Imaging A Plaques in Living Transgenic Mice with Multiphoton ...

Journal of Neuropathology and Experimental Neurology Copyright q 2002 by the American Association of Neuropathologists

Vol. 61, No. 9 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 early diagnosis and for monitoring the efficacy of newly developed anti-amyloid therapies. Methoxy-X04 is a derivative of Congo red and Chrysamine-G that contains no acid groups and is therefore smaller and much more lipophilic than Congo red or Chrysamine-G. Methoxy-X04 retains in vitro binding affinity for amyloid b (Ab) fibrils (Ki 5 26.8 nM) very similar to that of Chrysamine-G (Ki 5 25.3 nM). Methoxy-X04 is fluorescent and stains plaques, tangles, and cerebrovascular amyloid in postmortem 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 min after 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 produced high contrast images of plaques and cerebrovascular amyloid in PS1/APP mouse brain. Complementary quantitative studies using tracer doses of carbon-11-labeled methoxy-X04 show that it enters rat brain in amounts that suggest it is a viable candidate 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 tomography; Transgenic mice.

INTRODUCTION Amyloid plaques and neurofibrillary tangles (NFTs) are proposed to play key roles in the pathogenesis of Alzheimer disease (AD). Despite recent attempts (1), no accepted method currently exists to non-invasively quantify these b-sheet amyloid deposits in living patients. The preferred characteristics of such a biomarker have been outlined by Klunk (2) and the importance has been emphasized in a review by Selkoe (3). The availability of an in vivo amyloid probe could 1) facilitate early diagnosis, 2) allow clinico-pathological correlations of amyloid deposition with early cognitive symptoms over time, and 3) provide a surrogate marker of efficacy of antiamyloid therapies that are in early clinical trials (e.g. amyloid b [Ab] immunization, beta- and gamma-secretase inhibitors) (4–6). A surrogate marker of efficacy could From the L aboratory of Molecular Neuropharmacology, Department of Psychiatry (WEK, MLD), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Alzheimer’s Research Unit (BJB, STK, MEL, BTH), Massachusetts General Hospital, Charlestown, Massachusetts; PET Facility, Department of Radiology (CAM, DPH, YW), University 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 Pittsburgh, 705 Parran Hall-GSPH, 130 DeSoto Street, Pittsburgh, PA 15213. Support: This work was supported in part by grants from the Alzheimer’s Association (IIRG-95-076 and TLL-01-3381) and NIHAG01039 and AG20226 to WEK, a Pioneer Award from the Alzheimer’s Association and NIH-AG08487 to BTH, and NIH 5P01 AG15379 to MPF. We also thank the Walters Family Foundation (BTH). MPF is a 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 develop derivatives of Congo red and Chrysamine-G as in vivo amyloid probes (7), but we and others have been hampered by the marginal brain entry of these relatively large, acidic compounds (8–11). In this study, we report the properties of a promising compound, 1,4-bis(49-hydroxystyryl)-2-methoxybenzene (or methoxy-X04) (Fig. 1). Methoxy-X04 enters the brain much better than Congo red or ChrysamineG and retains good binding affinity for Ab. In addition, we took advantage of the fluorescent properties of methoxy-X04 to utilize a novel imaging technique, multiphoton microscopy (12), to directly demonstrate in vivo detection of individual plaques in living transgenic mice after systemic injection of this amyloid-binding probe. Multiphoton microscopy has been used for in vivo imaging of Ab deposits in amyloid b-protein precursor (AbPP) transgenic mice after direct application to the pial surface of either thioflavin-S (13, 14) or fluorescein-labeled Ab antibodies (15). Although these reagents visualize plaques using this technique, they must be administered topically because they do not cross the blood-brain barrier. Neither reagent can be used to detect amyloid deposits if administered systemically, preventing their use as in vivo imaging agents in humans. Our current work combines the use of multiphoton imaging technology along with systemic administration of an amyloid-imaging agent to demonstrate directly that methoxy-X04 can be utilized for in vivo visualization of individual plaques and cerebrovascular amyloid in living

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Chemical Synthesis

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

animals. In contrast to Congo red, thioflavin-S, and Ab antibodies, we also quantitatively demonstrate that carbon-11-labeled methoxy-X04 enters the brain relatively well. This combination of good brain entry and in vivo resolution of plaques after systemic injection make methoxy-X04 a potential positron emission tomography (PET) amyloid-imaging agent for human studies. MATERIALS AND METHODS Unless otherwise specified, reagents were of the highest grade available and were purchased from Aldrich Chemical Company (Milwaukee, WI). Yields reported were for crude product and were not optimized. Octanol-buffer partition coefficients (Poct) and logPoct values were determined as previously described using phosphate-buffered saline (PBS: 137 mM NaCl, 3 mM KCl, 10 mM sodium phosphate; pH 7.4) and 1octanol (16). The pKa of methoxy-X04 was determined spectrophotometrically by measuring the absorbance and lmax of a 25-mM solution of methoxy-X04 in H2O at a series of pH values from 9.0 to 12.5. The pKa was calculated by previously described methods (17, 18).

Methoxy-X04 [1,4-bis(49-hydroxystyryl)-2-methoxybenzene]: 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 dry DMSO and 640 ml (3.0 mmol) 25% sodium methoxide in methanol room temperature according to previously published procedures (19). Non-optimized yields varied from 80%–90%. The bis-MOMO compound was converted (deprotected) to the bishydroxy compound by heating a suspension of 25 mg of MOMO-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 58 mg (74% yield) of methoxy-X04, a yellow-green crystalline solid. 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 the 1- or 4-positions of the center ring, v1 and v2 refer to the vinyl protons 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); H24/64: 7.385 (d, J 5 8.64 Hz, 2H); H-v21: 7.199 (d, J 5 16.3 Hz, 1H); H-v24: 7.192 (d, J 5 16.3 Hz, 1H); H-3: 7.185 (d, J 5 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 8.64 Hz, 2H); H-2-OCH3: 3.905 (s, 3H). HRMS m/z calculated for C23H20O3 (M1) 344.1412, found 344.1415.

Radiochemical Synthesis [O-methyl-11C]methoxy-X04: Hydroxy-X04 [1,4-bis(49-hydroxystyryl)-2-hydroxybenzene] was synthesized by substituting 2-MOMO-p-xylylenediphosphonic acid tetraethyl ester for the 2-methoxy ester used in the synthesis of methoxy-X04. [11C]methylation of hydroxy-X04 was performed using no carrier added [11C]CH3I, 3.0 mmoles of hydroxy-X04, 7.2 mmoles K2CO3 in 400 ml of DMSO at 658C for 5 min according to previously published procedures (19) (Fig. 2). Chemical and radiochemical purities as well as specific activity were determined by analytical HPLC [k9 7.6 on a Prodigy ODS(3) column eluted with 50/50 acetonitrile/TEA buffer (TEA 5 50 mM trimethylammonium phosphate, pH 7.2)]. Radiochemical purity

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

J Neuropathol Exp Neurol, Vol 61, September, 2002

IN VIVO MULTIPHOTON/METHOXY-X04 IMAGING OF AMYLOID

was .95%, while chemical purity was .90% (determined using a Waters 996 photodiode array detector set at 369 nm) with the major chemical impurity being hydroxy-X04 (k9 3.3). Approximately 370–740 MBq (10–20 mCi) of [11C]methoxy-X04 was routinely produced having a specific activity .55.5 MBq/ nmole at end-of-synthesis (EOS). The radiochemical yield was 6.7% 6 1.2 at end-of-bombardment and 1.8% 6 0.5 at EOS based on [11C]CH3I (n 5 8). [11C]Methoxy-X34 was similarly prepared by [11C]methylation of the dimethyl ester of X34 (20), followed by rapid ester hydrolysis using potassium tert-butoxide (Aldrich), resulting in yields 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 concentration of 433 mg/ml (100 mM). This solution was magnetically stirred at 1,200 rpm for 3 days at room temperature, vortexing briefly when necessary to avoid gel formation at the meniscus. The initially clear solution gradually became homogeneously cloudy (without large clumps). After this treatment, approximately 2% of the protein remained in the supernatant after a 15-min centrifugation at 28,000 3 g. (21). The fibrillar nature of the Ab, thus prepared, was confirmed by the Congo red method (21, 22). The 100-mM PBS stock was then diluted 1:20 (to 5 mM) with 150 mM Tris-HCl, pH 7.0. The aggregated peptide suspension can be kept frozen at 2808C for at least 8 wk without noticeable change in its properties. Before the binding assay, the 5-mM stock was diluted 1:50 (to 100 nM) with binding buffer (150 mM Tris-HCl, pH 7.0, containing 20% ethanol). Aggregated Ab stock suspensions were continuously stirred to maintain a homogenous suspension during removal of aliquots for the binding assays. Binding Studies with Synthetic Ab(1–40) Fibrils: The appropriate concentrations of cold inhibitors to be tested were combined with [11C]methoxy-X04 (;700,000 cpm within 1 hours [h] of synthesis) in a volume of 950 ml of binding buffer (150 mM Tris-HCl, pH 7.0, containing 20% ethanol to enhance solubility of the test compounds). The assay was begun by addition of 50 ml of 100 nM Ab stock to achieve a final concentration of ;2 pM [11C]methoxy-X04, 5 nM Ab fibrils, and the appropriate concentration of test compound. After incubation for 30 min at room temperature (a time when equilibrium had been reached), the binding mixture was filtered through a Whatman GF/B glass filter via a Brandel M-24R cell harvester (Gaithersburg, MD) and rapidly washed twice with 3 ml binding buffer. The filters were placed in Cytoscint-ES (ICN Biomedical, Irvine, CA) and immediately counted. Complete (100%) inhibition of binding was defined as the number of counts displaced by 1 mM cold methoxy-X04. Higher concentrations of methoxy-X04 caused aggregate (micelle) formation that caused radioactivity to be trapped by the filter paper even in the absence of Ab. Specific binding varied between 70%–75% of total binding. All assays were done at least in triplicate. For determination of inhibition constants (Ki), inhibition curves were fit (using the RS/1 statistical package, version 6.1; Brooks Automation, Chelmsford, MA) to the following equation, which is derived from the Hill equation (23):

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F(x) 5 M(Ki)H/{[L]H 1 (Ki)H} Where M 5 maximal percent [11C]methoxy-X04 bound (typical fit gave 100%–104% for M), H is the Hill coefficient (typically 0.60–0.80), and [L] is the concentration of inhibitor compound.

Tissue Staining Tissue autofluorescence was quenched and staining was accomplished by the procedure used for X34 staining described in detail by Styren et al (20). Briefly, deparaffinized, quenched tissue sections were taken from PBS into a 100-mM solution of methoxy-X04 in (40% ethanol)/(60% distilled H2O) (adjusted to pH 10 with 0.1 N NaOH) for 10 min. The sections were then dipped briefly 5 times into tap water before differentiation in 0.2% NaOH in 80% ethanol for 2 min. The sections were then placed in tap water for 10 min prior to coverslipping with Fluoromount-G (Electron Microscopy Sciences, Fort Washington, PA). Pretreatment of selected slides with 99% formic acid for 5 min was performed prior to the PBS wash (20). Fluorescent sections were examined using an Olympus Vanox AHRFL-LB fluorescence microscope and were optimally viewed with a V-filter set (excites 400–410 nm, dichroic mirror DM455, 455 nm longpass filter).

In Vivo Pharmacokinetic Studies Brain Uptake Studies in Rats: Male Sprague-Dawley rats (n 5 3–5 at each time point) weighing 200 to 300 g were injected in a lateral tail vein with 50 to 100 mCi (50–100 pmol) of [11C]methoxy-X04 or [11C]methoxy-X34 (specific activity ;55 GBq/mmol or ;1500 Ci/mmol) contained in 0.1 ml of isotonic saline solution (containing ;5% ethanol from the Sep-Pak elution). Animals were anesthetized at 2 or 30 min following injection and killed by cardiac excision following cardiac puncture to obtain a terminal arterial blood sample. The brains were rapidly removed and dissected into cerebellum and remaining whole brain (including brain stem) fractions. Brain and blood samples were counted in a gamma well-counter (Packard Instruments Model 5003, Meridan, CT), and the counts were decay-corrected to the time of injection relative to C-11 standards prepared from the injection solution to determine the percent injected dose (% ID) in the samples. The samples were weighed to 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 concentration values (% ID index or % IDI, see Results) for each tissue 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 samples were obtained as described above at 2, 30, and 60 min following injection of [11C]methoxy-X04. The whole blood was centrifuged for 2 min to separate the plasma. Five hundred ml of plasma was added to an equal volume of acetonitrile and the mixture was vortexed for 1 min and then centrifuged (13,000 3 g) for an additional 2 min. A sample of the supernatant was analyzed by reverse phase HPLC using a Raytest Gabi RadioHPLC detector. A Phenomenex Prodigy ODS(3) 250 3 4.6 mm column was eluted with 50/50 acetonitrile/TEA buffer at 2 ml/ min.


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In Vivo Imaging Studies Animals examined were mice that carried both the Tg2576 APPsw (24) and the PS1(M146L) transgenes (25, 26). The PS1(M146L) line of mice was bred to homozygosity, and the animals for study were generated through breeding of these homozygous mice [PS1(M146L)/PS1(M146L)] with hemizygous Tg2576 mice to generate animals with single copies of each transgene. The genotype of all animals was determined through PCR-based analysis of tail DNA using previously reported protocols (24–26). The Tg2576 transgene was maintained in an SJL/B6 F1 background, while the PS1(M146L) transgene was maintained in an FVB/N background. This combination of transgenes has been observed to accelerate the development of Ab deposition in parenchyma and blood vessels (27). By 6 months of age, PS1/APP mice have extensive, congophilic, thioflavin-S-positive plaques that are predominantly composed of Ab(1–40) (28), which increase in number but do not change in character as the mouse ages. The cortical amyloid burden of a 12- month-old PS1/APP is similar to that of a typical AD case (29, 30). To fashion a thin-skull preparation for imaging, the mouse was anesthetized with Avertin (1.3% tribromoethanol, 0.8% tert-pentylalcohol in distilled water; 250 mg/kg, i.p.) and immobilized in custom-built, stage-mounted ear bars and a nosepiece, similar to a stereotaxic apparatus. Two circular regions of the skull (;1–1.2 mm in diameter located on either side of sagittal suture and just posterior to coronal suture) were thinned using a high-speed drill (Fine Science Tools, Foster City, CA) and a dissecting microscope (Leica, Wetzlar, Germany) for gross visualization. Heat and vibration artifacts were minimized during drilling by frequent application of artificial cerebrospinal fluid (ACSF: 125 mM NaCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 25 mM glucose). Seven- to 13-month-old PS1/APP mice (n 5 3) were injected i.v. with 5 to 10 mg/kg methoxy-X04 (1 mg/ml in normal saline adjusted to pH 5 12 with 0.1 N NaOH) immediately prior to imaging or were injected i.p. with 10 mg/kg methoxy-X04 (n 5 4; 5 mg/ml in 10% DMSO, 45% propylene glycol, 45% PBS, pH 7.5) 24 h prior to imaging. Multiphoton imaging and postmortem histology were performed as previously described (15, 31). Briefly, 2-photon fluorescence was generated with 750 nm excitation from a mode-locked Ti:Sapphire laser [Tsunami (Spectra-Physics, Mountain View, CA), driven by a 10- W Millenium Xs pump laser (Spectra-Physics), mounted on a commercially available multiphoton imaging system (Bio-Rad 1024ES; Bio-Rad, Hercules, CA). Custom-built external detectors containing 3 photomultiplier tubes (Hamamatsu Photonics, Bridgewater, NJ) collected emitted light in the range of 380 to 480, 500 to 540, and 560 to 650 nm. An ACSF reservoir was created within the opened scalp over the thin-skull preparations to accommodate the long working distance, water immersion dipping objectives (20 and 603, Olympus, Tokyo, Japan) of an Olympus BX-50 microscope. Three-dimensional volumes were acquired by collecting a stack of x-y sections starting at the surface of the thinned skull to several hundred microns deep into the cortex. Following imaging of the thin-skull sites, the thinned bone was removed using fine forceps (Fine Science Tools). Thioflavin-S (0.005% w/v in ACSF; Sigma, St. Louis, MO) was then


Fig. 3. Competitive binding assays using cold (non-radioactive) Chrysamine-G (closed circles) and methoxy-X04 (open squares) to compete for [11C]methoxy-X04 binding sites on Ab(1–40) fibrils. Complete inhibition of specific binding (i.e. 0% bound) was defined as the number of counts remaining in the presence of 1 mM cold methoxy-X04. Higher concentrations of methoxy-X04 caused aggregate (micelle) formation that caused radioactivity to be trapped by the filter paper even in the absence of Ab. Specific binding varied between 70%–75% of total binding. applied directly to the brain for 20 min to label dense core amyloid plaques. Sites were washed with ACSF and re-imaged. After image collection, the animal was killed and the brain fixed in 4% paraformaldehyde. Immunohistochemistry was performed using the BAM10 anti-Ab antibody (Sigma) as previously described (32).

RESULTS Physical Properties A systematic series of structural alterations of the Congo red pharmacophore resulted in the compound, methoxy-X04 (Fig. 1). Compared to the parent compound, Congo red, methoxy-X04 has a 47% lower molecular weight (MW 5 344), a value that falls into the optimal range for brain entry (33). The carboxylic acid groups found in Chrysamine-G and methoxy-X34 are removed, leaving only the weakly acidic phenols, which, with a pKa measured to be 10.82 6 0.07, would be uncharged at 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 compounds 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 radiolabeled [11C]methoxy-X04 and unlabeled (cold) methoxyX04 are compared to a similar competition between [11C]methoxy-X04 and Chrysamine-G in Figure 3. Unlabeled methoxy-X04 displaced [11C]methoxy-X04 binding to Ab(1–40) fibrils with a Ki if 26.8 6 10.0 nM. Displaceable (specific binding) ranged from 70%–75% of


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

total. Chrysamine-G displaced [11C]methoxy-X04 in a very similar fashion with a Ki of 25.3 6 10.1 nM. Along with the inhibition of [3H]Chrysamine-G binding to Ab(1–40) by methoxy-X04 (data not shown), these data indicate that methoxy-X04 and Chrysamine-G may share a common binding site on Ab(1–40) fibrils. Tissue Staining While the above binding studies using pure, synthetic Ab can assess binding affinity, this defined system does not address specificity for binding to amyloid deposits in the complex milieu of human brain. In order to gain some information regarding the specificity of methoxy-X04 for plaques and NFT in human brain, we exploited the fluorescent nature of this compound by using it to stain paraffin-embedded and frozen sections of postmortem AD brain. Staining was accomplished by a slight modification of the simple technique developed for staining human tissue with X34 (20) in which 100 mM methoxy-X04 was substituted for 100 mM X34. Figure 4 shows 2 sections from AD brain frontal cortex stained with methoxy-X04. Although methoxy-X04 is not as intensely fluorescent as X34, plaques, cerebrovascular amyloid, and NFT are clearly seen. Neuropil threads (deposits related to NFT within neurites) are also visible. Background staining of normal structures is minimal. Staining in control brain (not shown) is equivalent to the background seen in AD brain. The binding of methoxy-X04 to plaques, cerebrovascular amyloid, and NFT in AD brain tissue sections was abolished by pretreatment with 99% formic acid, suggesting that methoxy-X04 binds only to fibrillar bsheet deposits (20).

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Fig. 5. In vivo multiphoton microscopic images of a single plaque in the brain of a living, anesthetized 7-month-old PS1/ APP transgenic mouse. Left: Representative fields from 1 of 3 mice 1 h after i.v. injection of ;5 mg/kg methoxy-X04 into the lateral tail vein. Right: Same fields in the same mouse after topical addition of thioflavin-S (0.005% in artificial CSF for 20 min) directly to the surface of the brain. Only the same plaque demonstrated by methoxy-X04 is labeled by thioflavin-S. The plaques marked by arrows in the upper panels are enlarged 10fold in the lower panels. Asterisks in the upper panels demonstrate a larger, non-fluorescing vessel. The bar represents 100 mm in the upper panels and 10 mm in the lower panels. The plane of focus is ;35 mm below the brain surface.

In Vivo Detection of Methoxy-X04-Stained Plaques in Transgenic Mice In order for methoxy-X04 to be a useful in vivo imaging agent, it must not only bind to Ab with high affinity and specificity, but it must also be effective after systemic administration. PS1/APP mice were injected either intravenously (i.v.) or intraperitoneally (i.p.) with 5 to 10 mg/kg methoxy-X04. Distinguishable plaques could be detected 30 to 60 min after i.v. administration of 5 to 10 mg/kg methoxy-X04 as the initial blush of non-specific background fluorescence diminished, leaving the more slowly cleared plaque-bound compound (Fig. 5). Intraperitoneal injection in a buffered, lipophilic solvent avoided the difficulties of tail-vein injection in mice. Figure 6 shows a representative result from a PS1/APP mouse 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. Numerous plaques and cerebrovascular amyloid deposits were brightly fluorescent and could be individually imaged with high resolution (Fig. 6). No parenchymal labeling was observed when methoxy-X04 was injected into nontransgenic control mice. While the above images could be viewed ‘‘real-time’’ during acquisition, post-acquisition combination of several images acquired at progressively deeper x-y planes, starting at the surface of the thinned skull and progressing to several hundred microns deep into the cortex produced 3-dimensional representations of the data. Figure 7 shows one such 3-D representation after i.p. administration of J Neuropathol Exp Neurol, Vol 61, September, 2002

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Brain Entry of Radiolabeled Compounds

Fig. 6. In vivo multiphoton microscopic images of plaques and cerebrovascular amyloid in the brain of a living, anesthetized 13- month-old PS1/APP transgenic mouse after i.p. injection of 10 mg/kg methoxy-X04 24 h prior to imaging. The figure is a representative of field from 1 of 4 mice. Shown is a projection ;200-mm deep from the surface of the cortex. Note frequent amyloid plaques that appear to stain more intensely at their periphery. Also note the staining of the cerebrovascular amyloid. Scale bar 5 100 mm.

10 mg/kg methoxy-X04. This representation shows multiple plaques of various sizes and at various depths from the surface. Fluorescent deposits could be visualized through the thin-skull preparation several hundred microns deep from the cortical surface. The spherical nature of 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 bone was carefully removed and thioflavin-S (0.005%) was added to the surface of the brain to confirm the identity of the labeled deposits. Plaques stained by this relatively concentrated solution of topically applied thioflavin-S are easily distinguished from those stained by systemically administered methoxy-X04 by the greater fluorescence intensity of the topical thioflavin-S-stained plaques. Standard postmortem histochemistry has been used to confirm that topical thioflavin-S reproducibly labels amyloid deposits within 150 mm of the cortical surface (15). Within this depth, we observed a one-to-one correspondence between fluorescent structures stained by systemically administered methoxy-X04 and structures labeled with topical thioflavin-S (Fig. 5), confirming that they were amyloid deposits. Postmortem immunohistochemistry with an anti-Ab antibody (BAM10) confirmed that methoxy-X04 stained immunopositive plaques and amyloid angiopathy throughout the brain (data not shown). J Neuropathol Exp Neurol, Vol 61, September, 2002

The multiphoton microscopy results clearly show that systemic injections of methoxy-X04 can be used to image amyloid deposits in living mice. However, one can not directly extrapolate these results to in vivo human PET or single-photon emission computed tomography (SPECT) studies for several reasons. In particular, the 5to 10-mg/kg dose used in the multiphoton studies is ;10,000-fold higher than the tracer doses used in PET or SPECT studies. These doses were chosen to achieve the highest dose reasonably attainable for this preliminary study. Determination of the lowest dose detectable by multiphoton imaging was beyond the scope of this initial study, but even the lowest dose detectable by multiphoton microscopy will likely be several orders of magnitude higher than those used for PET and SPECT. Therefore, we performed the following quantitative brain uptake study using tracer doses of [11C]methoxy-X04 and compared this to the brain uptake of the dicarboxylic acid, [11C]methoxy-X34, a compound structurally related to both methoxy-X04 and Chrysamine-G (Fig. 1). It is useful to quantify brain entry in terms of the percent of injected radioactivity dose (% ID) in the brain, normalized to the brain:body weight ratio. Unlike units such as % ID/g brain, this normalized parameter is directly comparable across species of very different size; that is, very different ratios of brain mass to total body mass. We refer to this value as the percent (%) injected dose index (% IDI), where: % IDI 5

(% ID in an organ) (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/g brain. Useful PET imaging compounds typically have brain entry levels in the range of 100%–500% IDI at 2 to 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 levels of 81 6 5% IDI (;0.4% ID/g) at 2 min, just below our target value of 100% IDI. At 30 min post- injection, the brain radioactivity concentration of [11C]methoxy-X04 in rat brain decreased to 50 6 5% IDI, indicating clearance of radiotracer from normal (non-amyloid-containing) rat brain with an estimated clearance t1/2 of ;45 min. In contrast, the related dicarboxylic acid, [11C]methoxy-X34 showed a 2-min value of only 12 6 4% IDI, a value approximately twice that of compounds that do not cross the blood-brain barrier, but are restricted to the intravascular 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-X04 24 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 320 objective (NA 5 0.45, Olympus), which extends to ;300-um deep from the surface of the cortex. Autofluorescence from the thinned skull is seen as a solid plane at the top of the figure. Note the multiple, spherical amyloid plaques at various depths and the 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 rat plasma, unmetabolized [11C]methoxy-X04 accounted for 76% of the total radioactivity at 2 min post-injection, 24% at 30 min, and 15% at 60 min. All of the radiolabeled metabolites of [11C]methoxy-X04 at all 3 time points were extremely polar and eluted at the column void volume. The absence of lipophilic [11C]methoxy-X04 metabolites eluting near [11C]methoxy-X04 itself is important. If present, such lipophilic metabolites would be capable of readily entering brain tissue and would complicate analysis of future PET imaging studies. DISCUSSION Although many factors play a role in brain entry of a compound, molecular size, lipophilicity, and ionic charge all play key roles (33, 39–41). Studies suggest that the optimal lipophilicity range for brain entry, expressed in terms of the log10 of the octanol:aqueous buffer partition coefficient (logPoct), is between 1.5 and 2.5 (40, 42). Below a logPoct of 1.0, passive diffusion through the bloodbrain barrier is poor, and above a logPoct of 3.0, binding of radiotracer to blood components (e.g. red blood cells and albumin) is so great as to limit the amount available for brain entry. Even when the logPoct is optimized, the presence of a positive or negative charge in a molecule can impede brain uptake. Aromatic carboxylic acids, in particular, have been shown to have poorer than expected brain entry even when they possess near-optimal molecular weight and lipophilicity (41). As a result, brain-permeable compounds typically are relatively small, moderately 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 undesirable properties of highly charged acidic groups (pKa 2– 4). Radiolabeled derivatives of Chrysamine-G do not enter the brain in amounts sufficient for neuroimaging (8, 9, 11, 43). Compared to Chrysamine-G, methoxy-X04 is smaller (MW 5 344), lacks carboxylic acid groups, is neutral at physiologic pH (pKa 10.8), and is moderately lipophilic (logPoct 5 2.6). The affinity of [11C]methoxyX04 for Ab fibrils (Ki 5 26.8 nM) is equivalent to that of Chrysamine-G. Equally important, the brain entry of [11C]methoxy-X04 (81% IDI or ;0.4% ID/g) was 7-fold greater than the related dicarboxylic acid compound, methoxy-X34 (12% IDI). Using multiphoton imaging to study transgenic mice, individual plaques could be observed in a living animal with an intact cranium and blood-brain barrier within 1 h of i.v. administration of a single bolus injection of 5 to 10 mg/kg of methoxy-X04. Plaques were also well distinguished 24 h after i.p. administration. Intermediate time points were not investigated in this preliminary study but will be included in future studies. The 81% IDI brain entry level of [11C]methoxy-X04 is near the lower limit of agents found to be useful for neuroreceptor imaging. Determination of whether this level is sufficient for PET or SPECT amyloid imaging studies will require in vivo studies to be performed in humans or in amyloid-containing transgenic mouse brain with detection by recently developed small animal scanners J Neuropathol Exp Neurol, Vol 61, September, 2002

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(microPET or microSPECT) (44, 45). Compared to typical neuroreceptor imaging studies, it is advantageous that insoluble Ab deposits are present in micromolar concentrations in AD brain (46) whereas the receptor targets are present only in low nanomolar concentrations (47). In addition, it also is very promising that brain amyloid deposits in transgenic mice could be specifically labeled by systemically administered methoxy-X04 and detected in vivo by multiphoton microscopy. This finding strongly suggests that, at minimum, the signal-to-background ratio between methoxy-X04 levels in amyloid plaques and surrounding normal brain is sufficiently different that amyloid load could be quantified by PET if detectable levels of [11C]methoxy-X04 are achieved in brain. The multiphoton findings also suggest that the affinity of methoxyX04 is sufficient to allow retention in plaques while clearance occurs in normal brain tissue. Of course, the resolution of PET (and SPECT) is not sufficient to resolve individual plaques like the multiphoton microscopic technique. Quantitation of amyloid load by PET or SPECT would be on a global scale, similar to the quantitation of receptor density in classic receptor neuroimaging studies (without resolution of individual receptors). A somewhat similar finding of detectable plaques in Tg2576 mouse brain (24) after systemic administration of a brominated derivative of X34, termed BSB, was reported by Skovronsky et al (48). There are important methodological differences between the study of Skovronsky et al and the current study. Most significantly, Skovronsky et al did not demonstrate either in vivo detection of amyloid deposits or adequate brain uptake. Both of these parameters are critical for a potential brainimaging agent. Skovronsky et al used relatively large doses (;250 mg/kg) of BSB to produce labeling of plaques that could only be detected by ex vivo examination of 8- to 10-mm brain sections by the very sensitive technique of traditional fluorescence microscopy. Indeed, subsequent quantitative studies by this same group of investigators showed that radiolabeled derivatives of BSB showed relatively low permeability across the bloodbrain barrier. They concluded that ‘‘the potential usefulness of these agents for in vivo imaging after a bolus i.v. injection appears to be limited’’ (11). Other amyloid-binding compounds, not derived from Congo red, have recently been shown to hold potential as in vivo imaging agents (11, 16, 49–51). Agdeppa et al have reported a novel group of potential amyloid-imaging agents (49), and although they have not reported any in vivo data from transgenic mice, this group has already attempted preliminary human imaging studies. They found delayed clearance of their amyloid probe, [18F]FDDNP, from brain areas known to contain amyloid deposits. Unfortunately, these results are made difficult to interpret by large amounts of non-specific binding retained in brain areas known to be free of amyloid (1, 52). J Neuropathol Exp Neurol, Vol 61, September, 2002

The implications of the properties of methoxy-X04 described in this study are 2-fold. First, methoxy-X04 is a promising in vivo agent for imaging amyloid in experimental animal model systems using imaging techniques such as multiphoton microscopy or microPET (44, 45). Taking advantage of the high resolution of multiphoton microscopy, imaging could be repeated many times over the life span of a mouse, providing an ideal mechanism to examine the natural history of individual plaques, as well as tracking the fate of individual plaques during therapeutic interventions. Systemic administration and imaging through a thinned (but intact) cranium precludes possible artifacts created by repeated application of foreign substances directly onto the exposed brain. Second, [11C]methoxy-X04 is holds potential as a human PET amyloid-imaging agent for AD itself. Suitably labeled derivatives of methoxy-X04 also could be useful with other imaging modalities such as SPECT. These human uses could have diagnostic utility and could aid in the evaluation of anti-amyloid therapies now in early phases of development. REFERENCES 1. Shoghi-Jadid K, Small GW, Agdeppa ED, et al. Localization of neurofibrillary tangles and beta-amyloid plaques in the brains of living patients with Alzheimer disease. Am J Geriatr Psychiatry 2002;10:24–35 2. Klunk WE. Biological markers of Alzheimer’s disease. Neurobiol Aging 1998;19:145–47 3. Selkoe DJ. Imaging Alzheimer’s amyloid. Nature Biotechnol 2000; 18:823–24 4. Schenk DB, Seubert P, Lieberburg I, Wallace J. Beta-peptide immunization: A possible new treatment for Alzheimer disease. Arch Neurol 2000;57:934–36 5. Nunan J, Small DH. Regulation of APP cleavage by alpha-, betaand gamma-secretases. FEBS Letters 2000;483:6–10 6. Dovey HF, John V, Anderson JP, et al. Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J Neurochem 2001;76:173–81 7. Klunk WE, Debnath ML, Pettegrew JW. Development of small molecule probes for the beta-amyloid protein of Alzheimer’s disease. Neurobiol Aging 1994;15:691–98 8. Mathis CA, Mahmood K, Debnath ML, Klunk WE. Synthesis of a lipophilic radioiodinated ligand with high affinity to amyloid protein in Alzheimer’s disease brain tissue. J Label Compds Radiopharm 1997;40:94–95 9. Zhen W, Han H, Anguiano M, Lemere CA, Cho CG, Lansbury PT. Synthesis and amyloid binding properties of rhenium complexes: Preliminary progress toward a reagent for SPECT imaging of Alzheimer’s disease brain. J Med Chem 1999;42:2805–15 10. Dezutter NA, Dom RJ, de Groot TJ, Bormans GM, Verbruggen AM. 99mTc-MAMA-chrysamine G, a probe for beta-amyloid protein of Alzheimer’s disease. Eur J Nucl Med 1999;26:1392–99 11. Zhuang ZP, Kung MP, Hou C, et al. Radioiodinated styrylbenzenes and thioflavins as probes for amyloid aggregates. J Med Chem 2001;44:1905–14 12. Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy. Science 1990;248:73–76 13. Christie R, Yamada M, Moskowitz M, Hyman B. Structural and functional disruption of vascular smooth muscle cells in a transgenic mouse model of amyloid angiopathy. Am J Pathol 2001;158: 1065–71

IN VIVO MULTIPHOTON/METHOXY-X04 IMAGING OF AMYLOID 14. Kimchi EY, Kajdasz S, Bacskai BJ, Hyman BT. Analysis of cerebral amyloid angiopathy in a transgenic mouse model of Alzheimer disease using in vivo multiphoton microscopy. J Neuropathol Exp Neurol 2001;60:274–79 15. Bacskai BJ, Kajdasz ST, Christie RH, et al. Imaging of amyloid-beta deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy. Nat Med 2001;7:369–72 16. Klunk WE, Wang Y, Huang G-F, Debnath ML, Holt DP, Mathis CA. Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain. Life Sci 2001; 69:1471–84 17. Lewis KM, Archer RD. pKa values of estrone, 17 beta-estradiol and 2-methoxyestrone. Steroids 1979;34:485–99 18. Tam KY, Takacs-Novak K. Multiwavelength spectrophotometric determination of acid dissociation constants: Part II. First derivative vs. target factor analysis. Pharm Res 1999;16:374–81 19. Wang Y, Mathis CA, Huang G-F, Holt DP, Debnath ML, Klunk WE. Synthesis and 11C-labelling of (E,E)-1-(39,49-dihydroxystyryl)4-(39-methoxy-49-hydroxystyryl) bezene for PET imaging of amyloid deposits. J Label Compd Radiopharm 2002 (in press) 20. Styren SD, Hamilton RL, Styren GC, Klunk WE. X-34, a fluorescent derivative of Congo red: A novel histochemical stain for Alzheimer’s disease pathology. J Histochem Cytochem 2000;48: 1223–32 21. Klunk WE, Jacob RF, Mason RP. Quantifying amyloid b-peptide (Ab) aggregation using the Congo red-Abeta (CR-Ab) spectrophotometric assay. Anal Biochem 1999;266:66–76 22. Klunk WE, Pettegrew JW, Abraham DJ. Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J Histochem Cytochem 1989;37:1273–81 23. Bennett JP. Methods in binding studies. In: Yamamura HI, Enna SJ, Kuhar MJ, eds. Neurotransmitter receptor binding. New York: Raven Press, 1978:57–90 24. Hsiao K, Chapman P, Nilsen S, et al. Correlative memory deficits, abeta elevation, and amyloid plaques in transgenic mice. Science 1996;274:99–102 25. Kang DE, Soriano S, Frosch MP, et al. Presenilin 1 facilitates the constitutive turnover of beta-catenin: Differential activity of Alzheimer’s disease-linked PS1 mutants in the beta-catenin-signaling pathway. J Neurosci 1999;19:4229–37 26. Berezovska O, Frosch M, McLean P, et al. The Alzheimer-related gene presenilin 1 facilitates notch 1 in primary mammalian neurons. Brain Res Mol Brain Res 1999;69:273–80 27. Holcomb L, Gordon MN, McGowan E, et al. Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nat Med 1998; 4:97–100 28. McGowan E, Sanders S, Iwatsubo T, et al. Amyloid phenotype characterization of transgenic mice overexpressing both mutant amyloid precursor protein and mutant presenilin 1 transgenes. Neurobiol Dis 1999;6:231–44 29. Takeuchi A, Irizarry MC, Duff K, et al. Age-related amyloid beta deposition in transgenic mice overexpressing both Alzheimer mutant presenilin 1 and amyloid beta precursor protein Swedish mutant is not associated with global neuronal loss. Am J Pathol 2000; 157:331–39 30. Wengenack TM, Whelan S, Curran GL, Duff KE, Poduslo JF. Quantitative histological analysis of amyloid deposition in Alzheimer’s double transgenic mouse brain. Neuroscience 2000;101:939–44 31. Morgan ME, Lui K, Anderson BD. Microscale titrimetric and spectrophotometric methods for determination of ionization constants and partition coefficients of new drug candidates. J Pharm Sci 1998; 87:238–45 32. Gomez-Isla T, Hollister R, West H, et al. Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer’s disease. Ann Neurol 1997;97:17–24

805

33. Levin VA. Relationship of octanol/water partition coefficient and molecular weight to rat brain capillary permeability. J Med Chem 1980;23:682–84 34. Lemaire C, Cantineau R, Guillaume M, Plenevaux A, Christiaens L. Fluorine-18-altanserin: A radioligand for the study of serotonin receptors with PET: Radiolabeling and in vivo biologic behavior in rats. J Nucl Med 1991;32:2266–72 35. Scheffel U, Pogun S, Stathis M, Boja JW, Kuhar MJ. In vivo labeling of cocaine binding sites on dopamine transporters with [3H]WIN 35,428. J Pharmacol Exp Ther 1991;257:954–58 36. Hume SP, Myers R, Bloomfield PM, et al. Quantitation of carbon11-labeled raclopride in rat striatum using positron emission tomography. Synapse 1992;12:47–54 37. Suehiro M, Scheffel U, Ravert HT, Dannals RF, Wagner HN. [11C](1)McN5652 as a radiotracer for imaging serotonin uptake sites with PET. Life Sci 1993;53:883–92 38. Mathis CA, Simpson NR, Mahmood K, Kinahan PE, Mintun MA. [11C]WAY 100635: A radioligand for imaging 5-HT1A receptors with positron emission tomography. Life Sci 1994;55:PL403–7 39. Feher M, Sourial E, Schmidt JM. A simple model for the prediction of blood-brain partitioning. Int J Pharm 2000;201:239–47 40. Dishino DD, Welch MJ, Kilbourn MR, Raichle ME. Relationship between lipophilicity and brain extraction of C-11-labeled radiopharmaceuticals. J Nucl Med 1983;24:1030–38 41. Salminen T, Pulli A, Taskinen J. Relationship between immobilised artificial membrane chromatographic retention and the brain penetration of structurally diverse drugs. J Pharm Biomed Anal 1997; 15:469–77 42. Gupta SP. QSAR studies on drugs acting at the central nervous system. Chemical Reviews 1989;89:1765–1800 43. Dezutter NA, de Groot TJ, Busson RH, Janssen GA, Verbruggen AM. Preparation of 99mTc-N2S2 conjugates of chrysamine G, potential probes for the b-amyloid protein of Alzheimer’s disease. J Labelled Compounds Radiopharmaceuticals 1999;42:309–24 44. Cherry SR. Fundamentals of positron emission tomography and applications in preclinical drug development. J Clin Pharmacol 2001;41:482–91 45. Cherry SR, Gambhir SS. Use of positron emission tomography in animal research. ILAR J 2001;42:219–32 46. Naslund J, Haroutunian V, Mohs R, et al. Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA 2000;283:1571–77 47. Eckelman WC, Gibson RE. The design of site-directed radiopharmaceuticals for use in drug discovery. In: Burns HD, Gibson R, Dannals R, Siegl P, eds. Nuclear imaging in drug discovery, development and approval. Boston: Birkha¨user, 1993:113–34 48. Skovronsky DM, Zhang B, Kung M-P, Kung HF, Trojanowski JQ, Lee VM-Y. In vivo detection of amyloid plaques in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA 2000;97:7609–14 49. Agdeppa ED, Kepe V, Liu J, et al. Binding characteristics of radiofluorinated 6-dialkylamino-2-naphthylethylidene derivatives as positron emission tomography imaging probes for beta-amyloid plaques in Alzheimer’s disease. J Neurosci 2001;21:RC189 50. Mathis CA, Bacskai BJ, Kajdasz ST, et al. A lipophilic thioflavinT derivative for positron emission tomography (PET) imaging of amyloid in brain. Bioorg Medic Chem Lett 2002;12:295–98 51. Zhuang ZP, Kung MP, Hou C, et al. IBOX(2-(49-dimethylaminophenyl)-6-iodobenzoxazole): A ligand for imaging amyloid plaques in the brain. Nucl Med Biol 2001;28:887–94 52. Agdeppa ED, Kepe V, Shoghi-Jadid K, et al. In vivo and in vitro labeling of plaques and tangles in the brain of an Alzheimer’s disease patient: A case study. J Nucl Med 2001;42:65P Received February 8, 2002 Revision received May 20, 2002 Accepted May 22, 2002
