Prague 2008 1 Plasma vs. tissue concentration to predict antibiotic efficacy PL Toutain UMR 181 Physiopathologie et Toxicologie Experimentales INRA/ENVT.

Prague 2008 1

Plasma vs. tissue concentration to predict antibiotic efficacy

PL ToutainUMR 181 Physiopathologie et Toxicologie Experimentales

INRA/ENVT

ECOLENATIONALEVETERINAIRET O U L O U S E

Fourth International conference on AAVM Prague, Czech republic 24-28, 2008

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In veterinary medicine, there are many publications on tissular concentrations to promote the idea that some antibiotics having a high tissular concentration accumulate in biophase (quinolones, macrolides) and are more efficacious as suggested by their low or undetectable plasma concentrations

The inadequate tissue penetration hypothesis

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• Two false assumptions1. tissue is homogenous2. bacteria are evenly distributed through

tissue

spurious interpretation of all important tissue/serum ratios in predicting the antibacterial effect of AB

The inadequate tissue penetration hypothesis: Schentag 1990

Schentag, 1990

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Statements such as ‘concentrations in tissue x h after dosing are much higher than the MICs for

common pathogens that cause disease’ are meaningless

Mouton & al JAC 2007

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Objectives of the presentation:To address some basic questions

1. Where are located the bugs ?

• Extracellular vs. intracellular

2. Where is the biophase?

• Interstitial space fluid vs. intracellular cytosol vs. intracellular organelles

3. What is a tissue and what is a tissue concentration

4. How to assess the biophase antibiotic concentration

• Total tissular concentration vs. ISF concentration.

5. The issue of lung penetration

1. Epithelial lining fluid (ELF):????

2. The hypothesis of targeted delivery of the active drug at the infection site by phagocytes

6. Plasma as the best surrogate of biophase concentration for PK/PD interpretation

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Q1: Where are located the pathogens

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Where are located the pathogens

ISF

Most bacteria of clinical interest

•S. pneumoniae•E. coli•Klebsiella•Mannhemia ; pasteurella•Actinobacillius pleuropneumoniae•Mycoplasma hyopneumoniae•Bordetala bronchiseptia

Cell(most often in phagocytic cell)

• mycoplasma (some)• Chlamydiae• Brucella• Cryptosporidiosis• Listeria monocytogene• Salmonella• Mycobacteria• Rhodococcus equi

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Q2: Where is the biophase

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The interstitial space fluid is the biophase

1. Most bacterial infections are located in the extracellular compartment.

2. Except few cases, In acute infections in non-specialized tissues, where there is no abscess formation, interstitial space fluid (ISF) must be considered as the actual target space for anti-infective agents

3. ISF concentrations are of primary interest

Muller et al. AAC , 2004, 48: 1441-1453

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Q3: what is a tissue & what is a tissular concentration

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Historical definition of a tissue drug concentration

• In the past, it was used to characterize the total concentration in a homogenized biopsy sample– For vet medicine: a by-product of regulatory

residue studies

• It was assumed that:– tissue is homogenous– that antibiotics is evenly repartited in tissue – That bacteria are evenly repartited in tissue

– Each of these assumptions is false and can be very misleading

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Why a total drug tissue concentrations may be misleading?

1. Drug distributed mainly extracellularly• β-lactams and aminoglycosides,• grinding up the tissue means dilution of the drug by

mixing intracellular and extracellular fluids, resulting in underestimation of its concentrations at the site of infection.

2. Drug accumulated by cells • fluoroquinolones or macrolides• assay of total tissue levels will lead to gross

overestimation of the extracellular concentration.• The opposite is true for intracellular infections.

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Methods for studies of target site drug distribution in antimicrobial

chemotherapy

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“Tissue concentrations”

• Total tissue– homogenates– biopsies

• Extracellular fluids– implanted cages– implanted threads– wound fluid– blister fluid– ISF (Microdialysis, Ultrafiltration)

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The tissue cage model for in vivo and ex vivo investigations

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The tissue cage model

• Perforated hollow devices• Subcutaneous

implantation• development of a highly

vascularized tissue• fill up with a fluid with half

protein content of serum (delay 8 weeks)

•C.R. Clarke. J. Vet. Pharmacol. Ther. 1989, 12: 349-368

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The tissue cage model : PK limitations

• A foreign body– Not a physiological space

– Clinical counterparts?

• Ascitic fluid, effusions ( pericardial, pleural…)

• Interpretation may be difficult because PK

determined by:– diffusion capacity across the TC

– TC size and geometry

• surface area/volume ratio is the major determinant of peak and

trough drug level

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The tissue cage model

Drug administration

Slow equilibration

inoculation

Time

(C)

Time

(C)

T1/2 varies with the surface area / volume ratio of the tissue cage

Penicillin 5 to 20 hDanofloxacin 3 to 30 h

Greko, 2003, PhD Thesis

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Microdialysis & ultrafiltration Techniques

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What is microdialysis (MD)?

• Microdialysis, a tool to monitors free antibiotic concentrations in the fluid which directly surrounds the infective agent

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Microdialysis: The Principle

• The MD Probe mimics a "blood capillary".

•There is an exchange of substances via extracellular fluid

•Diffusion of drugs is across a semipermeable membrane at the tip of an MD probe implanted into the ISF of the tissue of interest.

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• the implanted MD probe is perfused with the perfusate, ie, a physiologic liquid at a very slow rate.

• Substances present in the interstitial space fluid of the investigated site can diffuse into the perfusate through a semipermeable membrane at the tip of the MD probe and appear in the dialysate.

• Afterward the concentration in the dialysate is chemically analyzed and the true concentration in the interstitial space fluid can be calculated.

Microdialysis: The Principle

Antibiotic

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Microdialysis materials

CMA60 MicrodialysisCMA60 Microdialysis CMA60 MicrodialysisCMA60 Microdialysis

1. Introducer with CMA 60 Microdialysis Catheter

2. Outlet tube

3. Vial holder

4. Microvial

5. Inlet tube

6. Luer lock connection

7. Puncture needle.

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Microdialysis : Limits

• MD need to be calibrated

• Retrodialysis method– Assumption: the diffusion process is quantitatively

equal in both directions through the semipermeable membrane.

– The study drugs are added to the perfusion medium and the rate of disappearance through the membrane equals in vivo recovery.

– The in vivo percent recovery is calculated (CV of about 10-20%)

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A small experimental error in recovery estimate results in a relatively larger error in

drug concentration estimates which is probably responsible for the greater

interanimal variability observed in lung tissue than in the other media

Marchand & al AAC June 2005

MD need to be calibrated:

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Ultrafiltration

• Excessive (in vivo) calibration procedures are required for accurate monitoring

• Unlike MD, UF-

sample concentrations are independent on probe diffusion characteristics

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Microdialysis vs. Ultrafiltration

Ultrafiltration

Vacuum

Microdialysis :a fluid is pumped

through a membrane;

The driving force is a pressure differential (a vacuum) applied

across the semipermeable membrane

The analyte cross the membrane by diffusion

The driving force is a concentration gradient

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Marbofloxacin : plasma vs.ISFIn vivo filtration

Bidgood & Papich JVPT 2005 28 329

Microdialysis•Not suitable for long term in vivo studies

Ultrafiltration•Suitable for long term sampling (in larger animals, the UF permits complete freedom of movement by using vacutainer collection method)

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What we learnt with animal and human microdialysis studies

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Plasma (total, free) concentration vs interstitial concentration (muscle, adipose

tissue) (Moxifloxacin)

Muller AAC, 1999 Time (h)

Total (plasma, muscle)free (plasma)interstitial muscleinterstitial adipose tissue

2 6 10 12 30 4020

100

1000

Co

nce

ntr

ati

on

(n

g/m

L)

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Plasma (total, free) concentration vs muscle (free) concentration

Total (plasma)free (muscle)free (plasma)

Liu J.A.C. 2002

cefpodoxine

cefixime

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What we learnt with animal and human MD studies

• MD studies showed that:1. the concentrations in ISF of selected

antibiotics correspond to unbound concentrations in plasma

2. They are generally much lower than total concentrations reported from whole-tissue biopsy specimens especially for macrolides and quinolones

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What we learnt with MD studies: Inflammation

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Tissue concentrations of levofloxacin in inflamed and healthy subcutaneous adipose tissue

Methods: Free Concentrations measured in six patients by

microdialysis after administration of a single intravenous dose of 500 mg.

Results:The penetration of levofloxacin into tissue appears to be unaffected by local inflammation.Same results obtained with others quinolones

Hypothesis: Accumulation of fibrin and other proteins, oedema, changed pH and altered capillary permeability

may result in local penetration barriers for drugs

Bellmann & al Br J Clin Pharmacol 2004 57

Inflammation

No inflammation

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What we learnt with MD studies: Inflammation

• Acute inflammatory events seem to have little influence on tissue penetration.

• “These observations are in clear contrast to reports on the increase in the target site availability of antibiotics by macrophage drug uptake and the preferential release of antibiotics at the target site a concept which is also used as a marketing strategy by the drug industry” Muller & al AAC May 2004

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In acute infections in non-specialized tissues, where there is no abscess formation, free serum

levels of antibiotics are good predictors of free levels in tissue

fluid

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The issue of lung penetration

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Animal and human studies MD: The issue of lung penetration

•Lung MD require maintenance under anesthesia, thoracotomy (patient undergoing lung surgery)..

•Does the unbound concentrations in muscle that are relatively accessible constitute reasonable predictors of the unbound concentrations in lung tissue (and other tissues)?

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Free muscle concentrations of cepodoxime were similar to free lung concentration and therefore provided a surrogate measure

of cefpodoxime concentraion at the pulmonary target site

Liu et al., JAC, 2002 50 Suppl: 19-22.

Cefpodoxime at steady state: plasma vs. ISF (muscle & Lung)

Plasma

Free plasma

Muscle Lung

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The issue of lung penetration:Imipem

• The major finding of this study was the observation of virtually superimposed free IPM concentration-versus-time profiles in the three media investigated,

• This result not only is in agreement with theory but also is consistent with most of the data in the literature.

imipenem distribution in muscle and lung interstitial fluids

Marchand & al AAC June 2005

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The issue of lung penetration

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Lung infections

• Uncertainty of the relevant actual location of proliferating bacteria– Alveoli, pulmonary interstitium, bronchioles, blood??

• What is the biophase??– Epithelium lining fluid (ELF)– Lung IF, alveolar macrophages, tisue biopsies, blood,

bronchial secretion, sputum??

• ELF seems the most relevant specimen but potential sources of error: dilution, release of AB from alveolar macrophage in the sample

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The blood-alveolar barrier

The alveolar epithelial cells would not be expected to permit passive diffusion of

antibiotics between cells, the cells being linked by tight junctions

•Fenestrated pulmonary capillary bed• expected to permit passive diffusion of antibiotics with a molecular weight 1,000

Epithelial lining fluid

ELF(protein:<10%)

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

space

Alveolarmacrophage

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

space

Alveolarmacrophage

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

space

Alveolarmacrophage

ABAB

AlveolarAlveolarAlveolar

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Drug passage in the lung

Drug passage through the alveolar epithelial cells will depend on the lipophilicity

and diffusibility of the antibiotics, similar to the

drug entry into the central nervous system.

Kiem & Schentag AAC 2008 Jan 24-36

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ELF

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ELF concentration: possible biais

• Measurement problems may confound the interpretation of the ELF concentrations of antibiotics.

• Cells, especially AM cells (that constitute 3.8 to 10.0% of ELF volume) are included in the composition of ELF

• The cells may be lysed during the measurement of antibiotic concentration in BAL-derived fluids.

Kiem & Schentag AAC 2008 Jan 24-36

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ELF

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BETA-LACTAMS

Measured ELF concentrations of the beta-lactams are well below serum concentrations, and their respective

concentrations in AM cells were negligible

The low measured ELF concentrations of betalactams in

comparison to their corresponding serum levels could be the result of low capacity of their unbound free

fractions for penetration through the alveolar epithelial cell barriers.

Kiem & Schentag AAC 2008 Jan 24-36

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ELF

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MACROLIDES AND KETOLIDES

Measured ELF concentrations of macrolides and ketolides and their derived AUCs were consistently

higher than serum levels by as much as 10-fold

the high ratios of ELF concentrationto serum concentration for macrolides and

ketolides could not be explained solely on the basis of good penetration across the alveolar

epithelium.

The high concentrations of macrolides and ketolides in ELF might be explained

by the possible contamination of intracellular antibiotics occurring during

the process of BAL.

Kiem & Schentag AAC 2008 Jan 24-36

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ELF

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FLUOROQUINOLONES

Fluoroquinolones achieved higher ELF levels than their free

serum concentrations

Kiem & Schentag AAC 2008 Jan 24-36

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

ISF

Capillarywall

AlveolarEpithelium

Thigh junctions

ABAB

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• The high ELF concentrations of some antibiotics, which were measured by the BAL technique, might be explained by possible contamination from high achieved intracellular concentrations and subsequent lysis of these cells during the measurement of ELF content.

• This effect is similar to the problem of measuring tissue content using homogenization

Kiem & Schentag’ Conclusions (1)

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• Fundamentally, ELF may not represent the lung site where antibiotics act against infection.

• In view of the technical and interpretive problems with conventional ELF and especially BAL, the lung microdialysis experiments may offer an overall better correlation with microbiological outcomes.

• We continue to express PK/PD parameters using serum concentration of total drug because these values do correlate with microbiological outcomes in patients.

Kiem & Schentag’ Conclusions (2)

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The site of infection: Intracellular pathogens

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Intracellular location of bacteria

Phagosome

Lysosome

Chlamydiae

Listeria

No fusion with lysosome

Phagolysosome

S.aureausBrucella

SalmonellaCoxiella burneti

pH=5.0

3

4

2

1

Fusion

pH=7.4

BB

B

B

B

B

B B

Cytosol

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Intracellular location of antibiotics

Phagolysosomevolume 1 to 5% of cell volume

pH=5.0

Macrolides (x10-50)Aminoglycosides (x2-4)

CytosolpH=7.2

Fluoroquinolones(x2-8)beta-lactams (x0.2-0.6)

Rifampicin (x2)Aminoglycosides (slow)

Ion trapping for weak base with high pKa value

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What are the antibiotic intracellular expressions of activity

Phagolysosome

Macrolides Aminoglycosides

CytosolpH=7.2

Fluoroquinolonesbeta-lactamsRifampicin

Aminoglycosides

Good Low or nul

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The hypothesis of targeted delivery of active drug at the active site by the phagocytes

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Drug delivery by the phagocytes

• Transport by "non professional" phagocytes (e.g. azithromycin by fibroblast)

• Fibroblast acts as a reservoir for drug and macrophages

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Neutrophils as antibiotic delivery system

The ability of neutrophils to migrate preferentially to sites of infection makes them attractive as a delivery mechanism for antibiotics; theoretically , with the proper cellular PK, an antibiotic could be taken up by neutrophils, which would then transport the drug and later release it . This could provide a mechanism for achieving higher levels of antibiotic in tissues i.e. directly at the nidus of infection.

Scorneaux & Shryock tilmicosine in pigs; JVPT 1998, 21: 257-258 & tilmicosine in cattle in: J. Dairy Sci. 1999, 82: 1202-1212)

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Macrolides: how to explain efficacy of low plasma concentrations?

M

M1.PK hypothesis

2-PD hypothesis

M

PMN

Nucleus

High local M concentration in the vicinity of the bug

Sink or reservoir???

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PK model for exposure to macrolides: The limits

M+ M

M

M

MMM

MM

2. Slow or very slow efflux(e.g. 20% within 3 h for

azithromycin) more rapid (90% within 1 h) for erythromycin)

Transit time in a (normal) lung capillary: 26 sec

No In vivo data to support the hypothesis

Mass balance considerations (Tylosine in piglet 10 Kg BW, dose: 10 mg/kg)• Total pool of PMN=2 mL/L of blood• Peak Plasma concentration: 1 µg/mL• Accumulation ratio: 50• Total amount of drug located in PMN :100µg in toto =1/1000 of the dose

Sink rather than reservoir

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Macrolides: how to explain efficacy of low plasma concentrations?

M

M1.PK hypothesis

2-PD hypothesis

M

PMN

Nucleus

MIC in vivo < MIC in vitroCytokine modulation, ERK signaling

High local M concentration in the vicinity of the bug

M

Sink or reservoir???

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Conclusions:

1. In acute infections in non-specialized tissues, where there is no abscess formation, free plasma levels of antibiotics are good predictors of free levels in interstitial fluid

2. PK/PD indices predictive of antibiotic efficacy should be based on free plasma concentration

3. People who truly understand tissue concentration work in corporate marketing departments (Apley, 1999)