part of the pharmaceutical market today, with Biologic License Applications

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making up approximately one-third of the new drugs approved within the last two years by the Food and

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Drug Administration (FDA) [1]. Technological advances in peptide and protein engineering have led to

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the emergence of a range of novel constructs, which can vary greatly in size and complexity. Monoclonal

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antibodies (~150 kDa), fusion proteins, protein and antibody fragments, and analogs to small proteins or

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peptides (<5 kDa) represent a significant component of the biological drug landscape. The broad

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description of a biologic is any material produced by a natural source such as an endogenous protein or

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any substance derived from bacterial or mammalian cells [2]. Their typical manufacture involves large-

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scale cell culture, filtration and purification of the protein. While processing requires protection of

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product, individual workers may still come in contact with the biologic drug through aerosol generation in

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positive pressure systems or in upset conditions. In addition, pharmacists and healthcare providers handle

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the finished drug to prepare and administer it to patients. Thus, there is a need to understand the hazard

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potential and develop occupational exposure limits (OEL) to establish appropriate exposure controls for

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the workplace.

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An OEL is an air concentration to which workers can be exposed for an 8-hour day, 5 days a week for a

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40-year career protecting them from experiencing pharmacological or toxicological effects of the

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substance. In order to assign an OEL, the preclinical and clinical safety data and pharmacokinetic (PK)

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properties are evaluated for each protein therapeutic. Though other approaches exist, an OEL is most

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commonly calculated by the following formula: OEL = PoD x α / FT x AF x V, where PoD is the point of

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departure (usually a No-Observed Adverse-Effect Level [NOAEL] or a Lowest-Observed Adverse Effect

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Level [LOAEL] for the critical effect); α is an adjustment factor to correct for differences in

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bioavailability (BA) between the route of administration from the study from which the PoD was selected

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and the inhalation route; FT is the composite adjustment factor which may account for 1) interspecies

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variation, 2) intraspecies variation, 3) study duration, 4) severity of the effect and database completeness,

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and 5) LOAEL to NOAEL extrapolation; AF is a factor used to account for potential accumulation

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following repeated exposure; and V is the breathing volume in an 8-hour workday. Additional details on

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these factors and how they are applied in OEL estimation can be found in several publications [3-5]. The

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research provided in this manuscript focuses on generation of α for protein therapeutics following

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inhalation exposure.

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Protein therapeutics are generally thought to be less hazardous for workers than traditional small molecule

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drugs because of their likeness to intrinsic human molecules (i.e., higher specificity, susceptibility to

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enzymatic degradation to amino acids, with minimal nonspecific toxicity), expected instability at room

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temperature, and negligible dermal bioavailability (generally considered to be poorly absorbed due to

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their large size); factors combined with the limited opportunity for inhalation (e.g., present as a dilute

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solution in primarily closed systems). However, since proteins can be systemically absorbed to some

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degree by the inhalation route [6-11], a more accurate assessment of their BA should be considered when

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assessing potential health effects in workers and in establishing OEL values. Since data on the inhalation

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BA of protein therapeutics are generally not available, most OELs for these substances are derived from

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clinical data using intravenous (IV) or subcutaneous injections and extrapolating to an inhalation BA of

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5% primarily based on studies in various species utilizing intratracheal dosing [6] or greater than 5%

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based on the NIOSH Hazardous Drug Alert List [12]. As a consequence, OELs for protein therapeutics

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likely over-predict the systemic presence of the drug following inhalation exposure, which can then lead

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to the implementation of overly conservative and potentially unnecessary environmental controls for

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workers. In addition, most of the published data on BA of protein therapeutics was developed using

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intratracheal delivery, specialized inhalation devices, and/or formulations.

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designed to enhance systemic drug delivery via the inhalation route; none of which accurately reflect

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normal breathing of an aerosolized protein in the workplace. Finally, whereas there are some reports that

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describe the inhalation delivery of peptide hormones like insulin (~6 kDa) and antibodies (~150 kDa), the

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BA of novel therapeutics that lie in a range of intermediate sizes (~10-150 kDa) is highly varied and

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These technologies are

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incomplete. Therefore, a critical question exists as to the most appropriate inhalation BA adjustment

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factor to be utilized for calculating an OEL for these types of molecules.

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In order to identify a default BA factor(s), the test proteins in the current study were selected to mimic

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typical protein therapeutics. Besides the molecular weight (MW) or size, the binding properties at the

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therapeutic target in the lung was considered in selection of the test proteins. Two protein features (i.e.,

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crystallization fragment [Fc] region present in Immunoglobulin G [IgG] and albumin) or modifications

157

(i.e., polyethylene glycol [PEG] conjugation) and their influence on pharmacokinetics were examined.

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The neonatal constant region fragment receptor (FcRn) facilitates IgG crossing of epithelial barriers (e.g.,

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placenta, neonatal intestinal epithelium, and adult lung) by receptor-mediated transcytosis with reduced

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lysosomal degradation [13, 14] leading to increased systemic absorption and circulating half-life after

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dosing to the lung [15, 16]. Likewise, protein PEGylation with its hygroscopic nature, increasing the

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overall molecule’s apparent size, has been shown to influence respiratory tract pharmacokinetics [17, 18].

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In essence, these factors were considered in selecting the protein therapeutics for the study.

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The work presented in this manuscript aims to provide information on BA for typical protein therapeutics

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with increasing molecular weight that can serve as a model to understand human lung and systemic

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exposure to an aerosolized drug solution. The application of these data to human inhalation BA estimates

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and its impact on the calculation of OELs will be discussed. Additionally, underlying transport properties

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were examined, as these relate to the lung absorption and disposition of different types of protein

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therapeutics. The potential for local effects following inhalation of proteins (e.g., specific binding to lung

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tissue, target receptor lung effects) are not addressed in this paper.

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2. Materials and Methods

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2.1. Materials

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NBCA, IL23 scFv, BTLA mAb and NCBA-PEG were synthesized, purified and verified in the Bristol-

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Myers Squibb laboratories. Description of the test proteins, their molecular weight and isoelectric point

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(pI) are listed in Table 1. BSA (essentially fatty acid and globulin free) was purchased from Sigma-

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Aldrich.

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Table 1

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Description of test proteins. Test Protein

Molecular Weight (kDa) 10.9

Isoelectric Pointb (pI) 5.14

FcRn binding c KD (µM) NA

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NBCA

Non-binding control adnectina

IL23 scFv

Anti-interleukin 23 single-chain variable fragment

30.1

6.18

NA

BSA

Bovine serum albumin

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5.40

Human = 3.7 Monkey = 2.2 Rat = NDBe Mouse = NDB

BTLA mAb (8A3)

Anti-B and T lymphocyte attenuator -human monoclonal IgG1antibody

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Heavy chain: 9.08 Light chain: 7.60 Intact mAb: 8.90

Human = 3.0 Monkey = 2.4 Rat = 1.2 Mouse = 0.9

NBCAPEG

non-binding control adnectina +40kDa branched polyethylene glycol (PEG)

10.9 + 40 (true)

5.14

NA

500 (apparent)d

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(c305)

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a

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fibronectin; b Calculated using the GPMAW algorithm; c NA – not measured since binding to FcRn

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not anticipated; d Apparent molecular weight; e no detectable binding.

Adnectins (e.g., NBCA) are a 10-12 kDa engineered form of the 10th repeat Type III domain of human

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2.2. Study dosing and exposure parameters

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Sprague-Dawley male rats (IV: 66 animals, age 7-8 weeks, weight 216-283 g; inhalation: 122 animals,

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age 7-9 weeks, weight 213-348 g) were obtained from Charles River Canada Inc. (St-Constant, QC,

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Canada), and randomly assigned to each group in such a way that 3 animals were utilized at each time

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point and no animal was bled more than 3-4 times depending on the time between bleeds. For IV studies,

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a single 2 mg/kg (BTLA mAb, BSA, and IL23 scFv) or 1 mg/kg (NBCA and NBCA-PEG) dose in

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aqueous buffer was filtered and then injected into the tail. For inhalation studies, rats were subjected to a

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single one-hour nose-only 40 mg test protein/m3 aerosol in aqueous vehicle or vehicle alone (Table 2).

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The test and vehicle control aerosols were generated using Pari LC Plus air-jet nebulizers.

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Table 2

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Inhalation study test protein air exposure conditions. IV and Inhalation Vehicle

NBCA

PBS, pH 7.4

Measured Air Concentration (mg/m3) 42

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Test Protein

IL23 scFv 20 mM Histidine and 125 mM NaCl pH 6.8

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BSA

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BTLA mAb

NBCAPEG

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20 mM citrate, 150 mM NaCl pH 6.0 buffer plus 8.3 µl dimethicone/mL 20 mM citrate, 150 mM NaCl pH 6.0 buffer

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PBS, pH 7.4

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Inhaled Dose (mg/kg)

MMAD+ GSDa (µm) Analytical

MMAD+ Aggregation (pre) GSD post nebulizationb (µm) HMW species Gravimetric

1.8

1.8 ± 1.9

1.9 ± 1.8

(0.7%) 7.6%

2.4

2.2 ± 2.2

(4.1%)

2.2 ± 1.9

3.9% 2.0

0.8 ± 2.0

(20.5%)

1.0 ± 1.9

24.5% 1.2

3.6 ± 1.6

(4.2%)

2.4 ± 2.0

4.5%

2.1

1.9 ± 2.3

(0.4%)

2.0 ± 2.2

0.3%

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a

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potential measured as high MW species by Size Exclusion Chromatography before and after 1-hour

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nebulization.

MMAD = mass median aerodynamic diameter; GSD = geometric standard deviation;

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b

Aggregation

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203 The inhaled dose with and without adjustment for deposited dose was calculated according to the method

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of Alexander and colleagues [19] to determine the estimated achieved dose (Table 3). Test protein

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aerosol concentrations and homogeneity were determined gravimetrically from an open-face

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polyvinylidene difluoride filter and calculated from the amount of test article collected on each filter

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sample divided by sample volume.

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gravimetrically at initiation of exposure and every 20 minutes for the duration of the exposure. Particle

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size distribution was performed using an 8-stage cascade impactor, which was followed by gravimetric or

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HPLC analysis. The stability (protein degradation or aggregation) of each test protein under study

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conditions was determined separately by collecting samples from nebulizer reservoirs before and after 60

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minutes of nebulization and analyzing by UPLC/MS. All animal studies were done in accordance with

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animal welfare guidelines and reviewed by an institutional animal care use committee.

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Actual chamber concentrations of aerosol were measured

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Estimated achieved dose calculation. [19]

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=

= respiratory minute volume, calculatedb

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Where RMV (L/min)

RMV × Active Concentration x T x IF BW

= chamber concentration of active ingredient determined by chemical analysis.

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Active Concentration (mg/L) T (min) IFa

= Exposure time (60 minutes) = inhalable fraction (proportion by weight of particles that are inhalable)

= mean body weight per group from the regular body weight occasion during exposure. Total body dose assuming an inhalable fraction of 100 unless stated otherwise; b 0.608 × [body weight BW (kg)

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a

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(kg)] 0.852 L/min. It is assumed that this parameter is unaffected by exposure to the test item.

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2.3. Sample collection

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After inhalation and IV exposures, animals were subjected to clinical signs examinations,

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pharmacokinetic serum collection and terminal procedures. Based on the different pharmacokinetic

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characteristics of test proteins, the sampling time points were selected to adequately define the

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concentration versus time curve (i.e., the absorption, distribution, and elimination phases) for each

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protein. In addition, total duration of sampling was generally extended to at least three to five expected

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half-lives to accurately determine the terminal elimination half-lives of test proteins. Approximately 0.3

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mL of blood was taken from the jugular vein at each time point (Table 4) except for terminal bleed

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samples, which were taken from the abdominal aorta. For all inhalation studies and IV studies with IL23

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scFv and BTLA mAb, the following postmortem lung procedures and evaluations were conducted. Body

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and lung weights were taken at necropsy. Given the likely differences in the drug disposition of test

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proteins between lung and blood, lung PK and tissue samples were collected at either 24 hours (NBCA)

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or 48 hours (all other proteins) post-dose to define the expected maximum concentrations. The BAL fluid

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was collected from the left lobe on select animals by lavaging three times with 1.5 mL of ice cold PBS-

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albumin, which was included to improve stability in certain samples. The BAL was centrifuged and the

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supernatant was collected for immediate cell typing and quantification, lactate dehydrogenase activity

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determination, and the remainder frozen for test protein analysis. The left lobe deplete of its BAL, was

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frozen, homogenized at a later date for test article analysis. The remainder of the lung lobes were fixed in

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10% neutral buffered formalin, processed with hematoxylin and eosin, and examined by microscopy.

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Table 4

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Pharmacokinetic sample collection times for the intravenous and inhalations studies. Treatment

IV Study Bleeding Timepoints (hours)

Inhalation Study Bleeding Timepoints (hours)

Inhalation Study BAL Timepoints (hours)

NBCA

0.03, 0.08, 0.25, 0.5, 1, 2, 4, 8

0.5, 1.0, 1.08, 1.25, 1.5, 2, 4, 5, 9

1, 25

IL23 scFv

0.03, 0.08, 0.25, 0.5, 1, 2, 4, 8

0.5, 1, 1.08, 1.25, 1.5, 2, 4, 5, 9

25, 49

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0.08, 0.5, 2, 4, 8, 24, 48, 72, 96

1.08, 1.5, 3, 5, 9, 25, 49, 73, 97, 121

1.08, 3, 5, 25, 49

BTLA mAb

0.5, 2, 6, 24, 48, 72, 168, 336, 504, 840, 1344

1.5, 3, 7, 25, 49, 73, 169, 337, 505, 832, 1344

1, 1.5, 3, 25, 49

NBCA-PEG

0.03, 0.5, 1, 2, 8, 24, 48, 72, 96, 168, 240, 312, 384

0.5, 1.0, 1.5, 2, 3, 9, 25, 49, 97, 145, 169

1, 25, 49, 169

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2.4. Assays for test protein, lactate dehydrogenase, immune cells, histology, FcRn binding

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2.4.1. Test protein analysis

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Concentrations of the test proteins were measured in serum, lung tissue, and BAL fluid using qualified

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Meso Scale Discovery (MSD, Gaithersburg, MD) electrochemiluminescent immunosorbent assays or

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chemiluminescent assay (for BSA). Biotinylated analyte-specific monoclonal or polyclonal antibodies

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were immobilized on the MSD streptavidin plate as the capture reagent. Samples, calibrators and quality

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controls were diluted in 1% BSA/PBS, 0.05% Tween 20. After incubation in the plates, unbound material

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was washed away and the captured analytes were detected with specific detection antibodies labeled with

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the ruthenium complex (HRP-labeled for BSA assay). Following addition of MSD read buffer,

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luminescence intensity was measured by a MSD reader [HRP Chemiluminescent substrate was used for

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BSA assay and luminscence was measured on Spectramax M5 (Molecular Devices)]. The concentration

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of each protein in serum or BAL samples was calculated using a 5-parameter logistic calibration curve

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generated from the corresponding protein calibrators. Additional assay parameters are listed in the Table

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5.

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Table 5

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Description of test protein bioanalytical parameters. Capture Absa

NBCA

Serum

BTLA mAb

NBCA-

PEG

Range (ng/mL)

a-Adnectin Rabbit pAb

a-Adnectin Rabbit pAb

4

4 - 4,000

BAL

a-Adnectin Rabbit pAb

a-Adnectin Rabbit pAb

2

4 - 4,000

Serum

a-Hu IL-23

a-VH Goat pAb

10

20 - 5,000

BAL/lung a-Hu IL-23

a-VH Goat pAb

2

10 - 1,250

Serum

a-BSA mAb

a-BSA Goat pAb

5

20 - 10,000

BAL/lung a-BSA mAb

a-BSA Goat pAb

2

31.3 - 500

a-Hu IgG mAb

a-Hu IgG Fc mAb

5

1.4 - 1,000

BAL/lung a-Hu IgG mAb

a-Hu IgG Fc mAb

2

2 - 500

Serum

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BSA

MRDc (fold)

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IL-23 scFv

Detection Absb

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Serum

a-Adnectin Rabbit pAb

a-PEG mAb

4

1 - 400

BAL

a-Adnectin Rabbit pAb

a-PEG mAb

2

0.2 - 200

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a

All capture Abs were biotinylated; b All detection Abs were rhutenylated except BSA, which was HRP

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conjugated; c Minimum required dilution.

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2.4.2. Lactate dehydrogenase and BAL cell assay 13

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Lactate dehydrogenase activity was determined by standard redox assay. BAL immune cell types and

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counts were measured by resuspending the BAL sample pellet into a volume of 2 mL of PBS-0.1% PBS-

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BSA, and cells counts were performed with a hematology analyzer (Advia 120). Additionally, cytospin

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slides were prepared, stained and a differential cell count was evaluated microscopically.

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2.4.3. FcRn binding

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To assess for potential differences in FcRn binding across species for BSA and BTLA mAb (compounds

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known to bind to FcRn), Biacore surface plasmon resonance (SPR) experiments were performed on a

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Biacore T100 instrument (Biacore/GE Healthcare). Sensor surfaces were prepared by immobilizing 30

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µg/ml BSA or BTLA mAb in 10 mM sodium acetate pH 4.5 on a CM5 sensor chip using standard

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ethyl(dimethylaminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide chemistry, with ethanolamine

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blocking, a running buffer of 10 mM sodium phosphate, 130 mM sodium chloride, 0.05% Tween 20

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(PBS-T) pH 7.1. FcRn binding experiments were performed by testing various concentrations of human-

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FcRn, cyno-FcRn, rat-FcRn or mouse-FcRn (produced in house) from 10 µM down to 0.1 µM in PBS-T

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pH 6.0, at either 25oC or 37oC, using association time of 240 s, dissociation time of 120 s, at a flow rate of

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30 µl/min. Surfaces were regenerated between cycles using two 10 s pulses of 50 mM Tris, 150 mM

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NaCl, pH 8.0, at a flow rate of 30 µl/min. Binding data were fit to a 1:1 steady state model using Biacore

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T100 evaluation software (Biacore/GE Healthcare) with KD binding affinity .

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2.5. Pharmacokinetic Analysis

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All pharmacokinetic parameters were calculated from a composite mean serum test protein concentration

297

versus time curve for each group only using non-compartmental methods by Phoenix WinNonlin

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(Certara, Princeton, NJ). The mean area under the concentration versus time curve (AUC0-T or AUC∞)

299

where T was the last measurable concentration or infinity was calculated using the linear trapezoidal rule.

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The mean maximum serum drug concentration (Cmax) and its corresponding post exposure time point

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(Tmax) were determined from each group composite concentration data. The elimination rate constant

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(λz) was determined by regression of the terminal log-linear portion of the curve. The apparent terminal

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half-life (T1/2) was calculated utilizing the equation T1/2 = ln2/λz). Inhalation bioavailability (%BA) was

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calculated as shown below. ( ℎ (

) × Dose( ) × Dose( ℎ

) )

× 100%

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To assess the amount of test protein unabsorbed from the bronchvaleolar space, the BAL was examined.

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The fluid collected from each rat was normalized to the weight of left lobe of rat lung. In addition,

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normalized drug concentrations in serum, lung, and BAL fluid were calculated by dividing the drug

308

concentrations by the respective IV dose or inhalation exposure dose levels.

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3. Results

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3.1. Systemic pharmacokinetic profiles following intravenous and inhalation exposures

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The systemic mean AUC, mean Cmax, mean Tmax, and apparent terminal T1/2 of the five test proteins

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following IV and one-hour nose-only inhalation exposures are shown in Table 6. Dose-normalized AUC

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and Cmax after inhalation exposure were substantially lower than those after IV administration. Reliable

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T1/2 values after inhalation exposure could not be assessed for NBCA, IL23 scFv, BSA or NBCA-PEG

315

due to insufficient time points at the terminal phase of the concentration profile or measurements below

316

the lower limit of quantitation (LLOQ; Figures 1 and 2). However, the BTLA mAb T1/2 after inhalation

317

exposure was generally comparable to that after IV administration, indicating similar systemic clearance

318

for both routes. In addition, BTLA mAb serum AUC after inhalation was greater by an order of

319

magnitude compared to the other test proteins, likely due to good serum stability and long half-life

320

presumably through FcRn binding and recycling [20]. Inhalation delivery of BSA resulted in a virtually

321

undetectable serum concentration, forcing an overestimation of %BA by assuming serum concentrations

322

equal to the LLOQ values. Overall, there was a low level of systemic BA after inhalation exposure for

323

each test protein. When compared to the respective IV study, systemic %BA was 1.4%, 0.55%, < 0.47%,

324

0.73%, and 0.05% for NBCA, IL23 scFv, BSA, BTLA mAb, and NBCA-PEG, respectively,

325

demonstrating generally similar values and thus a lack of MW dependence within this range of MWs,

326

with the exception of the pegylated NBCA-PEG.

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327 328 329

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333 334 Table 6

336

Comparison of systemic pharmacokinetic parameters after a single intravenous and 1-hour

337

nose-only inhalation exposure in rats.

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NBCA

IL23 scFv BSA

BTLA mAb

NBCA-PEG

10.9

30.1

67

144

500

1

2

2

2

1

6.57

4.25

410

4140

232

19.4

15.2

19.0

21.0

13.5

1.1

1.7

25

316

47

Inhaled Dose (mg/kg)

1.8

2.4

2.0

1.24

2.1

AUC0-Ta/Inhaled Dose

0.0939

0.0235

<1.94b

29.2

0.130

0.011

0.0067

<0.02

0.083

0.0048

Tmax (h)

2.0

1.5

-c

49

9.0

T1/2 (h)

5.5

<2

-

370

21

Route

MW (kDa)

Intravenous

Dose (mg/kg) a

-1

TE D

AUC0-T /Dose

-1

(µg•h•mL /mg•kg ) Cmax/Dose -1

-1

(µg•mL /mg•kg )

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Inhalation

EP

T1/2 (h)

-1

-1

(µg•h•mL /mg•kg ) Cmax/Inhaled Dose -1

-1

(µg•mL /mg•kg )

Inhalation F (%)

16

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Inhaled Dose

1.4

0.55

< 0.47

0.73

0.056

5%

15.9

9.2

9.5

22.6

0.5

10%

7.9

4.6

< 4.7

11.3

0.3

RI PT

Deposited Dosed

338

a

For AUC0-T, T = infinite time or where T is the time of the last measureable concentration; b Serum BSA

339

concentrations were less than LLOQ (0.04 µg/mL) at all time points (up to 97 hours); c Value could not

340

be determined;

341

(IF) or deposited dose as described in Table 3. The IF of 5% and 10% were based on estimations from the

342

literature [31, 35].

343

3.2. Lung Clearance and Tissue Dose after IV and Inhalation Exposure

344

For the two proteins in which lung concentrations were determined after intravenous exposure, IL23 scFv

345

and BTLA mAb, both diffused from systemic circulation across the lung barrier to the lung lining fluid as

346

demonstrated by a rapid peak in lung tissue and BAL concentrations (approximately 5 minutes to 2 hours

347

post-dose) and gradual decrease thereafter (Figure 2). Interestingly, this indicated that after IV injection,

348

test proteins rapidly distribute into the well-perfused lung for both the modest MW (i.e., the 30.9 kDa

349

IL23 scFv) and large MW (i.e., the 144 kDa BTLA mAb) proteins (Table 7). Moreover, the lung

350

exposures following IV administration (i.e., combined AUC in lung tissue and BAL) of IL23 scFv and

351

BTLA mAb were less than 10% of systemic exposures (i.e., AUC in serum), demonstrating that test

352

proteins localize in the lung at relatively low levels after IV dosing (Table 7).

353

After the one-hour inhalation exposure, there was a higher overall exposure of each test protein in the

354

lung than in the systemic circulation (Figure 1, Table 7). The concentrations peaked in BAL and lung

355

tissue immediately after the inhalation period and gradually decreased thereafter except for BTLA mAb

356

(Figure 1), which remained at approximately the same concentration over the 48-hour period examined,

357

consistent with the in vivo stability of monoclonal antibodies. The unabsorbed IL23 scFV, BSA and

358

BTLA mAb concentrations in the BAL and lung tissue were roughly equivalent on a nanomolar basis.

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%BA calculated utilizing an inhaled dose adjusted by a 5% or 10% inhalable fraction

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However, BTLA mAb had an order of magnitude greater serum AUC thus impacting the lung

360

tissue/serum and BAL/serum ratios dramatically compared to the other test proteins. When comparing

361

the test protein concentrations normalized by dose on a molar basis, there was a trend to a decreased

362

concentration in the BAL with increased in MW (Figure 1), which could reflect increased absorption or a

363

decreased molar administered dose for the higher MW test proteins used in the study.

364

The impact of PEGylation on pharmacokinetics was examined (Table 6). Compared to the NCBA

365

protein alone, the addition of a 40 kDa PEG increased the systemic AUC/dose 35-fold and the T1/2 40-fold

366

after IV injection, while after inhalation the systemic AUC of the two proteins were equivalent and the

367

T1/2 for the pegylated form was only four-fold greater. After inhalation, notably, the BA of NBCA-PEG

368

was drastically reduced to 0.04% compared to 1.4% for NBCA. However, in the lung (as BAL), there

369

were minimal differences in PK profiles (Figure 2). The BAL kinetic profiles for NBCA and NBCA-

370

PEG mirrored the serum profile with similar apparent T1/2 (4.5 and 7.2 hours in BAL, respectively; Table

371

7).

372

It is important to note that no clear inhalation MW-relationship was observed for the lung tissue or BAL

373

exposures or the inhalation BA values for the test proteins, with the exception of the low BA for the

374

PEGylated NBCA where size (MW of 500 kDa) did appear to impact absorption (Table 6).

377 378 379 380

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385 386 387

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394 395

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Table 7

397

Exposures in lung tissue, bronchioalveolar lavage (BAL) and serum after intravenous and 1-hour nose-

398

only inhalation exposure in rats.

399

A) Intravenous

0.228

BTLA mAb

74

AUC Ratio

BAL T1/2

Lung/Serum

BAL/Serum

h

0.131

4.25

0.054

0.031

1.0

110

4140

0.018

0.027

326 b

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IL23 scFv

400 401

Exposure AUC0-T a/Dose µg•h•mL-1/mg•kg-1 Lung BAL Serum

EP

Proteins

TE D

396

B) Inhalation Proteins

NBCA

Exposure AUC0-T a/Inhaled Dose µg•h•mL-1/mg•kg-1 Lung BAL Serum NA

1481

AUC Ratio

Lung/ Serum NA

0.0939

19

BAL/ Serum 15800

BAL T1/2 h 4.5

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15.5

126

0.0235

661

5360

5.9

BSA

5.70

79.5

<1.94b

<2.9

<41

19.0

BTLA mAb

10

201

29.2

0.34

6.9

-c

NBCA-PEG

NA

399

0.13

NA

3070

7.2

RI PT

IL23 scFv

NA: lung tissue was not collected; a Dose normalized AUC in µg•h•mL-1/mg•kg-1 where T = infinite time

403

or where T is the time of the last measureable concentration; b Serum BSA concentrations were less than

404

LLOQ (0.04 µg/mL) at all time points (up to 97 hours); c BAL BTLA mAb concentrations did not

405

decrease between 1 and 49 hours so T1/2 could not be calculated.

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407 408

3.3. Toxicological Impact of Test Protein Exposure

410

In all studies, there were no clinical signs observed that would indicate any detrimental effect of the test

411

proteins on the animals. In the inhalation studies, lung histology, BAL lactose dehydrogenase levels,

412

BAL immune cell type and counts were determined. With all proteins, minimal to no changes compared

413

to vehicle controls were observed after the one-hour inhalation exposure (data not shown). It should be

414

noted that the immunological hypersensitivity (Type I) potential, which would require chronic or repeat

415

exposure, was not assessed in this study.

416

3.4. Test protein description, physical integrity, and aerosolization of test proteins

417

The test proteins were selected to mimic typical protein therapeutics across a range of molecular weights.

418

As the binding properties at the therapeutic target may influence the pharmacokinetics, this specific target

419

binding in the rat was determined to be minimal to none for all proteins (data not shown) and thus was not

420

considered a factor for these proteins. However, the present study also assessed the FcRn binding

421

potential for both BSA and BTLA mAb, for which FcRn binding is known to play a role in protein

422

recycling and longevity in the circulation. While not all together surprising since human serum albumin

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was found to have poor FcRn binding in rodents [21], BSA did not bind to the rat FcRn as compared to

424

that in the human. As expected, there was strong binding of BTLA mAb to both human and rat FcRn.

425

As particle size influences deposition and concentration in the alveolar region, the nebulizer output was

426

assessed to ensure consistency and stability of the aerosol being delivered to the rat. The test protein

427

concentration in the inhaled air-stream, mass median aerodynamic diameter (MMAD) and the aerosol

428

droplet size was consistent across all test proteins (Table 2). Aerosol particle size estimation by two

429

different methods fell within a narrow range of 0.8-3.6 µm MMAD with a low standard deviation of 1.6-

430

2.2 µm for all test proteins (Table 2). Being aqueous buffer-based formulations that aerosolize in a

431

similar fashion, they are not likely to exhibit different aerodynamic deposition profiles. Therefore,

432

deposition was excluded from the interpretation of data. Additionally, the testing from the formulation

433

reservoir at the start and end of the nebulization period indicated that the test proteins were stable (Table

434

2), with the exception of BSA where the presence of 24.5% of a high MW species suggests a strong

435

tendency to agglomerate despite the presence of an antifoaming agent in the formulation. This increased

436

percentage of MW species likely decreased the concentration of molecules available for diffusion into the

437

systemic circulation, explaining the lack of detectable BSA in the serum after inhalation exposure.

438

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4. Discussion

440

4.1. Key findings

441

To more accurately determine the OEL and control workplace exposures of protein therapeutics, the fate

442

and disposition of proteins of different molecular weights and complexity were explored after inhalation

443

and IV exposure. The strategy was to determine the %BA of aerosolized proteins in a rat inhalation

444

model simulating nuisance dust air concentrations (e.g., PNOS – particles not otherwise specified [22]),

445

as a “worst case scenario” and utilizing an exposure length representing the time for typical open

446

processing in biologics manufacturing.

447

examining test proteins covering a MW range from 10 – 150 kDa, suggested a low systemic BA of

448

approximately 1% or less is reached after inhalation exposure. Second, from examination of lung and

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We identified three key findings.

21

First, the results from

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BAL, the test proteins were present at similar concentrations in the lung over the 48-hour observation

450

period, when considered on a molar basis. While negligible adverse impact to the integrity of the lung

451

was observed, there could be the potential for accumulation in the lung and low sustained systemic

452

release if there are repeated exposures. This could also allow residual protein to interact with therapeutic

453

target lung receptors, which was reported with the lung accumulation of cetuximab (152 kDa, IgG1

454

antibody for the treatment of cancer) after pulmonary delivery to mice [9, 23]. Lastly, following the fate

455

of the test proteins in the lung after IV exposure, there was clear evidence of the test proteins in the lung,

456

indicating that a low percentage, <10% of systemic AUC, of these higher MW proteins can pass from

457

circulation into BAL. Understanding that there is reasonable distribution of a test protein to the lung from

458

an IV exposure can provide an indication of potential for direct lung effects after an inhalation exposure.

459

4.2. Comparison to literature including mechanism of transfer

460

There have been a number of investigations on systemic BA of proteins after lung exposure; however

461

there is a lack of a solid consensus on a %BA value for proteins and understood absorption dependencies.

462

Several authors have provided an analysis of the literature focusing on MW with BA as a dependent

463

variable and have indicated an inverse relationship [11, 24-26]. More recently, Pfister and colleagues [6]

464

reinforced the MW dependence and provided further clarity by concluding that proteins greater than 40

465

kDa would not likely exhibit a systemic BA greater than 5% following lung exposure. The results of the

466

present study show that indeed proteins of >40 kDa and even down to 10.9 kDa have a BA lower than 5%

467

after true inhalation exposure. However, a clear difference in BA for test proteins in the MW range of 10

468

to 150 kDa was not demonstrated. The lack of a MW dependency can be explained by looking at the

469

mechanism of transfer. It is believable that as proteins increase in size, at some point, they would exceed

470

the pore size to transverse the alveolar junctions. The translocation being based on restricted, size-

471

dependent passive diffusion of molecules through aqueous pores that range in size from 5 to 20 nm [27,

472

28] wherein larger molecules experience greater hindrance entering and navigating across the alveolar

473

epithelial barrier. With the test proteins examined under the same experimental conditions, there were

474

low but similar test protein concentrations on a molar basis in lung tissue, BAL and serum (excluding the

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pegylated protein) (Table 7), thus supporting the concept that passive paracellular or transcellular

476

transport from the airways to vasculature is generally minimal. Looking at BTLA mAb absorption, the

477

data presented here do not differentiate whether absorption occurred only by the expected receptor

478

mediated (i.e., FcRn) active transport, passive diffusion, or a combination of both. Regardless, the low

479

systemic absorption of a full antibody with an active FcRn region following inhalation exposure was

480

similar to the presumed passive transport for the lower molecular weight test proteins. Guilleminault and

481

colleagues showed that passive diffusion can occur after intratracheal exposure of a Fc containing

482

antibody in a FcRn knockout mouse; while there was low systemic BA (7.9%), the overall exposure was

483

8% of that in the wildtype mouse under the same conditions [9]. While a definitive MW cut off for

484

epithelial membrane transfer was not examined, it can clearly be seen that the absorption drops off

485

dramatically with NSCA-PEG (apparent size of approximately 500 kDa), which demonstrated a BA of

486

0.05% as compared to the other proteins examined in this study. Other mechanisms of entry such as

487

through the lymphatic system may be in play in this case. Overall, there appears to be a plateau in

488

systemic BA after inhalation exposure for proteins in the MW range of 10 to 150 kDa.

489

The literature has sparse BA examples for proteins after lung exposure and the BA values are larger than

490

those observed in the present study. Proteins in the 9.4 – 22 kDa range have been reported to have much

491

higher BA at 10 – 56%; and as the MW increased to between 37 – 150 kDa, the BA decreased, and

492

appeared to fall into the < 5% range [11, 29, 30], supporting Pfister and colleagues’ conclusion that there

493

is a MW dependency. This elevated BA can be explained because these studies were conducted utilizing

494

intratracheal administration. We suspect the primary reason for the discrepancy between the approximate

495

1% BA of the present study and these reports is that intratracheal instillation represents a total deposited

496

dose; whereas here, the %BA is provided as an inhaled dose. A deposited dose was also calculated

497

(Table 6) and applied to an inhaled dose but multiple methods are available and depending on particle

498

size, the deposited dose could vary from <5% to 10% deposited in the rat pulmonary region [31-33],

499

impacting the BA result considerably (Table 6; e.g., NCBA inhaled %BA = 1.4% compared to NCBA

500

deposited %BA of 8% -16% assuming a 5% or 10% deposited dose, respectively). Thus the methodology

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for selecting the deposited dose would lead to varying BA increasing the uncertainty. It should be noted

502

that the %BA from the calculated deposited dose from the present study is greater than those %BA

503

published in the literature from similar sized proteins, suggesting that the calculated intratracheal %BA

504

may underestimate the true inhaled %BA. Overall, this comparison underscores the difference between

505

utilizing passive inhalation versus deposited intratracheal administration for understanding lung exposure,

506

indicating most results from the literature cannot be directly compared to the current study findings.

507

A similar sized protein to IL23 scFv of this work, a 29 kDa protein, dornase alpha (recombinant human

508

deoxyribonuclease I for the treatment of mucous accumulation in cystic fibrosis patients) was

509

administered to humans by nebulizer and pharmacological action in the lung was demonstrated. While

510

not specifically providing a %BA, there was no measurable change in background systemic DNAse

511

concentrations after three 10 mg doses for 6 consecutive days [34], behaving pharmacokinetically similar

512

to the low BA and short half-life observed with our 30.9 kDa protein, IL23 scFv.

513

Next, in looking at the BSA results, surprisingly, it was not measurable in the serum after inhalation

514

exposure. This is in contrast to Folkesson and colleagues’ [35] report of a BA of 6.4% (note: lung

515

deposition was incorporated in their %BA value), based on an one-hour nose-only inhalation exposure of

516

33 mg BSA/m3. This difference can be explained by first, the low serum concentration in the present

517

study due to a substantial percentage of a large molecular weight species reducing systemic absorption;

518

and, secondly, the lack of bovine albumin binding to the rat FcRn; both would minimize passive or active

519

transport translocation into the circulation.

520

Scientists have shown that Fc fusion proteins have enhanced absorption as compared to proteins of

521

equivalent size without an Fc region. The BA after intratracheal aerosol instillation of erythropoietin-Fc

522

fusion protein (112 kDa protein, functions to stimulate erythrocyte production) was 35%, which is much

523

higher than observed with any test protein in the current study. It is recognized that the %BA value of

524

erythropoietin-Fc fusion protein included an adjustment for 12.8% deposition [15]. With another protein,

525

an IgG antibody omalizumab (149 kDa; anti-IgE antibody for the treatment of asthma), Pfister and

526

colleagues [6] calculated the systemic BA from a published human nebulizer study [36]. The BA was

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527

low at 0.2 - 0.6% and in the same range as our BTLA mAb, even though those studies were designed to

528

optimize inhalation delivery as an inhaled product.

529

cetuximab (152 kDa, IgG1 chimeric antibody for the treatment of cancer) to mouse and non-human

530

primate resulted in what was described as low systemic exposure (i.e., 3.9% or 0.3%, respectively). In

531

addition, a similar persistent presence of cetuximab in the lung [9, 23] was observed in agreement with

532

the BTLA mAb lung clearance.

533

Finally, the addition of a large PEG group (40kDa) to the test protein NBCA drastically reduced the BA.

534

This observation is consistent with McLeod and colleagues’ [17] who reported that systemic BA after

535

intratracheal instillation of a 60 kDa PEGylated 19 kDa interferon protein was extremely low (< 0.4%).

536

Overall, the present study increases the body of evidence to support that the lung epithelium serves as an

537

effective barrier for minimizing the transport of proteins.

538

4.3. Evaluation of %BA value for human risk assessment

539

The present studies were conducted for human risk assessment, specifically to identify possible default

540

value(s) for the route-to-route modifying factor, α in the OEL calculation for proteins. The study design

541

was optimized with human exposure in mind. The target particle size of 2.0 µm maximized deep lung

542

deposition as well as an air concentration equivalent to the highest allowable level for particles of low

543

hazard concern (Particles Not Otherwise Specified –PNOS) according to American Conference of

544

Governmental Industrial Hygienists [22, 33, 37]. This 40 mg/m3 test protein aerosol concentration

545

provided to the rat included the standard adjustment of 4x to obtain a human equivalent deposited dose to

546

the pulmonary region [38, 39]. With a maximized concentration in the alveolar space (i.e., BAL),

547

according to Fick’s first law of diffusion, optimal test item flow into the circulation would be expected,

548

assuming similar mechanism for transfer across membranes between humans and rats. In addition, non-

549

binding proteins, with the exception of the FcRn binding of BTLA mAb, were selected to minimize

550

pharmacological dependent impact on the measured pharmacokinetics. Another key factor in accepting

551

this study’s %BA value is that this was an inhalation study. General practice in risk assessment for

552

inhalation studies is to utilize a minimal to no conversion factor for interspecies for rat to human

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Similarly, intratracheal aerosolized delivery of

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adjustment for local lung effects [38].

Likewise, the BA determined from the inhaled dose is

554

representative of occupational exposure and can be equated to the critical effect dose (Point of Departure)

555

utilized for the OEL determination where the predicted air concentration and the amount the individual

556

inhales over time comprises a human inhaled dose. While there is possibly some influence of MW at the

557

lower MW range of the test proteins examined and the differing mechanisms of movement from the lung

558

into the systemic circulation, the variability between the resulting BA values do not represent a

559

biologically meaningful difference.

560

supporting literature, we postulate that a default α of 1% to account for inhalation BA is reasonable for

561

similar protein therapeutics in the MW range of 10 – 150 kDa.

562

4.4. Impact to OEL calculation

563

Changing the absorption factor (α) in the OEL calculation can provide a dramatic difference in outcome.

564

For example, assuming all other factors and adjustments were the same, the application of an α of 100%

565

(default for no inhalation data), 5% [6] or 1% (present study), a protein therapeutic OEL would differ

566

from 1 µg/m3 to 20 µg/m3 to 100 µg/m3, respectively. Utilizing a protein therapeutic default approach

567

rather than the worst-case scenario of 100% may have far-reaching advantages. The National Institute of

568

Occupational Safety and Health (NIOSH) has described one characteristic for placing a drug on the

569

Hazardous Drug Alert List as having an OEL at ≤ 10 µg/m3. The decreased intrinsic hazard due to the

570

limited ability of proteins to be systemically bioavailable via inhalation as well as negligible through

571

dermal and oral routes should be considered when describing drugs as hazardous in this context; thus

572

minimizing application of excessive warnings to molecules of lesser risk and focusing efforts on drugs of

573

serious concern. Additionally, an increased OEL value, while ensuring protection of human health, may

574

also eliminate potentially unnecessary exposure controls, the ability of a facility to meet the OEL and

575

consequently, greater flexibility in manufacturing decisions.

576

4.5. Caveats to use of 1%

577

The study here chose model proteins with minimal interaction with the rat physiology with the purpose of

578

confirming the systemic BA of proteins after inhalation exposure. While the data presented here is based

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In light of these data presented here in conjunction with the

26

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on one representative protein of each size or type, it was assumed that proteins of similar size whether

580

native, modified or conjugated would have similar fate and BA in a normal human lung environment

581

experiencing mucociliary clearance, protease inactivation, physical entrapping and macrophage engulfing

582

of inhaled material, all of which may influence the extent of absorption. The systemic BA assumptions

583

presented here may not be applicable to all types of biologic therapeutics and would not be relevant when

584

conducting human risk assessment on proteins that have a MW of less than 10 kDa, have direct effects on

585

the lung (e.g. receptor-mediated pharmacology, respiratory sensitization - Type 1 hypersensitivity),

586

atypical protein chemistry (e.g., polycationic chain), or other biological materials (e.g., oligonucleutides,

587

RNA, DNA, viruses, etc). Additional studies may be needed to better understand specific circumstances.

588

Nonetheless, this study outlines a default BA criteria and recommendation in pursuing OEL estimation

589

for protein therapeutics, especially for compounds in pharmaceutical product development where a

590

limited body of knowledge is available.

591

5. CONCLUSION

592

Although there are several mechanisms to hinder proteins from entering the systemic circulation via

593

inhalation, some fraction of the amount of a protein that enters the lung is expected to become

594

bioavailable. Many of the experiments reported in the literature were conducted using conditions to

595

optimize deposition and enhance absorption. The method of delivery, inhalation device used, the type of

596

formulation (nebulized liquid or passively inhaled dry powder), intersubject variability, or the

597

pathophysiological state of the lung etc., all contribute to the BA of a protein. Since information on these

598

factors or their potential impact on inhalation BA in an occupational setting is limited, this study set out to

599

better understand fate and distribution of protein therapeutics ranging in MW from 10 to 150 kDa after

600

inhalation exposure.

601

approximately 1% or less. These pharmacokinetic properties suggest that compared to small molecule

602

drugs, there is an intrinsic decrease in hazard for proteins and the application of a default pharmacokinetic

603

factor to the OEL calculation should be considered for typical proteins. This estimate is expected to be

604

conservative and protective for the worker population. Furthermore, although in this study there was

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Overall, systemic bioavailability after inhalation exposure was low, at

27

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605

negligible to minimal lung effects from the test protein based on local evaluation, direct effects on the

606

lung should be considered when assessing the hazard potential for specific therapeutic proteins that have

607

lung targets.

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608 Conflicts of interest

610

The authors declare that they are employees of Bristol-Myers Squibb and the study was sponsored by

611

Bristol-Myers Squibb.

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609

612 Acknowledgements

614

Funding for this project was provided by Bristol-Myers Squibb. We would like to thank the scientists at

615

Bristol-Myers Squibb who supported this study: Guodong Chen, Sybille Herzer, Noah Ditto, Robert Li,

616

Mark Maurer, Thomas McDonagh, Jingjie Mo, Siegfried Rieble, Mark Rixon, Lumelle Schneeweis,

617

Lakshmi Sivaraman, Sally Thompson-Iritani, Caren Villano, Jack Valentine, Chunlei Wang, Hui Wei,

618

Hua Yao, Yan Yao, Chuan Zheng, Mian Gao, and Lin Cheng. In addition, the individuals responsible for

619

the conduct of the studies: Marilyne Boyer Johanne Laporte, Marie-Eve Rodrigue, Bassem Attalla, and

620

Sandra Gagnon.

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Figure legends

623

Figure 1. Comparison of dose-normalized mean concentrations (nM/mg•kg-1) in lung tissue (closed

624

triangle) and bronchioalveolar lavage (open square) with those in serum (closed circle) after a 1-hour nose

625

only inhalation exposures in rats with NBCA (A), IL23 scFv (B), BSA (C), BTLA mAb (D), and NBCA-

626

PEG (E). Serum BSA concentrations were < LLOQ at all time points in Panel C. Lung tissue NBCA and

627

NBCA-PEG concentrations were not assessed in Panels A and E. Units are presented in molar quantities

628

normalized by dose in order to account for differences in molecular weight and dose.

629

Figure 2. Comparison of dose-normalized mean concentrations (nM/mg•kg-1) in lung tissue (closed

630

triangle) and bronchioalveolar lavage (open square) with those in serum (closed circle) after

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28

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administration of a single IV dose in rats with IL23 scFv (A) and BTLA mAb (B). Units are presented in

632

molar quantities normalized by dose in order to account for differences in molecular weight and dose.

633

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634 635 636

SC

637 638

640 641 642 643 Appendix A. Supplemental Data

TE D

644

M AN U

639

645 Table A.1

647

Comparison of systemic pharmacokinetic parameters (in molar units) after a single intravenous and 1-

648

hour nose-only inhalation exposure in rats.

EP

646

AC C

649

NBCA IL23 scFv BSA

BTLA mAb

NBCA-PEG

Route

MW (kDa)

10.9

30.1

67

144

500

Intravenous

Dose (mg/kg)

1

2

2

2

1

603

141

6120

28800

464

1780

505

284

146

27.0

AUC0-Ta/Dose -1

(nM•h/mg•kg ) Cmax/Dose -1

(nM/mg•kg )

29

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1.1

1.7

25

316

47

Exposure Dose (mg/kg)

1.8

2.4

2.0

1.24

2.1

AUC0-Ta/Exposure Dose

8.61

0.78

<28.96

1.01

0.22

<0.30

Tmax (h)

2.0

1.5

-b

T1/2 (h)

5.5

short

-

F (%)

1.4

0.55

(nM•h/mg•kg-1) Cmax/Exposure Dose

a

651

not be determined.

< 0.47

M AN U

650

0.58

0.26

0.010

49

9.0

370

21

0.73

0.056

SC

(nM/mg•kg-1)

202

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Inhalation

T1/2 (h)

For AUC0-T, T = infinite time or where T is the time of the last measureable concentration; b Value could

652

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653

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655

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747 748 749 750 751

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(A) NBCA

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(B) IL23 scFv

(C) BSA

AC C

(E) NBCA-PEG

EP

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(D) BTLA mAb

1

Figure 1 1

ACCEPTED MANUSCRIPT

(B) BTLA mAb

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Figure 2

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2

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(A) IL23 scFv

2

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Highlights •

For typical large proteins (10-150 kDa), systemic bioavailability in the rat following inhalation is low, at ~≤1%. Addition of a PEG moiety on a protein can considerably decrease systemic bioavailability.

•

Direct effects on the lung should be considered when assessing the hazard potential for proteins

RI PT

•

that have lung targets.

SC

The application of a default PK factor adjusting for 1% bioavailability is warranted when

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calculating OELs.

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•