A Comparison of the High-Affinity Peripheral Benzodiazepine Receptor Ligands DAA1106 and (R)-PK11195...

A comparison of the high-affinity peripheral benzodiazepinereceptor ligands DAA1106 and (R)-PK11195 in rat modelsof neuroinflammation: implications for PET imagingof microglial activation

Sriram Venneti,* Brian J. Lopresti,� Guoji Wang,* Susan L. Slagel,� N. Scott Mason,�Chester A. Mathis,� Michelle L. Fischer,� Niccole J. Larsen,� Amanda D. Mortimer,�Teresa G. Hastings,� Amanda D. Smith,� Michael J. Zigmond,� Tetsuya Suhara,§Makoto Higuchi§ and Clayton A. Wiley*

*Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

�Department of Radiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

�Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

§Molecular Neuroimaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan

Abstract

Activated microglia are an important feature of many neurolo-

gical diseases and can be imaged in vivo using 1-(2-chlorophe-

nyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide

(PK11195), a ligand that binds the peripheral benzodiazepine

receptor (PBR). N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phen-

oxyphenyl) acetamide (DAA1106) is a new PBR-specific

ligand that has been reported to bind to PBR with higher

affinity compared with PK11195. We hypothesized that this

high-affinity binding of DAA1106 to PBR will enable better

delineation of microglia in vivo using positron emission

tomography. [3H]DAA1106 showed higher binding affinity

compared with [3H](R)-PK11195 in brain tissue derived from

normal rats and the rats injected intrastriatally with

6-hydroxydopamine or lipopolysaccharide at the site of the

lesion. Immunohistochemistry combined with autoradiography

in brain tissues as well as correlation analyses showed that

increased [3H]DAA1106 binding corresponded mainly to acti-

vated microglia. Finally, ex vivo autoradiography and positron

emission tomography imaging in vivo showed greater retent-

ion of [11C]DAA1106 compared with [11C](R)-PK11195 in

animals injected with either lipopolysaccaride or 6-hydroxy-

dopamine at the site of lesion. These results indicate that

DAA1106 binds with higher affinity to microglia in rat models

of neuroinflammation when compared with PK11195, sug-

gesting that [11C]DAA1106 may represent a significant

improvement over [11C](R)-PK11195 for in vivo imaging of

activated microglia in human neuroinflammatory disorders.

Keywords: DAA1106, microglia, neuroinflammation, periph-

eral benzodiazepine receptor, PK11195, positron emission

tomography imaging.

J. Neurochem. (2007) 102, 2118–2131.

Activated microglia are hypothesized to promote neurode-generation in several disorders such as Alzheimer’s disease,human immunodeficiency virus (HIV) associated dementiaand Parkinson’s diseases [reviewed in (Gonzalez-Scaranoand Baltuch 1999)]. On activation, microglia secretes severalneurotoxins, which are thought to trigger various cellular

Received January 11, 2007; revised manuscript received March 22,2007; accepted May 2, 2007.

Address correspondence and reprint requests to Dr Clayton A. Wiley,Presbyterian University Hospital, Neuropathology Division, 200 LothropStreet A515, Pittsburgh, PA 15213, USA. E-mail: [email protected]

Abbreviations used: 11C, carbon-11 isotope; 3H, tritium isotope; Bmax,

maximal bound receptors; BP, binding potential; CD68, rat lysosomalmarker for activated microglia; DAA1106, (N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide); GFAP, glial fibrillary acidicprotein; HIV, human immunodeficiency virus; KD, dissociation constant;LPS, lipopolysaccaride; MRI, magnetic resonance imaging; PBR, per-ipheral benzodiazepine receptor; PET, positron emission tomography;PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquino-linecarboxamide.

Journal of Neurochemistry, 2007, 102, 2118–2131 doi:10.1111/j.1471-4159.2007.04690.x

2118 Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 2118–2131� 2007 The Authors

processes including cell death cascades in neurons (Stoll andJander 1999). These harmful effects of microglia can bepotential therapeutic targets, which may be modulated todecrease CNS inflammatory pathology (Klegeris and McG-eer 2005). If microglial activation could be evaluated in vivo,it would be possible to identify dynamic neuroinflammatorychanges as well as monitor efficacy of therapies targeted atdecreasing neuroinflammation.

Several studies suggest that microglia can be imagedin vivo using positron emission tomography (PET) withligands specific to peripheral benzodiazepine receptor (PBR)(Banati 2002; Block et al. 2007). Unlike the central benzo-diazepine receptor, PBR is expressed at relatively low levelsin the normal brain on resting microglia and astrocytes(Cagnin et al. 2002; Casellas et al. 2002; Venneti et al.2006). Autoradiographic studies suggest increased expres-sion of PBR specifically in activated microglia in braintissues in neurological diseases such as multiple sclerosis andexperimental autoimmune encephalitis (Vowinckel et al.1997; Banati et al. 2000), rodent models of stroke (Myerset al. 1991; Stephenson et al. 1995), facial nerve axotomy(Banati et al. 1997), brain trauma (Raghavendra Rao et al.2000), and macaque models of HIVencephalitis (Mankowskiet al. 2003; Venneti et al. 2004).

Various ligands have been synthesized that bind specific-ally to PBR. Labeling these ligands with tritium (3H) andcarbon-11 (11C) has enabled their use in autoradiography andPET, respectively. Of these ligands 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide(PK11195), a lipid soluble isoquinoline carboxamide hasbeen extensively characterized (Banati 2002). 11C labeledPK11195 shows increased uptake to varying degrees (com-pared with control subjects) in human patients with glioma(Pappata et al. 1991), stroke (Ramsay et al. 1992; Gerhardet al. 2000, 2005; Price et al. 2006), multiple sclerosis(Banati et al. 2000; Debruyne et al. 2003; Versijpt et al.2005), Rasmussen’s encephalitis (Banati et al. 1999), herpesencephalitis (Cagnin et al. 2001b), Alzheimer’s disease(Cagnin et al. 2001a), multiple system atrophy (Gerhardet al. 2003), corticobasal degeneration (Gerhard et al. 2004;Henkel et al. 2004), amyotrophic lateral sclerosis (Turneret al. 2004), upper motor neuron syndromes (Turner et al.2005), Parkinson’s disease (Ouchi et al. 2005; Gerhard et al.2006), hepatic encephalopathy (Cagnin et al. 2006), andHuntington’s disease (Pavese et al. 2006).

While it is well established that [11C](R)-PK11195 showsincreased CNS retention in a wide array of neurologicaldisorders, the specific binding signal is generally very lowand challenging to quantitate with typical PET image noiselevels (Petit-Taboue et al. 1991; Groom et al. 1995; Banatiet al. 2000; Pappata et al. 2000). Furthermore, certaindiscrepancies in this body of work remain to be addressed.For example, [11C](R)-PK11195 shows increased binding inregions traditionally not associated with microglial activation

such as the thalamus in several diseases like Alzheimer’sdisease (Cagnin et al. 2001a), Parkinson’s disease (Ouchiet al. 2005), and Huntington’s disease (Pavese et al. 2006),and the occipital cortex in amyotrophic lateral sclerosis(Turner et al. 2004). As it is not possible to histopatholog-ically ascertain microglial activation in these regions inhuman PET studies, it is possible that these findings reflectregional variations in the constitutive PBR or some degree ofnon-uniform non-specific binding in the CNS. Theseconcerns highlight the significance of developing newerligands that show more specificity and sensitivity to activatedmicroglia for PET imaging.

A newer ligand, N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide (DAA1106), is an aryloxyan-ilide derivative that binds with higher affinity to PBRcompared with PK11195 (Chaki et al. 1999; Zhang et al.2003; Maeda et al. 2004). This is reflected by a signifi-cantly lower dissociation constant (KD) assessed in mitoch-ondrial fractions derived from the rat cerebral cortex (Chakiet al. 1999). Because of its high-affinity binding properties,it has been suggested that DAA1106 may provide greatersensitivity than PK11195 as a PET imaging agent for thePBR. DAA1106 in the resting brain is highest in theolfactory bulb and the cerebellum (Chaki et al. 1999;Zhang et al. 2003; Maeda et al. 2004), but the cell typeexpressing this receptor, such as in the cerebellum, has notbeen elucidated. Further, it is not known whether DAA1106preferentially labels activated microglia in neuroinflamma-tion. In this work we sought to characterize the bindingproperties of DAA1106 in normal rodent CNS tissues,verify the cellular component of DAA1106 binding, andultimately investigate whether or not the higher affinity ofDAA1106 to PBR results in improved in vivo imaging ofactivated microglia compared with PK11195 in rodentmodels of neuroinflammation using PET and ex vivoautoradiography.

Multiple investigational approaches were employed in tworodent models of neuroinflammation, which together permitthe comparison of in vivo PET data with histopathologicaland pharmacological analyses. We report that [3H]DAA1106displays higher binding affinity compared to[3H](R)-PK11195 in normal brain tissues and brain tissueobtained from rats injected with either lipopolysaccaride(LPS) or 6-hydroxydopamine (6-OHDA). Increases in[3H]DAA1106 binding corresponded mainly to activatedmicroglia. Ex vivo autoradiography showed that[11C]DAA1106 bound specifically at higher levels comparedwith [11C](R)-PK11195 at the site of lesion in LPS or6-OHDA-injected rats. Finally, PET imaging in lesioned ratsshowed increased [11C]DAA1106 retention compared with[11C](R)-PK11195 at the site of the lesion in vivo. These datasuggest that microglia in brain lesions bind and retain moreDAA1106 compared with PK11195 and may serve as abetter ligand to image activated microglia in vivo using PET.

PET imaging of microglia with DAA1106 2119

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 2118–2131

Materials and methods

Animals

All animals were housed and maintained according to standards of

the Association for Assessment and Accreditation of Laboratory

Animal Care, and experiments were approved by the University of

Pittsburgh Institutional Animal Care and Use Committee. Male

Sprague–Dawley rats (300–350 g) were injected with either 50 lgLPS (Sigma, St Louis, MO, USA) (total n = 13) or 20 lg 6-OHDA

(Sigma) (total n = 9) or vehicle (total n = 2) into the striatum.

Surgical procedures were conducted as described earlier (Hastings

et al. 1996). Briefly, animals were anesthetized with equithesin

[3.0 mg/kg; 258 mmol/L chloral hydrate/20% (vol/vol), propylene

glycol/86 mmol/L, MgSO4/20% (wt/vol), Nembutal (sodium pento-

barbital)] and were injected sterotaxically with either 6-OHDA or

LPS into the striatum with the help of a Hamilton syringe

(coordinates: +0.8 mm anterior–posterior; +2.7 mm medial–lateral

from bregma; )5.0 mm dorsal–ventral from dura). Animals were

allowed to recover after surgery and were housed for 4 days

following LPS injections or 21 days following 6-OHDA injections

prior to PET scanning and killed as maximal neuroinflammation was

seen at these time points in each of the two models (as assessed by

immunohistochemistry).

Tissue processing

Following PET scans, animals were perfused with normal saline,

killed, and the brain was removed. Brain tissue was either processed

to obtain frozen sections for immunohistochemistry/autoradiogra-

phy or homogenized for filtration binding assays. Brain tissue from

the cerebellum, occipital cortex, and basal ganglia was dissected

from control/normal rats (n = 3). In animals injected with LPS

(n = 3) and animals injected with 6-OHDA (n = 3) the ipsilateral

and contralateral striatum was dissected. Brain tissue from each of

these cases was then weighed and homogenized in ice-cold

50 mmol/L HEPES buffer (pH 7.4). Homogenates were washed

four times by centrifugation at 40 000 g for 20 min at 4�C and

protein values were estimated using the bicinchoninic acid protein

assay kit before using homogenates in filtration binding experiments

(Pierce, Rockford, IL, USA).

Filtration radioligand binding assays

Homogenized brain tissue samples (total protein concentration

ranging from 150 to 200 lg) were incubated with either 0.5–

64 nmol/L [3H](R)-PK11195 (sp. act., 89.9 Ci/mmol; NEN Life

Sciences Products, Boston, MA, USA) or 0.2–20 nmol/L [3H]-

DAA1106 (sp. act., 80 Ci/mmol; American Radiolabeled Chemical,

St Louis, MO, USA) at 4�C for 2 h in a final volume of 250 lL of

HEPES (4�C, pH 7.4). This was defined as total binding. Non-

specific binding was determined by the inclusion of 10 lmol/L

PK11195 or 10 lmol/L DAA11106, respectively. The reaction was

terminated by filtration through glass fiber filters (Whatman-GF/B

glass fiber filter paper, Catalogue # FP-100, Brandel, Gaithersburg,

MD, USA) pre-soaked in 0.3% polyethyleneimine by the addition

of ice-cold HEPES in a vacuum cell harvester (Brandel). Filter

bound radioactivity was counted in a liquid scintillation spectro-

meter (Tricarb liquid scintillation counter, Perkin Elmer Life

Sciences, Wellesley, MA, USA) after the addition of 6 mL of

liquid scintillation fluid (Perkin Elmer Life Sciences). Specific

binding at each concentration of 3H-ligand was defined as the

difference between total binding and non-specific binding and

ranged from 80% to 90% of total binding. All samples were run in

duplicate. Bmax in fmoles per mg protein and KD in nmol/L were

determined using PRISM software (Graphpad, San Diego, CA,

USA).

Immunohistochemistry

Immunostaining and laser confocal microscopic imaging was

performed as described before (Venneti et al. 2004). Frozen sections

obtained from lesioned rats were stained with antibodies to glial

fibrillary acidic protein (GFAP) (mouse monoclonal, DAKO,

Carpinteria, CA, USA), or anti-rat CD68 (mouse monoclonal,

Serotec, Raleigh, NC, USA), or tyrosine hydroxylase (rabbit

polyclonal, Covance Research Products, Denver, PA, USA) used

at concentrations 1 : 1000, 1 : 100 and 1 : 1000, respectively.

Sections were then incubated with Cy5-conjugated goat anti-mouse

or Cy3-conjugated anti-rabbit IgG at a concentration of 1 : 200

(Jackson Immunoresearch Laboratories Inc., West Grove, PA,

USA). Immunostained sections were then scanned and quantified

on a laser confocal microscope (LSM 510, Zeiss, Heidelberg,

Germany). This scope is equipped with an argon laser with 458,

477, 488, and 514 nm primary emission lines. Each section was

scanned along the z-axis to define the middle optical plane used in

quantification (262 144 pixels/plane; 1 pixel = 0.25 lm2). Image

analysis was performed on a Silicon Graphics computer (Windows

NT 4.0 operating system, Microsoft, Redmond, WA, USA) using the

LSM software (version 3.0, Zeiss). Scanning parameters such as

laser power aperture, gain, and photomultiplier tube settings were

kept constant for each wavelength.

An individual blinded to the experimental design imaged three

areas (40X) encompassing 106 100 lm2. For each cell phenotype

scanned, contribution to signal intensity from autofluorescence

was minimized using a threshold that was kept constant. In each

area the average pixel fluorescence along with the pixel counts for

a given cell phenotype marker that exceeded the threshold were

enumerated. The average pixel fluorescence was multiplied by the

total number of pixels to measure the total florescence for that cell

phenotype marker in that area. The total fluorescence values

determined from the 3 scanned areas in one brain region were

averaged to represent a measure of the cell phenotype in that

brain region.

In vitro autoradiography

Autoradiography was performed as described earlier (Venneti et al.2004). Briefly, 15 lm thick frozen brain sections were placed on

SuperfrostTM glass slides (Sigma) and incubated in ice-cold

50 mmol/L TRIS–HEPES (pH 7.4) containing 1 nmol/L [3H](R)-PK11195 or 1 nmol/L [3H]DAA1106 for 30 min. Specificity of

binding was ensured by the inclusion of 1 lmol/L PK11195 or

1 lmol/L DAA1106 in parallel sections. The sections were mounted

with a layer of autoradiographic LM-1 emulsion (Amersham, UK),

were developed after 4 weeks and imaged on the confocal

microscope.

To evaluate the cellular localization of [3H]DAA1106 in lesioned

rat brain sections, we combined immunostaining and autoradio-

graphy. Sections were first immunostained with either GFAP,

or CD68 or both as above and then processed for autoradio-

2120 S. Venneti et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 2118–2131� 2007 The Authors

graphy, following which they were imaged on the confocal

microscope.

PET imaging

High specific activity [11C](R)-PK11195 and [11C]DAA1106 were

produced at the University of Pittsburgh, PET facility using methods

similar to those previously described (Zhang et al. 2003; Vennetiet al. 2004). Briefly, [11C]DAA1106 was labeled with 11C by

O-[11C] methylation of a desmethyl precursor (DAA1123) (Zhang

et al. 2003) and [11C](R)-PK11195 was labeled with 11C by the

method of Shah et al. (1994). Chemical and radiochemical purities

were ‡95% with specific activities ‡2.0 Ci/lmol at the end of a

40 min synthesis. Typical end-of-synthesis yields were approxi-

mately 30% for both [11C]DAA1106 and [11C](R)-PK11195.Normal rats (n = 3) were anesthetized (isoflurane) and positioned

in a microPET P4 scanner (Concorde Microsystems, Knoxville, TN,

USA) to simultaneously image brain, heart, and lungs. Normal rats

were injected with either [11C]DAA1106 or [11C](R)-PK11195(750–1500 lCi) and emission data collected for 60–75 min. At

15 min post-injection, a displacing dose of unlabeled DAA1106

(1.0 mg/kg) was administered over 60 s.

Rats injected with LPS (n = 6) or 6-OHDA (n = 6) or vehicle

(n = 2) were imaged for 30 min, after which they were immediately

killed and their brains removed for ex vivo autoradiography.

Additional experiments to demonstrate blocking of [11C]DAA1106

or [11C](R)-PK11195 specific binding were also conducted using

5.0 mg/kg of PK11195 (n = 2) or 1.0 mg/kg of DAA1106 (n = 2)

administered 30 min prior to radiotracer injection. PET emission

data were reconstructed using 2D filtered back projection and

corrected for photon attenuation and scatter. Regional brain

radioactivity concentrations were summed over the scan frames at

various time points and were normalized to both the injected dose

and the body mass of the animal (percent injected dose of

radioligand normalized to body weight of the animal per gram of

brain tissue) to yield a semi-quantitative measure of radiotracer

binding.

Ex vivo autoradiography

Immediately following the 30 min PET imaging session, rats

injected with LPS (n = 6), 6-OHDA (n = 6) or vehicle (n = 2)

were killed and the brains were rapidly removed and placed on

a chilled sectioning block (Harvard Apparatus, Natick, MA,

USA). The brain was sectioned coronally into 2 mm sections

and mounted on a plastic film and apposed to a phosphor screen

(Fuji BAS-SR2025, Fuji Photo Film Co., Kanagawa, Japan) for

1 h, after which the exposed plated was imaged on a high-

resolution phosphorimager (Fuji BAS-5000, Fuji Medical Sys-

tems, Stamford, CT, USA). Brain sections were then fixed and

processed for immunohistochemistry to confirm the presence of

the lesion.

Statistical analysis

Data were analyzed using PRISM software (Graphpad). Student’s ttests or one-way ANOVA tests with post-test Bonferroni correction

and 95% CI were used to analyze data. Correlations using 95% CI

were performed to quantify the relationship between [3H]DAA1106

binding and microglia or astrocytes. Results from correlational

analyses are represented by ‘r’, the Pearson’s coefficient.

Results

[3H]DAA1106 shows lower KD values compared with

[3H](R)-PK11195 in normal rat brain tissue

We used saturation filtration binding to compare bindingparameters of [3H]DAA1106 with [3H](R)-PK11195 innormal rat (n = 3) brain tissue. Brain tissue from cerebellum,basal ganglia, and the occipital cortex of three adult rats wasdissected out. Saturation binding experiments were conduc-ted on homogenized brain tissue with either [3H](R)-PK11195 (representative curve Fig. 1a) or [3H]DAA1106(representative curve Fig. 1b) to obtain Bmax and KD valuesreflective of the total number of binding sites per mg proteinand binding affinity of the ligand to PBR, respectively.

Dissociation constant values for [3H]DAA1106 comparedwith [3H](R)-PK11195 were five- to sixfold lower in all brainregions assessed in ([3H](R)-PK11195 – occipital cortex:6.42 ± 2, cerebellum: 3.97 ± 0.7, and basal ganglia:5.86 ± 1.5; [3H]DAA1106 – occipital cortex: 0.4 ± 0.2,cerebellum: 0.5 ± 0.3, and basal ganglia: 0.4 ± 0.2) (Fig. 1d).Bmax values were uniformly lower with [3H]DAA1106 com-pared with [3H](R)-PK11195 ([3H](R)-PK11195 – Occipitalcortex: 144 ± 22, cerebellum: 429 ± 65, and basal ganglia:393 ± 87; [3H]DAA1106 – occipital cortex: 123 ± 11, cere-bellum: 349 ± 87, and basal ganglia: 225 ± 12) in all brainregions but did not approach statistical significance (Fig. 1c).As binding affinity is inversely proportionality to theKD, thesedata suggest that [3H]DAA1106 bindswith higher affinity than[3H](R)-PK11195.

Rat models of neuroinflammation

We used two rat models of neuroinflammation to determinewhether DAA1106 demonstrated an increase in specificbinding in neuroinflammation. Rats were injected with either50 lg of LPS into the striatum or 20 lg of 6-OHDA.Microglial activation was assessed by staining for thelysosomal activation marker CD68 and by changes from aramified, resting to an activated, amoeboid morphology.Astrocytosis was assessed by changes in GFAP staining.6-OHDA injected animals showed 26%, 78%, and 96%increase in CD68 staining and 23%, 79%, and 69% increasein GFAP staining compared with controls at 1, 2, and3 weeks post-injection. LPS injected animals showed 99%,160%, 73%, and 1% increase in CD68 staining and 37%,81%, 67%, and 33% increase in GFAP staining comparedwith controls at 2, 4, 7, and 14 days post-injection. The3 week and 4 day post-injection time points in 6-OHDA andLPS injected animals respectively were used for all experi-ments as maximum neuroinflammation was observed at thesetime points. In 6-OHDA lesioned animals, tyrosine hydroxy-lase staining was significantly decreased on the ipsilateralside compared with the contralateral side (Fig. 2a and b) alongwith increased ipsilateral staining for the activated microglial



marker (rat CD68) (Fig. 2c) at 3 weeks post-injection. LPS-injected rats showed greater ipsilateral infiltration of activatedmicroglia at the site of the lesion compared with 6-OHDAlesioned animals (Fig. 2c, d and i) at the specified time points.Activated microglias were not detected on the contralateralside in either model (data not shown).

[3H]DAA1106 shows lower KD values compared with

[3H](R)-PK11195 in brain tissue obtained from rats

lesioned with either 6-OHDA or LPS

We sought to determine whether DAA1106 showedincreased specific binding to activated microglia as comparedwith (R)-PK11195 in rodent models of neuroinflammation,

600(a)

(c) (d)

(b)

400

fmo

ls/m

gfm

ols

/mg

fmo

ls/m

g(n

mo

l/L)

[3H](R)-PK11195 (nmol/L) [3H]DAA1106 (nmol/L)

[3H](R)-PK11195

[3H]DAA1106

200

0

500 876543210

400

300

200

100

0CB BG OC CB BG OC

***** *

600

400

200

00 0 5 10 15 2010 20 30 40 50 60 70

Fig. 1 [3H]DAA1106 shows lower dissociation constant (KD) values

in normal brain tissues when compared with [3H](R)-PK11195. Sat-

uration filtration binding experiments were conducted in cerebellum

(CB), basal ganglia (BG), and occipital cortex (OC) of normal rat brain

tissue (n = 3) using [3H]DAA1106 (black bars) and [3H](R)-PK11195

(white bars). The Bmax (fmols/mg), reflective of the total number of

binding sites and the KD (nmol/L) reflective of the binding affinity of the

ligands to PBR was calculated using PRISM software. (a and b)

Representative saturation binding curves with [3H](R)-PK11195 (a)

and [3H]DAA1106 (b) from the cerebellum of normal rats. (c) Bmax with

[3H]DAA1106 (black bars) in all regions assessed in normal rat brain

tissue was uniformly lower than [3H](R)-PK11195 (white bars) but did

not approach statistical significance. (d) KD was significantly lower with

[3H]DAA1106 (black bars) than [3H](R)-PK11195 (white bars) indica-

ting a higher affinity in all regions assessed. Data was analyzed using

student’s t-test, ***p < 0.0001, **p < 0.001, *p < 0.01.

Fig. 2 [3H] DAA1106 shows lower dissociation constant (KD) values

in rats injected with either lipopolysaccaride (LPS) or 6-hydroxydop-

amine (6-OHDA) when compared with [3H](R)-PK11195. (a–d) Rats

were injected with either 20 lg of 6-OHDA (a–c) or 50 lg of LPS (d)

into the striatum. Brain sections were then stained with tyrosine hy-

droxylase (TH, marker for dopaminergic terminals, a and b) or CD68

(marker for rat activated microglia, c and d). Rats injected with 6-

OHDA showed a decrease in tyrosine hydroxylase staining in the

ipsilateral (b) compared with contralateral side (a). Rats injected with

6-OHDA (c) and LPS (d) showed activated microglia in the ipsilateral

striatum with more numbers of activated microglia in LPS injected rats

(d) compared with 6-OHDA (c) injected rats. Scale bar indicates

100 lm. (e–h) Filtration binding was assessed in rat striatal brain tis-

sue injected with either 20 lg 6-OHDA (n = 3, e and f) or 50 lg LPS

(n = 3, g and h) using [3H]DAA1106 (black bars) and [3H](R)-PK11195

(clear bars). (e and g) The Bmax (fmols/mg), reflective of the total

number of binding sites was significantly higher in the area ipsilateral

to the lesion compared with the area contralateral to the lesion with

both [3H]DAA1106 (black bars) and [3H](R)-PK11195 (clear bars) in

both 6-OHDA (e) and LPS (g) injected rats. Bmax with [3H]DAA1106

and [3H](R)-PK11195 did not differ significantly within the same area.

(f and h) The KD (nmol/L), reflective of the ligand-binding affinity was

not different between the ipsilateral and the contralateral regions with

the same ligand. However, the KD with [3H]DAA1106 (black bars) was

significantly lower than with [3H](R)-PK11195 (clear bars) in both ip-

sillateral and contralateral regions. Data was analyzed using student’s

t-test, ***p < 0.0001, **p < 0.001, *p < 0.01. (i) Microglia (CD68) and

astrocytes [glial fibrillary acidic protein (GFAP)] were quantified on the

side ipsilateral to the lesion in LPS (black bars), 6-OHDA (gray bars),

and sham (clear bars) lesioned animals. Microglial activation, but not

astrocytosis was significantly different in LPS and 6-OHDA lesioned

animals from sham-injected animals. CD68 (not detected) and GFAP

staining did not differ in both models on the side contralateral to the

lesion (data not shown). Data was analyzed using ANOVA, **p < 0.001,

*p < 0.01. (j) [3H]DAA1106 binding (Bmax, X-axis) correlated with the

abundance of microglia (CD68, black squares, r = 0.9437, filled line

p = 0.001), but not with the abundance of astrocytes (GFAP, black

triangles, dotted line, r = 0.3170, p = 0.3721).



(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i) (j)

nm

ol/L

nm

ol/L

6-OHDA 6-OHDATH TH

6-OHDA

700 10

[3H]DAA1106

[3H](R)-PK11195

8

6

4

2

0

10

8

6

4

2

0

1500 0001250 000

1000 000

500 000

750 000

250 000

0

LPS

6-OHDA

Sham1250 000

1000 000

Pix

el c

ou

nts

Pix

el c

ou

nts

750 000

500 000

250 000

00

CD68 GFAP500 1000

CD68

GFAP

r = 0.9437

r = 0.3170P = 0.3721

P = 0.0010

Bmax (fmols/mg) [3H]DAA11061500

*

** *

*600500

fmo

ls/m

gfm

ols

/mg

400300200100

140012001000

800600400200

0

0Ipsilateral Contralateral

Ipsilateral Contralateral



***

*

**

LPSCD68 CD68



and whether the higher binding affinity of DAA1106 toconstitutive PBR observed in normal rodent brain tissue isalso seen in neuroinflammatory conditions. Bmax valueswere significantly higher on the ipsilateral side comparedwith the contralateral side with either [3H](R)-PK11195(6-OHDA ipsilateral: 663.8 ± 15, contralateral: 393 ± 89;LPS ipsilateral: 1200 ± 77, contralateral: 401.9 ± 127) or[3H]DAA1106 (6-OHDA ipsilateral: 517.7 ± 62, contralat-eral: 225.1 ± 11, LPS ipsilateral: 1071 ± 267, contralateral:320.1 ± 113.3) in both models (Fig. 2e and g). On compar-ing the two ligands, Bmax values did not differ significantly inthe same brain regions, but KD values were significantlylower with [3H]DAA1106 (6-OHDA ipsilateral: 0.35 ± 0.1,contralateral: 0.43 ± 0.3; LPS ipsilateral: 0.33 ± 0.1, contra-lateral: 0.41 ± 0.2) than [3H](R)-PK11195 (6-OHDA ipsilat-eral: 7 ± 2.7, contralateral: 6.9 ± 1.7; LPS ipsilateral:6.6 ± 5, contralateral: 3.2 ± 2) (Fig. 2f and h). Further, Bmax

values in LPS injected rats were approximately twofoldhigher compared with 6-OHDA rats on the ipsilateral side(Fig. 2e and g), which was consistent with approximatelytwofold increase in activated microglia detected immuno-histochemically (Fig. 2i). These results suggest that theconcentration of [3H]DAA1106 binding sites (Bmax) isincreased within the site of inflammation in a manner similarto [3H](R)-PK11195 (Cicchetti et al. 2002). Further,[3H]DAA1106 displays lower KD values compared with[3H](R)-PK11195, suggesting a higher binding affinity inneuroinflammation.

[3H]DAA1106 binding corresponds mainly to activated

microglia

The peripheral benzodiazepine receptor is expressed largelyon astrocytes and microglia in the resting CNS (Banati et al.2000; Casellas et al. 2002). The relative contributions ofthese cell types to [3H]DAA1106 binding was determinedusing multiple techniques. First, we quantified the abundanceof microglia (CD68) and astrocytes (GFAP) in frozensections obtained from the same rat brain tissue used forfiltration binding analyses and correlated these values with[3H]DAA1106 Bmax values. [3H]DAA1106 binding correla-ted with the abundance of microglia (r = 0.9437,p = 0.001), but not with the abundance of astrocytes(r = 0.3170, p = 0.3721) (Fig. 2j). Next, we combinedimmunostaining for astrocytes (GFAP) and activated micro-glia (CD68) with [3H]DAA1106 autoradiography on frozenbrain sections obtained from rats injected with LPS (n = 3).[3H]DAA1106 binding (Fig. 3a and k) overlapped withactivated microglia (Fig. 3b, j and l) but not with GFAPlabeled astrocytes (Fig. 3e, f, i and l), suggesting that thepredominant cellular component of DAA1105 binding in theinjured CNS is to activated microglia. [3H]DAA1106 bindingto microglia was specific as it was displaced with 1 lmol/LDAA1106 (Fig. 3c, d, g, h, o and p). Similar results wereobtained in 6-OHDA lesioned rats (data not shown).

PET imaging of [11C]DAA1106 and [11C](R)-PK11195 in

normal rats

[11C]DAA1106 and [11C](R)-PK11195 were taken up innormal rodent brain equally well with maximal brainradioactivity concentrations between 0.08 and 0.17 percentinjected dose of radioligand normalized to body weight ofthe animal per gram of brain tissue approximately 2 minafter injection with [11C]DAA1106 exhibiting little or noclearance over the course of a 60 min emission scan(Fig. 4d, open gray squares). Uptake of both [11C](R)-PK11195 and [11C]DAA1106 was approximately fourfoldhigher in the heart and eightfold higher in the lung than inbrain where radiotracer delivery is not influenced by theblood–brain barrier (Fig. 4a–c). [11C](R)-PK11195 exhibitedmuch more rapid washout from lung (Fig. 4a) as comparedwith [11C]DAA1106 (Fig. 4b), while, both tracers showedsimilar clearance from the myocardium where PBR recep-tors are abundant (Fig. 4a and b). This difference could beexplained by an abundance of PBR in myocardium, and/orreduced efflux of tissue from myocardium. In rats injectedwith a displacing dose of unlabeled DAA1106,[11C]DAA1106 and [11C](R)-PK11195 were both effectivelydisplaced by unlabeled DAA1106 in lung (92% and 55% ofpre-displacement radioactivity concentration, respectively)(Fig. 4c, black and gray circles) and myocardium (80% and72% of pre-displacement radioactivity concentration,respectively) (Fig. 4c, black and gray triangles). In brain,approximately 75% of [11C]DAA1106 was effectivelydisplaced by unlabeled DAA1106 (Fig. 4d, filled graysquares) while [11C](R)-PK11195 brain radioactivity con-centrations showed a transient increase (�25%) afterDAA1106 displacement followed by renewed clearance atnominal rates (Fig. 4d, black squares). This transientincrease in brain radioactivity is likely from recirculationof [11C](R)-PK11195 displaced from peripheral tissues withabundant PBR expression, such as the heart and lung(Fig. 4c, black triangles and circles), by the high affinityDAA1106 ligand. These studies complement previouslydescribed blocking studies of [11C]DAA1106 with unlabe-led DAA1106 (1.0 mg/kg) and higher doses (5–10 mg/kg)of unlabeled PK11195 (Maeda et al. 2004).

Ex vivo binding analysis shows increased brain retention

of [11C]DAA1106 compared with [11C](R)-PK11195 in

rats lesioned with LPS and 6-OHDA

[11C]DAA1106 or [11C](R)-PK11195 was injected intra-venously into rats lesioned with LPS (n = 6, three perligand), 6-OHDA (n = 6, three per ligand), or vehicle(sham, n = 2). While both ligands showed increasedipsilateral retention in lesioned animals, [11C]DAA1106retention was qualitatively higher compared with [11C](R)-PK11195 in LPS (Fig. 5a and g) and 6-OHDA lesionedanimals (Fig. 6a and e). These results are consistent withthe filtration binding studies, suggesting that DAA1106 has



higher binding affinity compared with PK11195 inneuroinflammation. Blocking studies of [11C]DAA1106(n = 2) or [11C](R)-PK11195 (n = 2) in LPS lesionedanimals demonstrated that [11C](R)-PK11195 specificbinding was entirely blocked by either unlabeledPK11195 or DAA1106 (Fig. 5b and c). [11C]DAA1106binding was entirely blocked by unlabeled DAA1106(Fig. 5i), but incompletely blocked by PK11195 (Fig. 5h).Differences between 6-OHDA and sham-injected animalswere better delineated with [11C]DAA1106 (Fig. 6e and f)than [11C](R)-PK11195 (Fig. 6a and b), although to alesser extent than LPS-lesioned animals. These results

suggest that the increased retention of both radioligandsin the lesioned striatum is attributable to specific bindingto PBR.

PET imaging with [11C]DAA1106 shows increased brain

retention compared with [11C](R)-PK11195 in LPS and

6-OHDA lesioned rats in vivo

[11C]DAA1106 and [11C](R)-PK11195 were used to imagerats lesioned with LPS (n = 6, three per ligand), 6-OHDA(n = 6, three per ligand), or vehicle (sham, n = 2) in vivowith PET. PET images (Figs 5 and 6) are shown with whitesquares indicating the position of the rat brain relative to the

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

Fig. 3 [3H]DAA1106 binding corresponds mainly to microglia in li-

popolysaccaride (LPS)-injected rats. (a–h) Combined [3H]DAA1106

autoradiography (black grains) and immunostaining for activated

microglia (CD68) or astrocytes [glial fibrillary acidic protein (GFAP)]

was performed in rats injected with 50 lg of LPS (n = 3). Autoradi-

ographic images are shown in the panels a, c, e, and g while com-

bined autoradiography and immunohistochemistry are shown in the

panels b, d, f, and h. [3H]DAA1106 specific binding (a and e) over-

laps with CD68 labeled microglia (b) but not with GFAP immuno-

stained astrocytes (f). [3H]DAA1106 autoradiographic binding was

confirmed to be specific as it was displaced by unlabeled DAA1106

(c–d and g–h). Scale bar indicates 20 lm. (i–p) Combined

[3H]DAA1106 autoradiography (black grains) and immunostaining for

activated microglia (CD68, green) and astrocytes (GFAP, red) was

performed in rats injected with 50 lg of LPS (n = 3). (l and p)

Represent merged images of autoradiography and double label im-

munohistochemistry. [3H]DAA1106 Specific binding (k) overlaps with

CD68 labeled microglia (j and l) but not with GFAP immunostained

astrocytes (i and l). Unlabeled DAA1106 was included in parallel

sections to ensure specificity of binding (m–p). Scale bar indicates

100 lm.



head (outside the square). When compared with [11C](R)-PK11195, [11C]DAA1106 showed increased brain retentionin LPS (Fig. 5d and j) and 6-OHDA (Fig. 6c and g) lesionedanimals consistent with our filtration binding and ex vivoautoradiography data. Blocking experiments with 5.0 mg/kgof PK11195 (n = 2) or 1.0 mg/kg of DAA1106 (n = 2) inLPS-lesioned animals showed that [11C](R)-PK11195 retent-ion in the brain was blocked by either PK11195 or DAA1106(Fig. 5e and f), while [11C] DAA1106 was blocked effect-ively by DAA1106 (Fig. 5l) and to a lesser extent withPK11195 (Fig. 5k). Difference between 6-OHDA and sham-injected animals was better delineated with [11C]DAA1106(Fig. 6g and h) than [11C](R)-PK11195 (Fig. 6c and d).

Discussion

Activation of microglia is a common pathological feature ofseveral neurological disorders despite varied pathogenesis.Current hypotheses suggest that activation of microglia maybe a heterogeneous phenomenon depending on the type of

insult (Carson et al. 2004). The term ‘disease-associated’microglia to encompass these potentially different states ofactivation is perhaps more fitting. Although ‘disease-associ-ated’ microglia may not be a homogenous population asubstantial body of work suggests that activation of thesecells, albeit to different degrees, play a significant role in thepathogenesis of several neurologic disorders. Activatedmicroglia can be imaged in vivo by taking advantage ofincreased expression of PBR expressed at low levels in thenormal brain, but enriched in activated microglia in neuro-logical disorders (Cagnin et al. 2001a; Casellas et al. 2002)Venneti et al. 2006). By utilizing PK11195, a ligand thatspecifically binds to PBR, activated microglia can be imagedusing PET in a number of neurodegenerative disorders[reviewed in (Cagnin et al. 2002)]. In this study, we showthat activated microglia can be better delineated in vivo withPET using [11C]DAA1106, which has an approximately five-to 10-fold higher affinity for PBR than PK11195. We usedfiltration binding analyses to compare the pharmacologicproperties of [3H]DAA1106 with [3H](R)-PK11195 in

(a) (b)

(c) (d)

(a) [11C](R)-PK11195 [11C]DAA1106

[11C]DAA1106 lung[11C]DAA1106(no displacement)[11C]DAA1106

[11C](R)-PK11195

[11C]DAA1106 myo

[11C](R)-PK11195 lung

[11C](R)-PK11195 myo

myo

lung

myo

lung

Late (30–60 min)Early (0–5 min)

0.9 0.10

0.08

0.06

0.04

0.02

DAA1106

DAA11060.6

[11C

] lig

and

%(I

D*k

g)/

g

[11C

] lig

and

%(I

D*k

g)/

g

0.3

0.00 10 20 30

Time (min)40 50 60 0 10 20 30

Time (min)

40 50 60

Late (30–60 min)Early (0–5 min)

Min

Max

Min

Max

Fig. 4 Positron emission tomography (PET) imaging with

[11C]DAA1106 and [11C](R)-PK11195 in normal rats. The kinetics of

[11C]DAA1106 or [11C](R)-PK11195 injected in normal rats was fol-

lowed in the heart, lungs (a–c) and brain (d), in vivo using PET. (a)

[11C](R)-PK11195 was taken up in the lungs by 0–5 min and was

cleared in the lungs by 30–60 min. [11C](R)-PK11195 retention was

seen in the myocardium in the late time frames (30–60 min). (b)

[11C]DAA1106 was taken up in the lungs by 0–5 min and remained in the

lungs by 30–60 min. [11C]DAA1106 retention was seen in the myocar-

dium in the late time frames (30–60 min). (c and d) A displacing does of

1 mg/kg DAA1106 was injected 15 min (indicated by the dotted black

line) after initial the 11C-ligand administration. Time-activity curves of

radioactivity concentrations are expressed as percent-injected dose per

gram normalized by body mass (%ID*kg/g, Y-axis) plotted against time

(minutes, X-axis). (d) [11C]DAA1106 was taken up in the brain and

showed retention over time (gray open squares). [11C]DAA1106 was

displaced by DAA1106 (gray filled squares), indicating that

[11C]DAA1106 binding was specific. [11C](R)-PK11195 binding (black

squares) showed an initial displacement by DAA1106 followed by brief

upswing before renewed clearance from brain. (c) [11C]DAA1106

retention in the lung (gray circles) and myocardium (gray triangles) was

displaced by DAA1106 indicating specific binding in both these organs.

Similarly, [11C](R)-PK11195 binding was displaced by DAA1106 in

both the lung (black circles) and myocardium (black triangles).



normal rat brain tissues, as well as brain tissue obtained fromrats lesioned with either LPS or 6-OHDA. In all of theseexperiments the KD of [3H]DAA1106 was five to sixfoldlower than [3H](R)-PK11195, reflecting a significantly higherbinding affinity to PBR (Figs 1 and 2). The Bmax, whichreflects the number of binding sites, was uniformly lowerwith [3H]DAA1106 than [3H](R)-PK11195, but this findingdid not achieve statistical significance. These observationsare consistent with previous observations suggesting that the

binding domains of PK11195 and DAA1106 do notcompletely overlap (Okuyama et al. 1999; Culty et al.2001). [3H]DAA1106 bound mainly to activated microgliaas determined by correlational analyses (Fig. 2) and com-bined autoradiography and immunohistochemistry (Fig. 3).Ex vivo autoradiography experiments showed increasedbrain retention of [11C]DAA1106 compared with[11C](R)-PK11195 at the site of the lesion in LPS and6-OHDA treated animals (Figs 5 and 6) and [11C]DAA1106

[11C](R)-PK11195

[11C]DAA1106

No block PK11195 DAA1106

No block PK11195 DAA1106

Min

Max

Min

Max

Min

Min

Max

Max

(a) (b) (c)

(d)

(g) (h) (i)

(j) (k) (l)

(e) (f)

Fig. 5 [11C]DAA1106 is retained at greater levels at the site of the

lesion compared with [11C](R)-PK11195 in lipopolysaccaride (LPS)-

lesioned animals. Rats lesioned with 50 lg LPS (n = 6, total) were

injected with either [11C]DAA1106 or [11C](R)-PK11195 (1–2 mCi)

intravenously (n = 3 each). To confirm that the ligand retention was

specific 5.0 mg/kg of PK11195 (n = 2) or 1.0 mg/kg of DAA1106

(n = 2) was administered 30 min prior to the radiotracer injection. A

30-min uptake period was allowed after radiotracer injection, following

which animals were scanned in the microPET imager. Animals were

then killed and brain tissue was processed for ex vivo autoradiogra-

phy. PET data was then compared with ex vivo autoradiography data.

(a–c) Ex vivo autoradiography images obtained from animals injected

with [11C](R)-PK11195. Uptake at the site of lesion (a), which is

blocked by either PK11195 (b), or DAA1106 (c). (d–f) Positron emis-

sion tomography (PET) images obtained from animals injected with

[11C](R)-PK11195. White squares indicate the position of the rat brain

relative to the rat cranium (outside the square). Retention at the site of

lesion (d), which is blocked by either PK11195 (e), or DAA1106 (f). (g

and h) Ex vivo autoradiography images obtained from animals injected

with [11C]DAA1106. Uptake of [11C]DAA1106 at the site of lesion (g) is

greater than [11C](R)-PK11195 uptake (a). [11C]DAA1106 uptake is

blocked partially by PK11195 (h), or completely by DAA1106 (i). (j–l)

PET images obtained from animals injected with [11C]DAA1106.

In vivo retention of [11C]DAA1106 at the site of lesion (j) is greater

than [11C](R)-PK11195 retention (d). [11C]DAA1106 uptake is blocked

partially by PK11195 (k), or completely by DAA1106 (l).



was effectively displaced by DAA1106 in the brain suggest-ing that [11C]DAA1106 binding was specific and reversible(Figs 4 and 5). Finally, in vivo PET imaging with[11C]DAA1106 showed greater specific uptake at the site

of the lesion in both LPS and 6-OHDA injected animalscompared with [11C](R)-PK11195 (Figs 5 and 6). These datatogether suggest that DAA1106 is a specific marker foractivated microglia, which may better delineate neuroinflam-mation when compared with PK11195 in rat models in vivousing PET imaging.

[11C](R)-PK11195 has been extensively used to studymicroglial activation non-invasively using PET in a varietyof neurologic disorders with an inflammatory component(Cagnin et al. 2002). [11C](R)-PK11195 binding in most ofthese conditions is seen in regions traditionally associatedwith the histopathological presence of activated microglia.However, [11C](R)-PK11195 binding is also seen in regionsusually not associated with pathology and microglial activa-tion such as the thalamus in several human neurologicaldisorders disease (Cagnin et al. 2001a), (Ouchi et al. 2005)(Pavese et al. 2006), (Pappata et al. 2000). These findingshave been interpreted as a result of microglial activation and‘synaptic stripping’ in regions connected to areas of primarypathology. As it is not possible to confirm the histologicalpresence of activated microglia in these regions in humanstudies, it is possible that these increases reflect regionalvariations in the constitutive PBR population that areindependent of the disease pathology or a non-uniformelement of non-specific [11C](R)-PK11195 binding. Anotherimportant consideration is regarding the extent of microglialactivation that needs to be present in the CNS before a signalcan be detected using [11C](R)-PK11195. No significantdifferences are detected in [11C](R)-PK11195 bindingbetween controls and patients diagnosed with mild cognitiveimpairment (Schuitemaker et al. 2004, 2006), a conditionthat may progress to Alzheimer’s disease. Also, no signifi-cant differences are noted in [11C](R)-PK11195 PET imagingin HIV-infected patients with or without dementia(Hammoud et al. 2005; Wiley et al. 2006). These studiessuggest that low levels of microglial activation may not bedetected using [11C](R)-PK11195 and underscore the import-ance in developing ligands that bind to microglia with highspecificity and sensitivity. The high-affinity PBR agentDAA1106 may be able to address some of these concerns.

We choose to examine some of these questions in two rodentmodels of neuroinflammation. In the first model, rats injectedintrastriatally with LPS showed robust infiltration of activatedmicroglial similar to previous studies (Montero-Menei et al.1996). Rats injected intrastriatally with another neurotoxin,6-OHDA are frequently employed as a rodent model forParkinson’s disease and may also be a useful model ofneuroinflammation as it has been demonstrated that microglialactivation is observed in the lesioned tissue (Akiyama andMcGeer 1989; Cicchetti et al. 2002; Sanchez-Pernaute et al.2004). Rats lesioned with either toxin also show severalbehavioral abnormalities including altered feeding habits,hyperactivity, rotational movements, and motor disturbances(Mokry 1995; Hsieh et al. 2002; Smith et al. 2002).

(a) (b)

(c) (d)

(e) (f)

(g) (h)

[11C](R)-PK11195

[11C]DAA1106

Max

Max

Max

Max

Min

Min

Sham6-OHDA

Sham6-OHDA

Min

Min

Fig. 6 [11C]DAA1106 is retained at greater levels at the site of the

lesion compared to [11C](R)-PK11195 in 6-hydroxydopamine (6-

OHDA)-lesioned animals. Rats lesioned with 20 lg 6-OHDA (n = 6,

total) or sham lesioned (n = 2) were injected with either [11C]DAA1106

or [11C](R)-PK11195 (n = 3, each) (1–2 mCi) intravenously. A 30-min

uptake period was allowed after radiotracer injection, following which

animals were scanned in the microPET imager. Animals were then

killed and brain tissue was processed for ex vivo autoradiography.

Positron emission tomography (PET) data was then compared with

ex vivo autoradiography data. (a and b) Ex vivo autoradiography

images obtained from 6-OHDA lesioned animals (a) and sham-injected

animals (b) injected with [11C](R)-PK11195. (c and d) PET images

obtained from 6-OHDA lesioned animals (c) compared with sham-

injected animals (d) injected with [11C](R)-PK11195. White squares

indicate the position of the rat brain relative to the rat cranium (outside

the square). (e and f) Ex vivo autoradiography images obtained from

animals injected with [11C]DAA1106. Uptake of [11C]DAA1106 at the

site of lesion (e) is greater than [11C](R)-PK11195 uptake (a) or uptake

with either sham-injected animal (b and f). (g and h) PET images

obtained from animals injected with [11C]DAA1106. In vivo retention of

[11C]DAA1106 at the site of lesion (g) is greater than [11C](R)-PK11195

retention (c) or uptake with either sham-injected animal (d and h).



The relative contributions of astrocytes versus microglia toPBR-ligand binding in neuroinflammation are a subject ofcontinuing debate. Our results suggesting that [3H]DAA1106binds better to activated microglia compared with astro-cytes similar to results obtained with [3H](R)-PK11195from rat models of stroke (Myers et al. 1991) and ischemia(Stephenson et al. 1995), experimental autoimmune enceph-alitis (Vowinckel et al. 1997), multiple sclerosis (Vowinckelet al. 1997; Banati et al. 2000), facial nerve axotomy inrats (Banati et al. 1997), brain trauma in rats (RaghavendraRao et al. 2000), simian immunodeficiency virus enceph-alitis in macaques (Mankowski et al. 2003; Venneti et al.2004), and hippocampal lesions in mice (Pedersen et al.2006). Some studies report increased astrocyte expressionof PBR following an initial increase in microglia in braintissues obtained from rodents treated with the neurotoxintrimethyltin (Kuhlmann and Guilarte 2000) and cuprizone(Chen et al. 2004). However, our studies indicate thatincreased [3H]DAA1106 binding corresponded better toactivated microglia.

Our PET imaging studies in normal rodents have elucidatedsome significant differences in the relative brain kinetics of[11C]DAA1106 and [11C](R)-PK11195 that have importantimplications for their utility as PET imaging agents. Thesuccessful displacement of both [11C]DAA1106 and [11C](R)-PK11195 in heart and lung tissue, where PBR receptors areknown to be in higher abundance compared with brain,suggests that a significant component of the heart and lungtissue radioactivity following the injection of both agents canbe attributable to specific binding to PBR. In contrast, only[11C]DAA1106 was effectively displaced in brain, suggestingthat the majority of brain radioactivity in rats following theinjection of [11C](R)-PK11195 is non-displaceable non-spe-cific binding while the majority of radioactivity following theinjection of [11C]DAA1106 is bound to constitutive PBR.This is consistent with our in vitro binding studies thatdemonstrate DAA1106 to have approximately five to sixfoldhigher affinity for PBR in both rat brain tissues. This is ofsome importance as PET binding potential (BP); a commonlyemployed index of specific binding of PET radiotracers, isproportional to the ratio of Bmax to KD (Mintun et al. 1984).Therefore, there is an important dependence on KD in the PETBP measure. Indeed, the data presented in this work showsthat the ratio of Bmax/KD determined for DAA1106 usingconventional filtration binding methods was substantiallyhigher than (R)-PK11195. However, some caution must beexercised in interpreting these results in the context of PETBP. For example, the factors influencing radioligand kineticssuch as the tissue free-fraction f2, cannot be easily uncoupledfrom the PET BP measures and a low tissue free-fractioncould offset the advantages of a higher-affinity ligand in thedetermination of the BP.

The pharmacological properties of these ligands suggestthat DAA1106 binds with greater affinity to PBR compared

with PK11195. It is possible that the filtration bindingexperiments conducted using membrane-enriched are notlikely to be predictive of in vivo properties of the ligand.Moreover, in the ex vivo studies brains were block-sectioned manually into 2 mm thick slices, which yieldeda slice thickness approximately twice that of the microPETimages that could not be precisely co-registered with themicroPET data. The relatively small size of the rodentbrain and limited resolution of the microPET system addsfurther layers of complexity in making comparisonsbetween these various experimental systems. Further,PK11195 resulted in only partial blockade of[11C]DAA1106 in the LPS model and ex vivo autoradiog-raphy in 6-OHDA lesioned animals [11C]DAA1106 seemedto show a more diffuse pattern of uptake compared with[11C](R)-PK11195. Histopathological examination showsthat microglial activation does not fall off abruptly as onetraverses the lesion. It is possible that [11C](R)-PK11195 isless sensitive to this increasingly weak penumbra ofinflammation radiating from the lesion. Further, the relat-ively small size of the rodent brain and limited resolutionof the microPET system, coupled with the rapid brainclearance of [11C](R)-PK11195 and the resulting loss ofsignal-to-noise complicates quantification of the lesion onthe microPET images. Nevertheless, the assertion that aPET radioligand with potential clinical utility is binding tothe intended cellular component and that changes inmeasured Bmax values determined using a standardizedassay are observed in a manner that is consistent with theknown disease pathology is an important validation that isoften overlooked or bypassed in the progression to humaninvestigational studies.

In conclusion, DAA1106 exhibits many favorable proper-ties that could potentially yield improvements over [11C](R)-PK11195 in the delineation of activated microglia in vivousing PET. Among these properties are high in vitro bindingaffinities, cellular localization to activated microglia, andincreased specific binding in activated microglia in responseto CNS insults. MicroPET and autoradiography studiesdemonstrated that [11C]DAA1106 effectively crossed theblood–brain barrier and was retained in a specific manner athigher levels in rats lesioned with either LPS or 6-OHDA incomparison with [11C](R)-PK11195. While these resultssuggest that [11C]DAA1106 may represent a significantimprovement over [11C](R)-PK11195 for imaging activatedmicroglia in human neurological disorders in vivo using PET,further studies in human neuroinflammation are neededto fully characterize the potential enhancements of[11C]DAA1106.

Conflict of interest

All the authors declare no proprietary interest or any conflictof interest related to this study.



Acknowledgements

We thank James Kasenchak and Jason Nugyen for technical help

and James Ruskiewicz for help with the PET scans. The National

Institutes of Health supported this work: RO1 MH64921 (CAW),

K24 MH01717 (CAW).

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A Comparison of the High-Affinity Peripheral Benzodiazepine Receptor Ligands DAA1106 and (R)-PK11195...

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