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Journal of Neuropathology and Experimental Neurology Vol. 61, No. 9Copyright q 2002 by the American Association of Neuropathologists September, 2002

pp. 797 805

Imaging Ab Plaques in Living Transgenic Mice with Multiphoton Microscopy and Methoxy-X04,a Systemically Administered Congo Red Derivative

WILLIAM E. KLUNK, MD, PHD, BRIAN J. BACSKAI, PHD, CHESTER A. MATHIS, PHD, STEPHEN T. KAJDASZ, BA,MEGAN E. MCLELLAN, BA, MATTHEW P. FROSCH, MD, PHD, MANIK L. DEBNATH, MS, DANIEL P. HOLT, BS,

YANMING WANG, PHD, AND BRADLEY T. HYMAN, MD, PHD

Abstract. The identification of amyloid deposits in living Alzheimer disease (AD) patients is important for both earlydiagnosis and for monitoring the efficacy of newly developed anti-amyloid therapies. Methoxy-X04 is a derivative of Congored and Chrysamine-G that contains no acid groups and is therefore smaller and much more lipophilic than Congo red orChrysamine-G. Methoxy-X04 retains in vitro binding affinity for amyloid b (Ab) fibrils (Ki 5 26.8 nM) very similar to thatof Chrysamine-G (Ki 5 25.3 nM). Methoxy-X04 is fluorescent and stains plaques, tangles, and cerebrovascular amyloid inpostmortem sections of AD brain with good specificity. Using multiphoton microscopy to obtain high-resolution (1 mm)fluorescent images from the brains of living PS1/APP mice, individual plaques could be distinguished within 30 to 60 minafter a single i.v. injection of 5 to 10 mg/kg methoxy-X04. A single i.p. injection of 10 mg/kg methoxy-X04 also producedhigh contrast images of plaques and cerebrovascular amyloid in PS1/APP mouse brain. Complementary quantitative studiesusing tracer doses of carbon-11-labeled methoxy-X04 show that it enters rat brain in amounts that suggest it is a viablecandidate as a positron emission tomography (PET) amyloid-imaging agent for in vivo human studies.

Key Words: Alzheimer disease; Amyloid; Chrysamine-G; Imaging; Multiphoton microscopy; Positron emission tomogra-phy; Transgenic mice.

INTRODUCTION

Amyloid plaques and neurofibrillary tangles (NFTs)are proposed to play key roles in the pathogenesis ofAlzheimer disease (AD). Despite recent attempts (1), noaccepted method currently exists to non-invasively quan-tify these b-sheet amyloid deposits in living patients. Thepreferred characteristics of such a biomarker have beenoutlined by Klunk (2) and the importance has been em-phasized in a review by Selkoe (3). The availability ofan in vivo amyloid probe could 1) facilitate early diag-nosis, 2) allow clinico-pathological correlations of amy-loid deposition with early cognitive symptoms over time,and 3) provide a surrogate marker of efficacy of anti-amyloid therapies that are in early clinical trials (e.g. am-yloid b [Ab] immunization, beta- and gamma-secretaseinhibitors) (4–6). A surrogate marker of efficacy could

From the L aboratory of Molecular Neuropharmacology, Departmentof Psychiatry (WEK, MLD), University of Pittsburgh School of Medi-cine, Pittsburgh, Pennsylvania; Alzheimer’s Research Unit (BJB, STK,MEL, BTH), Massachusetts General Hospital, Charlestown, Massachu-setts; PET Facility, Department of Radiology (CAM, DPH, YW), Uni-versity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;Center for Neurologic Diseases, Department of Pathology (MPF),Brigham and Women’s Hospital and Harvard Medical School, Boston,Massachusetts.

Correspondence to: William E. Klunk, MD, PhD, University of Pitts-burgh, 705 Parran Hall-GSPH, 130 DeSoto Street, Pittsburgh, PA15213.

Support: This work was supported in part by grants from the Al-zheimer’s Association (IIRG-95-076 and TLL-01-3381) and NIH-AG01039 and AG20226 to WEK, a Pioneer Award from the Alzhei-mer’s Association and NIH-AG08487 to BTH, and NIH 5P01 AG15379to MPF. We also thank the Walters Family Foundation (BTH). MPF isa Paul Beeson Physician Faculty Scholar in Aging Research.

help bring these therapies into clinical use more quickly.Our laboratory has worked for several years to developderivatives of Congo red and Chrysamine-G as in vivoamyloid probes (7), but we and others have been ham-pered by the marginal brain entry of these relativelylarge, acidic compounds (8–11).

In this study, we report the properties of a promisingcompound, 1,4-bis(49-hydroxystyryl)-2-methoxyben-zene (or methoxy-X04) (Fig. 1). Methoxy-X04 entersthe brain much better than Congo red or Chrysamine-G and retains good binding affinity for Ab. In addition,we took advantage of the fluorescent properties of me-thoxy-X04 to utilize a novel imaging technique, mul-tiphoton microscopy (12), to directly demonstrate invivo detection of individual plaques in living transgen-ic mice after systemic injection of this amyloid-bindingprobe.

Multiphoton microscopy has been used for in vivo im-aging of Ab deposits in amyloid b-protein precursor(AbPP) transgenic mice after direct application to the pialsurface of either thioflavin-S (13, 14) or fluorescein-la-beled Ab antibodies (15). Although these reagents visu-alize plaques using this technique, they must be admin-istered topically because they do not cross theblood-brain barrier. Neither reagent can be used to detectamyloid deposits if administered systemically, preventingtheir use as in vivo imaging agents in humans.

Our current work combines the use of multiphoton im-aging technology along with systemic administration ofan amyloid-imaging agent to demonstrate directly thatmethoxy-X04 can be utilized for in vivo visualization ofindividual plaques and cerebrovascular amyloid in living

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Fig. 1. Structures of prototype compounds from 4 genera-tions of Congo red derivatives. Abbreviations: Gen. 5 genera-tion; M.W. 5 molecular weight; logPoct 5 log10 of the octanol:buffer partition coefficient.

Fig. 2. Reaction scheme for C-11 labeling of hydroxy-X04 to produce [11C]methoxy-X04. By-products labeled in the 49-position of the side rings were also obtained, but eluted later by HPLC (k9 5 11.3 vs 7.6 for methoxy-X04) and were identifiedby 49-methoxy cold standards.

animals. In contrast to Congo red, thioflavin-S, and Abantibodies, we also quantitatively demonstrate that car-bon-11-labeled methoxy-X04 enters the brain relativelywell. This combination of good brain entry and in vivoresolution of plaques after systemic injection make me-thoxy-X04 a potential positron emission tomography(PET) amyloid-imaging agent for human studies.

MATERIALS AND METHODS

Unless otherwise specified, reagents were of the highestgrade available and were purchased from Aldrich ChemicalCompany (Milwaukee, WI). Yields reported were for crudeproduct and were not optimized. Octanol-buffer partition co-efficients (Poct) and logPoct values were determined as previouslydescribed using phosphate-buffered saline (PBS: 137 mMNaCl, 3 mM KCl, 10 mM sodium phosphate; pH 7.4) and 1-octanol (16). The pKa of methoxy-X04 was determined spec-trophotometrically by measuring the absorbance and lmax of a25-mM solution of methoxy-X04 in H2O at a series of pH valuesfrom 9.0 to 12.5. The pKa was calculated by previously de-scribed methods (17, 18).

Chemical Synthesis

Methoxy-X04 [1,4-bis(49-hydroxystyryl)-2-methoxybenze-ne]: 2-Methoxy-p-xylylenediphosphonic acid tetraethyl ester(480 mg; 1.2 mmol) was coupled to 4-methoxymethoxy(MOMO)-benzaldehyde (600 mg; 3.6 mmol) in 4 ml dryDMSO and 640 ml (3.0 mmol) 25% sodium methoxide in meth-anol room temperature according to previously published pro-cedures (19). Non-optimized yields varied from 80%–90%. Thebis-MOMO compound was converted (deprotected) to the bis-hydroxy compound by heating a suspension of 25 mg ofMOMO-protected compound per 5 ml of 4:1 glacial acetic acid:water in 5 ml screw cap vials at 90–958C for 90 min giving 58mg (74% yield) of methoxy-X04, a yellow-green crystallinesolid. Five hundred MHz 1H NMR (DMSO-d6; Bruker AM500)of methoxy-X04 (for proton numbering, superscripts ‘‘1’’ and‘‘4’’ refer to protons on the styryl moieties substituted on the1- or 4-positions of the center ring, v1 and v2 refer to the vinylprotons with v1 being attached to the center ring): H-6: 7.587(d, J 5 8.14 Hz, 1H); H-21/61: 7.440 (d, J 5 8.64 Hz, 2H); H-24/64: 7.385 (d, J 5 8.64 Hz, 2H); H-v21: 7.199 (d, J 5 16.3Hz, 1H); H-v24: 7.192 (d, J 5 16.3 Hz, 1H); H-3: 7.185 (d, J5 1.53 Hz, 1H); H-5: 7.133 (dd, J 5 1.53/8.14 Hz, 1H); H-v11:7.118 (d, J 5 16.3 Hz, 1H); H-v14: 7.017 (d, J 5 16.3 Hz, 1H);H-31/51: 6.782 (d, J 5 8.64 Hz, 2H); H-34/54: 6.772 (d, J 5 8.64Hz, 2H); H-2-OCH3: 3.905 (s, 3H). HRMS m/z calculated forC23H20O3 (M1) 344.1412, found 344.1415.

Radiochemical Synthesis

[O-methyl-11C]methoxy-X04: Hydroxy-X04 [1,4-bis(49-hy-droxystyryl)-2-hydroxybenzene] was synthesized by substitut-ing 2-MOMO-p-xylylenediphosphonic acid tetraethyl ester forthe 2-methoxy ester used in the synthesis of methoxy-X04.[11C]methylation of hydroxy-X04 was performed using no car-rier added [11C]CH3I, 3.0 mmoles of hydroxy-X04, 7.2 mmolesK2CO3 in 400 ml of DMSO at 658C for 5 min according topreviously published procedures (19) (Fig. 2). Chemical andradiochemical purities as well as specific activity were deter-mined by analytical HPLC [k9 7.6 on a Prodigy ODS(3) columneluted with 50/50 acetonitrile/TEA buffer (TEA 5 50 mM tri-methylammonium phosphate, pH 7.2)]. Radiochemical purity

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was .95%, while chemical purity was .90% (determined us-ing a Waters 996 photodiode array detector set at 369 nm) withthe major chemical impurity being hydroxy-X04 (k9 3.3). Ap-proximately 370–740 MBq (10–20 mCi) of [11C]methoxy-X04was routinely produced having a specific activity .55.5 MBq/nmole at end-of-synthesis (EOS). The radiochemical yield was6.7% 6 1.2 at end-of-bombardment and 1.8% 6 0.5 at EOSbased on [11C]CH3I (n 5 8).

[11C]Methoxy-X34 was similarly prepared by [11C]methyla-tion of the dimethyl ester of X34 (20), followed by rapid esterhydrolysis using potassium tert-butoxide (Aldrich), resulting inyields and specific activity very similar to those obtained with[11C]methoxy-X04.

In Vitro Binding Studies

Preparation of Ab Fibrils: Ab(1–40) (Bachem Bioscience,Inc., King of Prussia, PA) was dissolved in PBS to a final con-centration of 433 mg/ml (100 mM). This solution was magnet-ically stirred at 1,200 rpm for 3 days at room temperature, vor-texing briefly when necessary to avoid gel formation at themeniscus. The initially clear solution gradually became homo-geneously cloudy (without large clumps). After this treatment,approximately 2% of the protein remained in the supernatantafter a 15-min centrifugation at 28,000 3 g. (21). The fibrillarnature of the Ab, thus prepared, was confirmed by the Congored method (21, 22). The 100-mM PBS stock was then diluted1:20 (to 5 mM) with 150 mM Tris-HCl, pH 7.0. The aggregatedpeptide suspension can be kept frozen at 2808C for at least 8wk without noticeable change in its properties. Before the bind-ing assay, the 5-mM stock was diluted 1:50 (to 100 nM) withbinding buffer (150 mM Tris-HCl, pH 7.0, containing 20% eth-anol). Aggregated Ab stock suspensions were continuouslystirred to maintain a homogenous suspension during removal ofaliquots for the binding assays.

Binding Studies with Synthetic Ab(1–40) Fibrils: The appro-priate concentrations of cold inhibitors to be tested were com-bined with [11C]methoxy-X04 (;700,000 cpm within 1 hours[h] of synthesis) in a volume of 950 ml of binding buffer (150mM Tris-HCl, pH 7.0, containing 20% ethanol to enhance sol-ubility of the test compounds). The assay was begun by additionof 50 ml of 100 nM Ab stock to achieve a final concentrationof ;2 pM [11C]methoxy-X04, 5 nM Ab fibrils, and the appro-priate concentration of test compound. After incubation for 30min at room temperature (a time when equilibrium had beenreached), the binding mixture was filtered through a WhatmanGF/B glass filter via a Brandel M-24R cell harvester (Gaithers-burg, MD) and rapidly washed twice with 3 ml binding buffer.The filters were placed in Cytoscint-ES (ICN Biomedical, Ir-vine, CA) and immediately counted. Complete (100%) inhibi-tion of binding was defined as the number of counts displacedby 1 mM cold methoxy-X04. Higher concentrations of me-thoxy-X04 caused aggregate (micelle) formation that caused ra-dioactivity to be trapped by the filter paper even in the absenceof Ab. Specific binding varied between 70%–75% of total bind-ing. All assays were done at least in triplicate. For determina-tion of inhibition constants (Ki), inhibition curves were fit (us-ing the RS/1 statistical package, version 6.1; BrooksAutomation, Chelmsford, MA) to the following equation, whichis derived from the Hill equation (23):

F(x) 5 M(K )H/{[L]H 1 (Ki)H}i

Where M 5 maximal percent [11C]methoxy-X04 bound (typicalfit gave 100%–104% for M), H is the Hill coefficient (typically0.60–0.80), and [L] is the concentration of inhibitor compound.

Tissue Staining

Tissue autofluorescence was quenched and staining was ac-complished by the procedure used for X34 staining describedin detail by Styren et al (20). Briefly, deparaffinized, quenchedtissue sections were taken from PBS into a 100-mM solution ofmethoxy-X04 in (40% ethanol)/(60% distilled H2O) (adjustedto pH 10 with 0.1 N NaOH) for 10 min. The sections werethen dipped briefly 5 times into tap water before differentiationin 0.2% NaOH in 80% ethanol for 2 min. The sections werethen placed in tap water for 10 min prior to coverslipping withFluoromount-G (Electron Microscopy Sciences, Fort Washing-ton, PA). Pretreatment of selected slides with 99% formic acidfor 5 min was performed prior to the PBS wash (20). Fluores-cent sections were examined using an Olympus Vanox AH-RFL-LB fluorescence microscope and were optimally viewedwith a V-filter set (excites 400–410 nm, dichroic mirrorDM455, 455 nm longpass filter).

In Vivo Pharmacokinetic Studies

Brain Uptake Studies in Rats: Male Sprague-Dawley rats (n5 3–5 at each time point) weighing 200 to 300 g were injectedin a lateral tail vein with 50 to 100 mCi (50–100 pmol) of[11C]methoxy-X04 or [11C]methoxy-X34 (specific activity ;55GBq/mmol or ;1500 Ci/mmol) contained in 0.1 ml of isotonicsaline solution (containing ;5% ethanol from the Sep-Pak elu-tion). Animals were anesthetized at 2 or 30 min following in-jection and killed by cardiac excision following cardiac punc-ture to obtain a terminal arterial blood sample. The brains wererapidly removed and dissected into cerebellum and remainingwhole brain (including brain stem) fractions. Brain and bloodsamples were counted in a gamma well-counter (Packard In-struments Model 5003, Meridan, CT), and the counts were de-cay-corrected to the time of injection relative to C-11 standardsprepared from the injection solution to determine the percentinjected dose (% ID) in the samples. The samples were weighedto determine the percent injected dose per gram (g) tissue (%ID/g), and this value was multiplied by the whole body weight(in g) to determine body weight normalized radioactivity con-centration values (% ID index or % IDI, see Results) for eachtissue sample at 2 or 30 min time points after i.v. injection of[11C]methoxy-X04 or [11C]methoxy-X34.

Metabolite Determinations: Terminal arterial blood sampleswere obtained as described above at 2, 30, and 60 min follow-ing injection of [11C]methoxy-X04. The whole blood was cen-trifuged for 2 min to separate the plasma. Five hundred ml ofplasma was added to an equal volume of acetonitrile and themixture was vortexed for 1 min and then centrifuged (13,0003 g) for an additional 2 min. A sample of the supernatant wasanalyzed by reverse phase HPLC using a Raytest Gabi Radio-HPLC detector. A Phenomenex Prodigy ODS(3) 250 3 4.6 mmcolumn was eluted with 50/50 acetonitrile/TEA buffer at 2 ml/min.

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Fig. 3. Competitive binding assays using cold (non-radio-active) Chrysamine-G (closed circles) and methoxy-X04 (opensquares) to compete for [11C]methoxy-X04 binding sites onAb(1–40) fibrils. Complete inhibition of specific binding (i.e.0% bound) was defined as the number of counts remaining inthe presence of 1 mM cold methoxy-X04. Higher concentrationsof methoxy-X04 caused aggregate (micelle) formation thatcaused radioactivity to be trapped by the filter paper even inthe absence of Ab. Specific binding varied between 70%–75%of total binding.

In Vivo Imaging StudiesAnimals examined were mice that carried both the Tg2576

APPsw (24) and the PS1(M146L) transgenes (25, 26). ThePS1(M146L) line of mice was bred to homozygosity, and theanimals for study were generated through breeding of thesehomozygous mice [PS1(M146L)/PS1(M146L)] with hemizy-gous Tg2576 mice to generate animals with single copies ofeach transgene. The genotype of all animals was determinedthrough PCR-based analysis of tail DNA using previously re-ported protocols (24–26). The Tg2576 transgene was main-tained in an SJL/B6 F1 background, while the PS1(M146L)transgene was maintained in an FVB/N background. This com-bination of transgenes has been observed to accelerate the de-velopment of Ab deposition in parenchyma and blood vessels(27). By 6 months of age, PS1/APP mice have extensive, con-gophilic, thioflavin-S-positive plaques that are predominantlycomposed of Ab(1–40) (28), which increase in number but donot change in character as the mouse ages. The cortical amyloidburden of a 12- month-old PS1/APP is similar to that of a typ-ical AD case (29, 30).

To fashion a thin-skull preparation for imaging, the mousewas anesthetized with Avertin (1.3% tribromoethanol, 0.8%tert-pentylalcohol in distilled water; 250 mg/kg, i.p.) and im-mobilized in custom-built, stage-mounted ear bars and a nose-piece, similar to a stereotaxic apparatus. Two circular regionsof the skull (;1–1.2 mm in diameter located on either side ofsagittal suture and just posterior to coronal suture) were thinnedusing a high-speed drill (Fine Science Tools, Foster City, CA)and a dissecting microscope (Leica, Wetzlar, Germany) forgross visualization. Heat and vibration artifacts were minimizedduring drilling by frequent application of artificial cerebrospinalfluid (ACSF: 125 mM NaCl, 26 mM NaHCO3, 1.25 mMNaH2PO4, 2.5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 25mM glucose).

Seven- to 13-month-old PS1/APP mice (n 5 3) were injectedi.v. with 5 to 10 mg/kg methoxy-X04 (1 mg/ml in normal salineadjusted to pH 5 12 with 0.1 N NaOH) immediately prior toimaging or were injected i.p. with 10 mg/kg methoxy-X04 (n5 4; 5 mg/ml in 10% DMSO, 45% propylene glycol, 45% PBS,pH 7.5) 24 h prior to imaging. Multiphoton imaging and post-mortem histology were performed as previously described (15,31). Briefly, 2-photon fluorescence was generated with 750 nmexcitation from a mode-locked Ti:Sapphire laser [Tsunami(Spectra-Physics, Mountain View, CA), driven by a 10- W Mil-lenium Xs pump laser (Spectra-Physics), mounted on a com-mercially available multiphoton imaging system (Bio-Rad1024ES; Bio-Rad, Hercules, CA). Custom-built external detec-tors containing 3 photomultiplier tubes (Hamamatsu Photonics,Bridgewater, NJ) collected emitted light in the range of 380 to480, 500 to 540, and 560 to 650 nm. An ACSF reservoir wascreated within the opened scalp over the thin-skull preparationsto accommodate the long working distance, water immersiondipping objectives (20 and 603, Olympus, Tokyo, Japan) of anOlympus BX-50 microscope. Three-dimensional volumes wereacquired by collecting a stack of x-y sections starting at thesurface of the thinned skull to several hundred microns deepinto the cortex.

Following imaging of the thin-skull sites, the thinned bonewas removed using fine forceps (Fine Science Tools). Thiofla-vin-S (0.005% w/v in ACSF; Sigma, St. Louis, MO) was then

applied directly to the brain for 20 min to label dense coreamyloid plaques. Sites were washed with ACSF and re-imaged.After image collection, the animal was killed and the brain fixedin 4% paraformaldehyde. Immunohistochemistry was per-formed using the BAM10 anti-Ab antibody (Sigma) as previ-ously described (32).

RESULTS

Physical Properties

A systematic series of structural alterations of the Con-go red pharmacophore resulted in the compound, me-thoxy-X04 (Fig. 1). Compared to the parent compound,Congo red, methoxy-X04 has a 47% lower molecularweight (MW 5 344), a value that falls into the optimalrange for brain entry (33). The carboxylic acid groupsfound in Chrysamine-G and methoxy-X34 are removed,leaving only the weakly acidic phenols, which, with apKa measured to be 10.82 6 0.07, would be unchargedat physiologic pH. The logPoct of methoxy-X04 is 2.6,higher than that of the less lipophilic Chrysamine-G(logPoct 5 1.8) and much higher than the polar com-pounds methoxy-X34 (logPoct 5 0.19) and Congo red(logPoct 5 20.18).

Binding Affinity to Ab(1–40) Fibrils

The results of a competition assay between radiola-beled [11C]methoxy-X04 and unlabeled (cold) methoxy-X04 are compared to a similar competition between[11C]methoxy-X04 and Chrysamine-G in Figure 3. Un-labeled methoxy-X04 displaced [11C]methoxy-X04 bind-ing to Ab(1–40) fibrils with a Ki if 26.8 6 10.0 nM.Displaceable (specific binding) ranged from 70%–75% of

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Fig. 4. AD frontal cortex (8-mm-thick paraffin sections)stained with methoxy-X04. Bar represents 100 mm. An amy-loid-laden vessel is marked by an asterisk in the left image.Arrows on the right mark 2 NFTs. Many fine neuropil threadsare faintly stained. The remaining bright, round structures areplaques of various sizes. Equivalent results were seen usingfrozen sections (not shown).

Fig. 5. In vivo multiphoton microscopic images of a singleplaque in the brain of a living, anesthetized 7-month-old PS1/APP transgenic mouse. Left: Representative fields from 1 of 3mice 1 h after i.v. injection of ;5 mg/kg methoxy-X04 into thelateral tail vein. Right: Same fields in the same mouse aftertopical addition of thioflavin-S (0.005% in artificial CSF for 20min) directly to the surface of the brain. Only the same plaquedemonstrated by methoxy-X04 is labeled by thioflavin-S. Theplaques marked by arrows in the upper panels are enlarged 10-fold in the lower panels. Asterisks in the upper panels dem-onstrate a larger, non-fluorescing vessel. The bar represents 100mm in the upper panels and 10 mm in the lower panels. Theplane of focus is ;35 mm below the brain surface.total. Chrysamine-G displaced [11C]methoxy-X04 in a

very similar fashion with a Ki of 25.3 6 10.1 nM. Alongwith the inhibition of [3H]Chrysamine-G binding toAb(1–40) by methoxy-X04 (data not shown), these dataindicate that methoxy-X04 and Chrysamine-G may sharea common binding site on Ab(1–40) fibrils.

Tissue Staining

While the above binding studies using pure, syntheticAb can assess binding affinity, this defined system doesnot address specificity for binding to amyloid deposits inthe complex milieu of human brain. In order to gain someinformation regarding the specificity of methoxy-X04 forplaques and NFT in human brain, we exploited the fluo-rescent nature of this compound by using it to stain par-affin-embedded and frozen sections of postmortem ADbrain. Staining was accomplished by a slight modificationof the simple technique developed for staining humantissue with X34 (20) in which 100 mM methoxy-X04 wassubstituted for 100 mM X34. Figure 4 shows 2 sectionsfrom AD brain frontal cortex stained with methoxy-X04.Although methoxy-X04 is not as intensely fluorescent asX34, plaques, cerebrovascular amyloid, and NFT areclearly seen. Neuropil threads (deposits related to NFTwithin neurites) are also visible. Background staining ofnormal structures is minimal. Staining in control brain(not shown) is equivalent to the background seen in ADbrain. The binding of methoxy-X04 to plaques, cerebro-vascular amyloid, and NFT in AD brain tissue sectionswas abolished by pretreatment with 99% formic acid,suggesting that methoxy-X04 binds only to fibrillar b-sheet deposits (20).

In Vivo Detection of Methoxy-X04-Stained Plaques inTransgenic Mice

In order for methoxy-X04 to be a useful in vivo im-aging agent, it must not only bind to Ab with high affin-ity and specificity, but it must also be effective after sys-temic administration. PS1/APP mice were injected eitherintravenously (i.v.) or intraperitoneally (i.p.) with 5 to 10mg/kg methoxy-X04. Distinguishable plaques could bedetected 30 to 60 min after i.v. administration of 5 to 10mg/kg methoxy-X04 as the initial blush of non-specificbackground fluorescence diminished, leaving the moreslowly cleared plaque-bound compound (Fig. 5). Intra-peritoneal injection in a buffered, lipophilic solventavoided the difficulties of tail-vein injection in mice. Fig-ure 6 shows a representative result from a PS1/APPmouse that was injected i.p. with 10 mg/kg methoxy-X04(5 mg/ml solution in 10% DMSO, 45% propylene glycol,45% PBS, pH 7.5) 24 h prior to imaging. Numerousplaques and cerebrovascular amyloid deposits werebrightly fluorescent and could be individually imagedwith high resolution (Fig. 6). No parenchymal labelingwas observed when methoxy-X04 was injected into non-transgenic control mice.

While the above images could be viewed ‘‘real-time’’during acquisition, post-acquisition combination of sev-eral images acquired at progressively deeper x-y planes,starting at the surface of the thinned skull and progressingto several hundred microns deep into the cortex produced3-dimensional representations of the data. Figure 7 showsone such 3-D representation after i.p. administration of

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Fig. 6. In vivo multiphoton microscopic images of plaquesand cerebrovascular amyloid in the brain of a living, anesthe-tized 13- month-old PS1/APP transgenic mouse after i.p. injec-tion of 10 mg/kg methoxy-X04 24 h prior to imaging. Thefigure is a representative of field from 1 of 4 mice. Shown is aprojection ;200-mm deep from the surface of the cortex. Notefrequent amyloid plaques that appear to stain more intensely attheir periphery. Also note the staining of the cerebrovascularamyloid. Scale bar 5 100 mm.

10 mg/kg methoxy-X04. This representation shows mul-tiple plaques of various sizes and at various depths fromthe surface. Fluorescent deposits could be visualizedthrough the thin-skull preparation several hundred mi-crons deep from the cortical surface. The spherical natureof the plaques is clearly shown in this view. In addition,amyloid-laden pial vessels also are visible (arrow).

After acquiring images, the remaining layer of bonewas carefully removed and thioflavin-S (0.005%) wasadded to the surface of the brain to confirm the identityof the labeled deposits. Plaques stained by this relativelyconcentrated solution of topically applied thioflavin-S areeasily distinguished from those stained by systemicallyadministered methoxy-X04 by the greater fluorescenceintensity of the topical thioflavin-S-stained plaques. Stan-dard postmortem histochemistry has been used to confirmthat topical thioflavin-S reproducibly labels amyloid de-posits within 150 mm of the cortical surface (15). Withinthis depth, we observed a one-to-one correspondence be-tween fluorescent structures stained by systemically ad-ministered methoxy-X04 and structures labeled with top-ical thioflavin-S (Fig. 5), confirming that they wereamyloid deposits. Postmortem immunohistochemistrywith an anti-Ab antibody (BAM10) confirmed that me-thoxy-X04 stained immunopositive plaques and amyloidangiopathy throughout the brain (data not shown).

Brain Entry of Radiolabeled Compounds

The multiphoton microscopy results clearly show thatsystemic injections of methoxy-X04 can be used to imageamyloid deposits in living mice. However, one can notdirectly extrapolate these results to in vivo human PETor single-photon emission computed tomography(SPECT) studies for several reasons. In particular, the 5-to 10-mg/kg dose used in the multiphoton studies is;10,000-fold higher than the tracer doses used in PETor SPECT studies. These doses were chosen to achievethe highest dose reasonably attainable for this preliminarystudy. Determination of the lowest dose detectable bymultiphoton imaging was beyond the scope of this initialstudy, but even the lowest dose detectable by multiphotonmicroscopy will likely be several orders of magnitudehigher than those used for PET and SPECT. Therefore,we performed the following quantitative brain uptakestudy using tracer doses of [11C]methoxy-X04 and com-pared this to the brain uptake of the dicarboxylic acid,[11C]methoxy-X34, a compound structurally related toboth methoxy-X04 and Chrysamine-G (Fig. 1).

It is useful to quantify brain entry in terms of the per-cent of injected radioactivity dose (% ID) in the brain,normalized to the brain:body weight ratio. Unlike unitssuch as % ID/g brain, this normalized parameter is di-rectly comparable across species of very different size;that is, very different ratios of brain mass to total bodymass. We refer to this value as the percent (%) injecteddose index (% IDI), where:

(% ID in an organ)% IDI 5

(organ weight in g/total body weight in g)

In a 200 g rat, 100% IDI is equivalent to 0.5% ID/g brain.In a 25 g mouse, 100% IDI is equivalent to 4.0% ID/gbrain. Useful PET imaging compounds typically havebrain entry levels in the range of 100%–500% IDI at 2to 5 min following injection (34–38).

When injected i.v. into the lateral tail vein of rats,[11C]methoxy-X04 successfully entered the brain at levelsof 81 6 5% IDI (;0.4% ID/g) at 2 min, just below ourtarget value of 100% IDI. At 30 min post- injection, thebrain radioactivity concentration of [11C]methoxy-X04 inrat brain decreased to 50 6 5% IDI, indicating clearanceof radiotracer from normal (non-amyloid-containing) ratbrain with an estimated clearance t1/2 of ;45 min. In con-trast, the related dicarboxylic acid, [11C]methoxy-X34showed a 2-min value of only 12 6 4% IDI, a valueapproximately twice that of compounds that do not crossthe blood-brain barrier, but are restricted to the intravas-cular space of vessels inside the brain.

Metabolism of [11C]methoxy-X04 in Rats

Plasma samples from rats at 2, 30, and 60 min after i.v.injection of [11C]methoxy-X04 were taken and analyzed

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Fig. 7. Three-dimensional reconstruction of data acquired at multiple depths after i.p. injection of 10 mg/kg methoxy-X0424 h prior to imaging in a 13-month-old PS1/APP mouse. The figure is a side view of the imaging volume obtained with a 320objective (NA 5 0.45, Olympus), which extends to ;300-um deep from the surface of the cortex. Autofluorescence from thethinned skull is seen as a solid plane at the top of the figure. Note the multiple, spherical amyloid plaques at various depths andthe resolution of individual plaques. Also note amyloid-laden vessels at the surface (arrows). Scale bar 5 100 um.

using HPLC separation and radioactivity detection. In ratplasma, unmetabolized [11C]methoxy-X04 accounted for76% of the total radioactivity at 2 min post-injection, 24%at 30 min, and 15% at 60 min. All of the radiolabeledmetabolites of [11C]methoxy-X04 at all 3 time points wereextremely polar and eluted at the column void volume.The absence of lipophilic [11C]methoxy-X04 metaboliteseluting near [11C]methoxy-X04 itself is important. If pre-sent, such lipophilic metabolites would be capable of read-ily entering brain tissue and would complicate analysis offuture PET imaging studies.

DISCUSSION

Although many factors play a role in brain entry of acompound, molecular size, lipophilicity, and ionic chargeall play key roles (33, 39–41). Studies suggest that theoptimal lipophilicity range for brain entry, expressed interms of the log10 of the octanol:aqueous buffer partitioncoefficient (logPoct), is between 1.5 and 2.5 (40, 42). Be-low a logPoct of 1.0, passive diffusion through the blood-brain barrier is poor, and above a logPoct of 3.0, bindingof radiotracer to blood components (e.g. red blood cellsand albumin) is so great as to limit the amount availablefor brain entry. Even when the logPoct is optimized, thepresence of a positive or negative charge in a moleculecan impede brain uptake. Aromatic carboxylic acids, inparticular, have been shown to have poorer than expectedbrain entry even when they possess near-optimal molec-ular weight and lipophilicity (41). As a result, brain-per-meable compounds typically are relatively small, mod-erately lipophilic compounds that are uncharged at

physiologic pH (i.e. pKa for acidic compounds is .8; pKa

for amines is ,6).Congo red and Chrysamine-G both share the undesir-

able properties of highly charged acidic groups (pKa 2–4). Radiolabeled derivatives of Chrysamine-G do not en-ter the brain in amounts sufficient for neuroimaging (8,9, 11, 43). Compared to Chrysamine-G, methoxy-X04 issmaller (MW 5 344), lacks carboxylic acid groups, isneutral at physiologic pH (pKa 10.8), and is moderatelylipophilic (logPoct 5 2.6). The affinity of [11C]methoxy-X04 for Ab fibrils (Ki 5 26.8 nM) is equivalent to thatof Chrysamine-G. Equally important, the brain entry of[11C]methoxy-X04 (81% IDI or ;0.4% ID/g) was 7-foldgreater than the related dicarboxylic acid compound, me-thoxy-X34 (12% IDI). Using multiphoton imaging tostudy transgenic mice, individual plaques could be ob-served in a living animal with an intact cranium andblood-brain barrier within 1 h of i.v. administration of asingle bolus injection of 5 to 10 mg/kg of methoxy-X04.Plaques were also well distinguished 24 h after i.p. ad-ministration. Intermediate time points were not investi-gated in this preliminary study but will be included infuture studies.

The 81% IDI brain entry level of [11C]methoxy-X04 isnear the lower limit of agents found to be useful for neu-roreceptor imaging. Determination of whether this levelis sufficient for PET or SPECT amyloid imaging studieswill require in vivo studies to be performed in humansor in amyloid-containing transgenic mouse brain with de-tection by recently developed small animal scanners

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(microPET or microSPECT) (44, 45). Compared to typ-ical neuroreceptor imaging studies, it is advantageous thatinsoluble Ab deposits are present in micromolar concen-trations in AD brain (46) whereas the receptor targets arepresent only in low nanomolar concentrations (47). Inaddition, it also is very promising that brain amyloid de-posits in transgenic mice could be specifically labeled bysystemically administered methoxy-X04 and detected invivo by multiphoton microscopy. This finding stronglysuggests that, at minimum, the signal-to-background ratiobetween methoxy-X04 levels in amyloid plaques and sur-rounding normal brain is sufficiently different that amy-loid load could be quantified by PET if detectable levelsof [11C]methoxy-X04 are achieved in brain. The multi-photon findings also suggest that the affinity of methoxy-X04 is sufficient to allow retention in plaques whileclearance occurs in normal brain tissue. Of course, theresolution of PET (and SPECT) is not sufficient to re-solve individual plaques like the multiphoton microscopictechnique. Quantitation of amyloid load by PET orSPECT would be on a global scale, similar to the quan-titation of receptor density in classic receptor neuroim-aging studies (without resolution of individual receptors).

A somewhat similar finding of detectable plaques inTg2576 mouse brain (24) after systemic administration ofa brominated derivative of X34, termed BSB, was re-ported by Skovronsky et al (48). There are importantmethodological differences between the study of Sko-vronsky et al and the current study. Most significantly,Skovronsky et al did not demonstrate either in vivo de-tection of amyloid deposits or adequate brain uptake.Both of these parameters are critical for a potential brain-imaging agent. Skovronsky et al used relatively largedoses (;250 mg/kg) of BSB to produce labeling ofplaques that could only be detected by ex vivo exami-nation of 8- to 10-mm brain sections by the very sensitivetechnique of traditional fluorescence microscopy. Indeed,subsequent quantitative studies by this same group of in-vestigators showed that radiolabeled derivatives of BSBshowed relatively low permeability across the blood-brain barrier. They concluded that ‘‘the potential useful-ness of these agents for in vivo imaging after a bolus i.v.injection appears to be limited’’ (11).

Other amyloid-binding compounds, not derived fromCongo red, have recently been shown to hold potentialas in vivo imaging agents (11, 16, 49–51). Agdeppa etal have reported a novel group of potential amyloid-im-aging agents (49), and although they have not reportedany in vivo data from transgenic mice, this group hasalready attempted preliminary human imaging studies.They found delayed clearance of their amyloid probe,[18F]FDDNP, from brain areas known to contain amyloiddeposits. Unfortunately, these results are made difficultto interpret by large amounts of non-specific binding re-tained in brain areas known to be free of amyloid (1, 52).

The implications of the properties of methoxy-X04 de-scribed in this study are 2-fold. First, methoxy-X04 is apromising in vivo agent for imaging amyloid in experi-mental animal model systems using imaging techniquessuch as multiphoton microscopy or microPET (44, 45).Taking advantage of the high resolution of multiphotonmicroscopy, imaging could be repeated many times overthe life span of a mouse, providing an ideal mechanismto examine the natural history of individual plaques, aswell as tracking the fate of individual plaques duringtherapeutic interventions. Systemic administration andimaging through a thinned (but intact) cranium precludespossible artifacts created by repeated application of for-eign substances directly onto the exposed brain. Second,[11C]methoxy-X04 is holds potential as a human PET am-yloid-imaging agent for AD itself. Suitably labeled de-rivatives of methoxy-X04 also could be useful with otherimaging modalities such as SPECT. These human usescould have diagnostic utility and could aid in the evalu-ation of anti-amyloid therapies now in early phases ofdevelopment.

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Received February 8, 2002Revision received May 20, 2002Accepted May 22, 2002