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Physicochemical Determinants of Beryllium Toxicity using in vitro and in vivo Models. Gregory L. Finch, PhD Drug Safety Evaluation Pfizer Global Research & Development Groton, CT June 25/26, 2002 Beryllium Research Symposium, Bethesda MD

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physicochemical determinants of beryllium toxicity using in vitro and in vivo models

Physicochemical Determinants of Beryllium Toxicity using in vitro and in vivo Models

Gregory L. Finch, PhD

Drug Safety Evaluation

Pfizer Global Research & Development

Groton, CT

June 25/26, 2002

Beryllium Research Symposium, Bethesda MD

Acknowledgement: Most of the research presented today was conducted at Inhalation Toxicology Research Institute [LRRI], Albuquerque, NM

Be Research Symposium, GL Finch

cbd an occupational health mystery
CBD: An occupational health mystery

Who?

  • current screening reveals many sensitized and diseased pts
  • only “susceptible” individuals appear to get CBD

What?

  • granulomatous lesions with pronounced TH lymphocytic component, with pronounced Be-specific reactivity
  • a debilitating lung disease

When?

  • a widely varying latency period following Be exposure; preceded by sensitization

Where?

  • mostly occupational following exposure to various Be forms
  • no clear dose-response relationship has been defined

Why?

  • an MHC-II restricted response
  • component of genetic susceptibility
  • importance of role of Be physicochemical form

Be Research Symposium, GL Finch

slide3

Model of Be Interaction with Immune System

From Newman, 1993

Be Research Symposium, GL Finch

selected observations in role of physicochemical form in cbd
Selected observations in role of physicochemical form in CBD
  • There are a wide variety of physicochemical forms encountered
    • Natural occuring mineral
    • Various “soluble”/”insoluble” forms in processing
    • Mostly insoluble forms delivered to end users
  • Early experience:
    • More soluble forms generally lead to acute Be disease
    • More insoluble forms generally lead to CBD
    • No exposure-dose-response apparent
  • Exposure-response relationships are now being revealed
    • CBD more likely following exposure to relatively insoluble forms
    • apparent excess risk for certain occupations/processes

Be Research Symposium, GL Finch

how can in vitro in vivo models help
How can in vitro/in vivo models help?
  • Understanding exposure-dose-response relationships
    • role of physicochemical form
    • acute, episodic, or chronic exposures
    • Linkage to health effects
  • Understanding pathogenesis of response
    • detailed characterization
    • manipulated and/or knock-in/out models
  • Seeking therapeutic intervention

Be Research Symposium, GL Finch

slide6

Lovelace database on the properties and health effects of beryllium aerosols

Be Research Symposium, GL Finch

slide7

Lovelace database on the properties and health effects of beryllium aerosols

Be Research Symposium, GL Finch

physicochemical properties and in vitro characteristics
Physicochemical properties and in vitro characteristics
  • Laboratory-produced preparations
    • BeO: produced with 7Be radiolabel and fired at 500 or 1000oC
    • Be metal: size-fractionated using an aerosol cyclone
  • “Field” preparations
    • Sawing/milling of alloys
    • Laser vaporization of Be metal

Be Research Symposium, GL Finch

slide9

Be Metal

Ni-Be Alloy

Cu-Be Alloy

Softer alloys yielded relatively more fine particles with identical machining processes

Be Research Symposium, GL Finch

slide10

Beryllium Metal Particles

Separated by an Aerosol Cyclone

Similarities: particle morphology

Differences: physical and aerodynamic size; specific surface area

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slide11

Be Metal Particles Have an Oxide Surface Layer

Initial dissolution behavior

might be similar for

Be and BeO

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slide12

Solubility in an acidic environment

Low fired BeO is more soluble than high fired or metal in an acidic environment

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slide13

Solubility in a simulated lung extracellular fluid

BeO is more soluble than Be in simulated lung fluid

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slide14

Toxicity to Canine Alveolar Macrophages

Toxicity increases with solubility of the Be material

Be Research Symposium, GL Finch

slide15

In Vitro Toxicity based on:

Mass Surface Area

Normalization by “surface area dose” resulted in comparable toxicity

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large animal models of be biokinetics and be induced toxicity studies in dogs
Large animal models of Be biokinetics and Be-induced toxicity – Studies in Dogs
  • Respirable preparations of 7BeO fired at either 500o or 1000oC were used
  • Dogs were exposed once by inhalation to achieve either low [17 g/kg] or high [50 g/kg] initial lung burdens [ILBs]
  • Dogs were sacrificed up through 365 days post-exposure
    • Biokinetic evaluation
    • Lung histopathology
  • A companion group was evaluated through 730 days post-exposure
    • Period lung lavage for cytology and lymphocyte simulation
    • Dogs were re-exposed at 2-yr then followed for an additional 210 days

Be Research Symposium, GL Finch

slide17

BeO Clearance/Translocation in Dogs

LUNGS

Lower-fired BeO cleared more rapidly from lung, and persisted at higher levels in extrapulmonary compartments

Be Research Symposium, GL Finch

lung lesion in beo exposed dogs
Lung lesion in BeO-exposed dogs

Be Research Symposium, GL Finch

interstitial granuloma in beo exposed dogs
Interstitial granuloma in BeO-exposed dogs

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slide21

Influence of BeO Temperature History

on the Influx of Neutrophils

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lymphocyte numbers and sis in dogs
Lymphocyte numbers and SIs in Dogs

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slide23
Lymphocyte SIs following re-exposure to BeOGrouped by first exposure to high or low ILBs of 500 or 1000oBeO

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summary of results in dogs
Summary of results in dogs
  • Compared to high fired BeO, low-fired BeO:
    • cleared from lungs more rapidly
    • produced more marked inflammatory response
    • Increased numbers of lymphocytes
    • Increased lymphocyte stimulation indices
  • Responses peaked several months after exposure
  • Previous exposure history did not influence responses to a 2nd exposure to low-fired BeO, and the effects were not cumulative

Be Research Symposium, GL Finch

comparative toxicity of be metal vs beo in monkeys
Comparative toxicity of Be metal vs. BeO in monkeys
  • Study used low-fired BeO [500oC] and size fractionated Be metal
  • Animals were exposed by bronchoscopic, intrabronchiolar instillation
  • Regimen 1:
    • Graded doses of BeO [saline, 2.5, 12.5, 37.5 g] or Be metal [saline, 1, 50, 150 g] into different lung lobes
    • Doses based on estimated dissolution over 80 dpe
    • Histological evaluation of granulomas guided dose selection for regimen 2
  • Regimen 2:
    • Single doses of 12.5 g BeO or 50 g Be metal
    • Lavages through 120 dpe and sacrifice at 180 dpe

Be Research Symposium, GL Finch

be induced lesions in monkey
Be-induced lesions in monkey

Be Research Symposium, GL Finch

be induced lesion in monkey
Be-induced lesion in monkey

Be Research Symposium, GL Finch

lymphocytes recovered by lavage
Lymphocytes recovered by lavage

Be Research Symposium, GL Finch

lymphocyte proliferation
Lymphocyte proliferation

Be Research Symposium, GL Finch

large animal studies summary
Large animal studies - summary
  • Clear differences between BeO [and temperature history] and Be metal demonstrated
  • Biologically:
    • Granulomatous lesions were produced
    • Lymphocytes were increased in number
    • Increased lymphocyte proliferation demonstrated
  • However:
    • Biological responses were not progressive
    • Additional efforts were devoted to murine studies using Be metal

Be Research Symposium, GL Finch

summary of importance of physicochemical form
Summary of importance of physicochemical form
  • It is important for “relatively insoluble” particles
    • The amount of surface presented appears to control dissolution and toxicity
    • Form and preparation influences disposition, biokinetics, and in vivo toxicity
  • Exposure-dose-response need not be rejected
    • Just need to look in the right place
    • Compare “equivalent” exposures
    • Account for host factors, genetic susceptibility

Be Research Symposium, GL Finch

conclusion a hypothesis
Conclusion – a hypothesis
  • A hypothesis: there is a critical balance in lung between solubility and retained or newly deposited dose
    • Solubility/stimulus: needed to release Be++ to produce antigenic stimulus and induction of sensitization
    • Retention/re-challenge: needed to provide long term challenge depot of Be++ once sensitization is achieved
    • Both form/solubility and chronicity of exposure undoubtably work in concert – with host factors - to drive CBD

Be Research Symposium, GL Finch

acknowledgements
LRRI Principal Investigators:

Greg Finch

Mark Hoover

Pat Haley

LRRI Scientists

Ed Barr

Bill Bechtold

Dave Bice

Fletcher Hahn

Charles Hobbs

Tom March

Bruce Muggenburg

Kris Nikula

Bill Griffith

Janet Benson

Steve Belinsky

Technical Support Staff:

Lee Blair

Dee Esparza

Anna Holmes

Applied Toxicology Group

Exposure Operations Group

Animal Care Unit

Necropsy/Histology Lab

Lung Cancer Program

Collaborations:

Bill Carlton, DVM, Purdue

Alan Rebar, DVM, Purdue

Funding from the US Department of Energy

Acknowledgements

Be Research Symposium, GL Finch

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