WO2001074374A1 - Methods for detecting plaques in vivo - Google Patents

Abstract

Methods for detecting extracellular deposits in the brain of a living mammal are described that include administering a polyamine modified, labeled polypeptide having specific binding affinity for the extracellular deposit, to the living mammal. Isolated β-amyloid peptides that are polyamine modified and labeled with a radioisotope or contrast agent also are described.

Description

METHODS FOR DETECTING PLAQUES IN VIVO

TECHNICAL FIELD

This invention relates to detecting extracellular deposits such as plaques in the brain of a living mammal, and more particularly to using a polyamine-rnodifϊed, labeled polypeptide to detect the extracellular deposits.

BACKGROUND

Alzheimer's Disease (AD) is characterized neuropathologically by neuritic plaques and neurofibrillary tangles. Neuritic, or senile plaques contain a dense core consisting largely of several species of amyloid-β (Aβ) peptide. Aβ is a 39-43 amino acid peptide derived from amyloid precursor protein. Aβ is highly hydrophobic and spontaneously aggregates in vitro to form β pleated sheets. Maggio, J.E. and Mantyh, P.W., Brain PathoL, 6:147-162 (1996). Aβ also has been reported to be neurotoxic in vitro and in vivo. The main link between AD and Aβ is based on genetic mutations that have been discovered in familial forms of AD and Down's syndrome that result in aberrant processing or increased levels and deposition of Aβ. Selkoe, D.J., Science, 275:630-631 (1997). Furthermore, transgenic mice overexpressing the same mutations have been shown to develop amyloid deposits like those in AD as well as significant behavioral deficits. Hsiao, K. et al., Science, 274:99-102 (1996); and Holcomb, L. et al, Nature Med., 4:97-100 (1998). There also appears to be a significant correlation between amyloid burden and dementia in AD patients.

Currently, there is no definitive diagnosis for AD except by post-mortem observation of these deposits and a process of elimination of other neurodegenerative disorders.

SUMMARY

The invention is based on a technique for visualizing extracellular deposits in vivo, which includes using labeled and polyamine modified polypeptides that can cross the blood-brain barrier (BBB) and that have affinity for the extracellular deposits. As described herein, the permeability of Aβ at the BBB was increased by at least two-fold in different brain regions through covalent modification with the naturally occurring polyamine, putrescine. Following intravenous injection of radiolabeled, putrescine- modified Aβ, amyloid deposits were labeled in vivo in a transgenic mouse model of Alzheimer's disease. This technique, when applied to humans, may detect extracellular deposits in vivo, allowing early diagnosis of Alzheimer's disease and other neurological disorders with extracellular deposits such as Creutzfeld- Jakob disease, as well as therapeutic intervention before cognitive decline occurs.

In one aspect, the invention features a method for detecting extracellular deposits, such as β-amyloid plaques, in the brain of a living mammal. The method includes administering (e.g., intravenously) an amount of polypeptide to the mammal effective to detectably bind the extracellular deposits, wherein the polypeptide is labeled, polyamine- modified, and has specific binding affinity for the extracellular deposits; and detecting the polypeptide bound to the extracellular deposits. The detecting step can include diagnostic imaging, such as positron emission tomography, gamma-scintigraphy, single photon emission computerized tomography, magnetic resonance imaging, functional magnetic resonance imaging, or magnetoencephalography. Magnetic resonance imaging is a particularly useful technique. The polypeptide can be, for example, a β-amyloid peptide such as β-amyloid peptide_1-4o. The polyamine can be selected from the group consisting of putrescine, spermine, and spermidine. Putrescine is particularly useful. The label can be a radiosotope such as ¹²³1, ¹⁸F, ^ulIn, ⁶⁷Ga, or ^99mTc or a contrast agent such as Gd, dysprosium, or iron. Gd is a particularly useful contrast agent. In some embodiments, the contrast agent can be radiolabeled.

Isolated β-amyloid peptides also are featured. The β-amyloid peptide is polyamine-modified and labeled with a radioisotope or contrast agent suitable for diagnostic imaging. The polyamine can be selected from the group consisting of putrescine, spermine, and spermidine. Examples of radiosiotope and contrast agents are described above.

In yet another aspect, an isolated antibody having specific binding affinity for a β- amyloid peptide (e.g., β-amyloid peptide_1-40) or a prion protein (e.g., protease resistant prion protein) is featured. The antibody is polyamine modified and labeled with a radioisotope or contrast agent suitable for diagnostic imaging. The contrast agent can be selected from the group consisting of Gd, dysprosium, and iron. Gd is a particularly useful contrast agent.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

Figures 1 A and IB are RP-HPLC chromatograms of unlabeled Aβ_1-4o (A) and PUT- Aβ_{1- 0} (B). The abscissa plots the retention time in min. The left ordinate plots the absorbance at 214 nm. The right ordinate plots the gradient as percent of Buffer B (80% ACN/0.05% TF A/19.95% HPLC water).

Figures 2 A and 2B are autoradiographs that depict ¹²⁵I- Aβ_{1- 0} and ¹²⁵I-PUT- Aβi. ₀ labeling of amyloid deposits in vitro: AD temporal lobe sections were incubated with I- Aβ_{1- 0} and exposed for 6 days (A) or with I-PUT- Aβ_{1- 0} and exposed for 1 day

(B). Scale bars, 5 mm.

Figures 3 A-3F are photomicrographs of ¹²⁵I- Aβι-40 and ¹²⁵I-PUT- Aβ_1- o labeling of amyloid deposits in vitro with equivalent radioactivity. Figures 3A-3C represent adjacent sections incubated with buffer alone (A), or 5 x 10⁵ cpm of either ¹²⁵I- Aβ_{1- 0} (B) or ¹²⁵I-PUT- Aβι.₄₀ (C) and processed for anti-Aβ IH and emulsion autoradiography with an equal exposure time of 6 days. Scale bars, 200 μm. Figures 3D-3F are higher magnifications of amyloid deposits indicated by arrows in Figures 3A-3C. Scale bars, 50 μm.

Figures 4A-4F are photomicrographs of ¹²⁵I- Aβ_{1- 0} and ¹²⁵I-PUT- Aβ_1-40 labeling of amyloid deposits in vitro with equivalent peptide concentration. Figures 4A-4C represent adjacent sections incubated with buffer alone (A), or 100 pM of either ¹²⁵I- Aβi o (B) or 125 I-PUT- Aβ_1-40 (C) and processed for anti-Aβ IH and emulsion autoradiography with an equal exposure time of 6 days. Scale bars, 200 μm. Figures 4D-4F represent higher magnification of amyloid deposits indicated by arrows in Figures 4A-4C. Scale bars, 50 μm.

Figures 5 A-5H are photomicrographs of ¹²⁵I- Aβ_1-40 and ¹²⁵I-PUT- Aβι.₄0 labeling of amyloid deposits in vitro in the absence or presence of 10-fold excess unbound putrescine. In Figures 5 A and 5C, sections were incubated with 100 pM of either I- Aβ_{1- 0} (5A) or I-PUT- Aβι-₄₀ (5C) in the absence of unbound putrescine. Figures 5B and 5D represent adjacent section incubated with 100 pM of either I- Aβ_1-40 (5B) or ¹²⁵I-PUT- Aβ_1-40 (5D) in the presence of 10-fold excess unbound putrescine. All sections were processed for anti-Aβ IH and emulsion autoradiography with an equal exposure time of 6 days. Scale bars (5A-5D), 100 μm. Figures 5E-5H represent higher magnification of amyloid deposits indicated by arrows in 5A-5D. Scale bars, 10 μm.

Figures 6A-6D are photomicrographs of ¹²⁵I-PUT-Aβ_1-4o labeling of amyloid deposits in vivo in APP, PS1 transgenic mouse brain. Figure 6 A is a section through medial septum processed for anti-Aβ IH and emulsion autoradiography with 8 weeks of exposure exhibiting several labeled deposits. Figure 6B is an adjacent section showing the same deposits stained with thioflavin S. Figures 6C and 6D represent higher magnification of deposit #2. Scale bars (6A, 6B), 100 μm; (6C, 6D), 10 μm.

Figures 7A-7F are photomicrographs of ¹²⁵I-PUT-Gd-Aβ₁. ₀ labeled amyloid deposits in vitro. Figures 7A-7C are sections of AD temporal lobe incubated with buffer alone (A) or lxl 0⁶ cpm of either ¹²⁵I-Gd-Aβ_1-40 (B) or ¹²⁵I-PUT-Gd-Aβ_1-40 (C) and processed for anti-Aβ iminunohistochemistry and emulsion autoradiography with exposure times of 9 days (B) or 1 day (C). Scale bars = 200 μm. Figures 7D-7F represent higher magnification of amyloid deposits indicated by arrows in Figures 7A-7C. Scare bars = 50 μm.

DETAILED DESCRIPTION

The invention features a method for detecting extracellular deposits in the brain of a living mammal. Extracellular deposits that can be detected include amyloid deposits, neuritic plaques, and diffuse plaques. Amyloid deposits include, for example, deposits of an Aβ polypeptide or of a prion protein (PrP). In some embodiments, the methods of the invention can be used to detect tumors in the brain. Methods of the invention include administering an amount of a polypeptide to the mammal effective to detectably bind to the extracellular deposits. Suitable polypeptides have specific binding affinity for the extracellular deposits and are at least 10 amino acid residues in length. For example, amyloid β peptide (Aβ), which has affinity for amyloid deposits, can be used. Non- limiting examples of Aβ polypeptides that can be used include Aβ_1-40 and Aβι-₄₂. Beta- sheet blockers, i.e., short peptides that are homologous to the central region of Aβ and include residues that inhibit beta-sheet formation (e.g., proline residues) can be used, as well as peptidyl modulators of the recognition sequence of β-amyloid aggregation. Poduslo et al, J. Neurobiol.. 39:371-382 (1999). Peptidyl modulators typically are peptides that contain a portion of amino acid residues from the Aβ recognition sequence (within residues 15-25 of Aβ and in particular, residues KLVFF) and six lysine residues at the C-terminus as a disrupting element. Pallito et al., Biochemistry, 38:3570-3578 (1999). For example, the peptidyl modulator can be a peptide that includes residues 16- 20 or 15-25 of Aβ with 6 lysine residues at the C-terminus. Other derivatives of amyloid precursor protein that have affinity for Aβ plaques also are suitable.

In addition, antibodies having specific binding affinity for polypeptides within the extracelluar deposits (e.g., affinity for Aβ_{1- 0} or Aβ_1-4 , or the protease resistant form of PrP) can be used. See, U.S. Patent No. 5,231 ,000 and U.S. Patent No. 5,262,332 for examples of antibodies having specific binding affinity for Aβ. See, Zanusso et al. Proc. Natl. Acad. Sci. USA, 95:8812-8816 (1998) for examples of antibodies having specific binding affinity for the protease resistant form of PrP. As used herein, antibodies include polyclonal or monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab) fragments. Monoclonal antibodies are particularly useful. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Chimeric antibodies can be produced through standard techniques. Antibody fragments that have specific binding affinity for Aβ_1-40 and Aβ_1-4 or for the protease resistant form of PrP can be generated by known techniques. Such fragments include, but are not limited to, F(ab')₂ fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')₂ fragments. Alternatively, Fab expression libraries can be constructed. See, for example, Huse et al., Science, 246:1275 (1989). Single chain Fv antibody fragments are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., 15 to 18 amino acids), resulting in a single chain polypeptide. Single chain Fv antibody fragments can be produced through standard techniques. See, for example, U.S. Patent No. 4,946,778.

Labeling and Polyamine Modification of Polypeptides

Polypeptides that are administered to the living mammal are labeled and polyamine modified. The use of a labeled peptide with greater permeability at the BBB increases sensitivity and allows the use of lower quantities of radioisotope. Typical labels that are useful include radioisotopes and contrast agents used for imaging procedures in humans. Non-limiting examples of labels include radioisotope such as ¹²³I (iodine), ¹⁸F (fluorine), ^99mTc (technetium), ^lllIn (indium), and ⁶⁷Ga (gallium), and contrast agents such as, gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotopes (¹⁵³Gd) also are available and suitable for imaging procedures in non-human mammals. Polypeptides can be labeled tlirough standard techniques. For example, polypeptides can be iodinated using chloramine T or l,3,4,6-tetrachloro-3α,6α-diphenylglycouril. For fluorination, polypeptides are synthesized and fluorine is added during the synthesis by a fluoride ion displacement reaction. See, Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Dmg Carrier Svst., 16(2):209-244 (1999) for a review of synthesis of proteins with such radioisotopes.

Polypeptides also can be labeled with a contrast agent tlirough standard techniques. For example, polypeptides can be labeled with Gd by conjugating low molecular Gd chelates such as Gd diethylene triarnine pentaacetic acid (GdDTPA) or Gd tetraazacyclododecanetetraacetic (GdDOTA) to the polypeptide. See, Caravan et al., Chem. Rev. 99:2293-2352 (1999) and Lauffer et al. J. Magn. Reson. Imaging 3:11-16 (1985). Antibodies can be labeled with Gd by, for example, conjugating polylysine-Gd chelates to the antibody. See, for example, Curtet et al., Invest. Radiol. 33(10):752-761 (1998). Alternatively, antibodies can be labeled with Gd by incubating paramagnetic polymerized liposomes that include Gd chelator lipid with avidin and biotinylated antibody. See, for example, Sipkins et al. Nature Medicine, 4 623-626 (1998).

Polypeptides are modified with polyamines that are either naturally-occurring or synthetic. See, for example, U.S. Patent No. 5,670,477. Useful naturally-occurring polypeptides include putrescine, spermidine, spermine, 1,3-diaminopropane, norspermidine, syn-homospermidine, thermine, thermospermine, caldopentamine, homocaldopentamine, and canavalmine. Putrescine, spermidine, and spermine are particularly useful. Synthetic polyamines are composed of the empirical formula C_XH_VN_Z, and can be cyclic or acyclic, branched or unbranched, hydrocarbyl chains of 3-12 carbon atoms that further include 1-6 NR or N(R)₂ moieties, wherein R is H, (C₁-C₄) alkyl, phenyl, or benzyl.

As described herein, putrescine modification of Aβ_1-40 significantly increased its permeability at the blood brain barrier (BBB) an average of two-fold. Permeability at the BBB of putrescine modified and Gd labeled Aβ_{1- 0} also was significantly increased 1.5- 2.0 fold relative to native Aβ_1-4o. It should be noted that the permeability coefficient- surface area product (PS) values for Aβ_1-40 are relatively high already and compare to that of insulin, whose PS value in rat cortex is 15.78 x 10^"6 ml/g/s, and BBB uptake is known to occur by receptor-mediated transport. Poduslo, J.F. et al., Proc. Natl. Acad. Sci. USA, 5 9:5705-5709 (1994). As a basis for comparison, the PS value for albumin in rat cortex is

0.15 x 10^"6 ml/g/s, and is thought to cross the BBB by passive diffusion. The high PS value for Aβ_{1- 0} coupled with stereospecifϊc BBB permeability data for L-Aβ_1-40 indicate that the BBB transport likely occurs by a receptor-mediated mechanism. Thus, a doubling of the already high PS value for Aβι_-40 by putrescine modification represents a o further dramatic increase in its permeability at the BBB with important physiological implications for enhanced delivery into the CNS.

There are many approaches for the chemical cross-linking or "linkage" of polypeptides to polyamines. Significant advancement in the application of these cross- linking agents has led to the synthesis of cleavable bifunctional compounds. There are 5 over 300 cross-linkers now available. It is desirable that the linkage of polypeptide to polyamine allows the polypeptide to maintain the ability to bind the extracellular deposit and the polyamine to facilitate increased permeability across the BBB.

Numerous considerations, such as reactivity, specificity, spacer arm length, membrane permeability, cleavability and solubility characteristics need to be evaluated 0 when choosing an appropriate cross-linker. See, for example, "Chemistry of Protein Conjugation and Cross-Linking", Shan S. Wong, CRC Press, Ann Arbor, 1991. Functional groups that are available for conjugation are not involved in the binding of the polypeptide to the extracellular deposit.

Linking reagents have at least two reactive groups and can be either 5 homobifunctional with two identical reactive groups or heterobifunctional with two or more different reactive groups. Trifunctional groups also exist and can contain three functional groups. Most homobifunctional cross-linkers react with primary amines commonly found on proteins. Other homobifunctional cross-linkers couple through primary sulfhydryls. Homobifunctional cross-linkers can be used in a one step reaction 0 procedure in which the compounds to be coupled are mixed and the cross-linker is added to the solution. The resulting cross-linking method may result in self-conjugation, intermolecular cross-linking, and/or polymerization. The following are examples of cross- linking approaches and are not meant to be inclusive. Imido esters are the most specific acylating reagents for reaction with amine groups whereby in mild alkaline pH, imido esters react only with primary amines to form imidoamides. The product carries a positive charge at physiological pH, as does the primary amine it replaces and therefore, does not affect the overall charge of the protein. Homobifunctional N-hydroxysuccinimidyl ester conjugation is also a useful crosslink approach to crosslink amine-containing proteins. Homobifunctional sulfhydryl reactive cross-linkers include bismaleimidhexane (BMH), l,5-difluoro-2,4-dinitrobenzene (DFDNB), and l,4-di-(3',2*-ρyridyldithio) propionamido butane (DPDPB).

Many heterobifunctional cross-linkers are commercially available with the majority containing an amine-reactive functional group on one end and a sulfhydryl- reactive group on the other end. Multiple heterobifunctional haloacetyl cross-linkers are available, as are pyridyl disulfide cross-linkers. Carbodiimides are a classic example of heterobifunctional cross-linking reagents for coupling carboxyls to amines resulting in an amide bond.

Administration of Labeled, Polyamine Modified Polypeptides

The labeled, polyamine modified polypeptides are formulated with a pharmaceutically acceptable earner and administered to the living mammal. In general, the polypeptides are administered intravenously (i.v.), although other parenteral routes of administration, including subcutaneous, intramuscular, intrarterial, intracarotid, and intrathecal also can be used.

Formulations for parenteral administration may contain pharmaceutically acceptable carriers such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, and the like.

The dosage of labeled, polyamine modified polypeptide to be administered will be determined by the attending physician talcing into account various factors known to modify the action of drugs. These include health status, body weight, sex, diet, time and route of administration, other medications, and any other relevant clinical factors. Typically, about l-3000μg/kg body weight are administered. For example, the dosage can range from about 10-1000μg/kg body weight or 50-500μg/kg body weight. Therapeutically effective dosages may be determined by either in vitro or in vivo methods. Detecting Polypeptides Bound to Extracellular Deposits

Imaging techniques that can be used to detect labeled deposits in vivo include positron emission tomography (PET), gamma-scintigraphy, magnetic resonance imaging (MRI), functional magnetic resonance imaging (FMRI), magnetoencephalography (MEG), and single photon emission computerized tomography (SPECT). MRI is particularly useful as the spatial resolution and signal-to-noise ratio provided by MRI (30 microns) is suitable for detecting amyloid deposits, which can reach up to 200 microns in size. Aβ plaques were detected in vivo using a Gd-labeled and polyamine modified (putrescine) Aβ polypeptide. As described herein, labeled, polyamine modified Aβ peptide detectably bound to amyloid deposits in vivo in APP, PS1 transgenic mouse brain. Cerebrovascular and pial labeling were observed, but more importantly, many parenchymal amyloid deposits were labeled. The labeled deposits were most prevalent in the dorsal hippocampus, medial septum, and fimbria/fornix. A few labeled deposits were observed in the cortex, where the majority of amyloid deposits are found. This may be related to the density of vascularization. Most of the labeled deposits in the hippocampus were situated along the hippocampal fissure, which typically contains some large blood vessels. Some labeled deposits were associated with cerebrovascular amyloid, but most had no apparent relation to blood vessels, particularly those in the fimbria/fornix and medial septum. Increased brain uptake of ¹²⁵I-PUT- Aβ_1-40 or ¹²⁵I- Aβ_1-40 and labeling might result if general disruption of the BBB occurred in APP, PS1 transgenic mice, as has been proposed by some to occur in AD. No labeled deposits were observed, however, after injection of ¹²⁵I- Aβ 1-40, even after 12 weeks of exposure.

One experiment using aged squirrel monkeys, known to exhibit amyloid deposits with advanced age, reported labeling of cerebrovasculature amyloid deposits following injection of radiolabeled Aβ_{1- 0} into the carotid artery. Labeled parenchymal deposits were not observed. Ghilardi, J.R. et ah, NeuroReport, 7:2607-2611 (1996). The preponderance of amyloid deposits in aged squirrel monkeys is associated with the cerebrovasculature and the few deposits found within the parenchyma tend to be diffuse rather than neuritic plaques.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES Example 1 -PS and V_p measurements of radioiodinated Aβ proteins.

Permeability of Aβ_1-40 or PUT- Aβ_1-40 at the BBB was determined using an i.v. bolus injection technique that has been described in detail. Poduslo, J.F. and Curran, G.L., Proc. Natl. Acad. Sci.USA 89:2218-2222 (1992); Poduslo, J.F. and Curran, G.L., Molec. Brain Res.. 23:157-162 (1994); and Poduslo, J.F.et al, Proc. Natl. Acad. Sci. USA, 9:5705-5709 (1994). Putrescine modification of synthetic human Aβ_1-4o was performed by covalent linkage of the polyamine to carboxylic acid groups using carbodiimide at a pH of 6.7. Poduslo, J.F. and Curran, G.L., J. Neurochem.. 66:1599-1609 (1996); and Poduslo, J.F. and Curran, G.L., J. Neurochem., 67:734-741 (1996).

Separate aliquots of native (Aβ 1-40) or putrescine-modified (PUT-Aβ 1-40) peptides then were labeled with ¹²⁵I and ¹³¹I (Amersham) using a modified chloramine-T procedure. PS and V_p values were detemiined in normal adult male Sprague-Dawley rats (400-450 g) obtained from Harlan. All procedures performed were in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. Briefly, a bolus of 0.9% NaCl containing Aβ_1-40 or PUT- Aβ_1-40 labeled with ¹²⁵I was injected rapidly into the catheterized brachial vein of an anesthetized rat (sodium pentobarbital, 25 mg/kg, i.p.). Blood (200 μl) was sampled from the brachial artery at several intervals during the next 15 min. An aliquot of the peptide labeled with ¹³¹I was then injected into the brachial vein 15 sec prior to sacrifice of the animal to serve as a measure of residual plasma volume (V_p; μl/g). After collection of the final blood sample, the anesthetized animal was sacrificed. Several brain regions and plasma samples were assayed for ¹²⁵I and ¹³¹I radioactivity in a two-channel gamma counter (Cobra II, Packard) with the activity corrected for background and crossover of I activity into the I channel. The permeability coefficient x surface area products (PS; 10^"6 ml/g/s) for Aβ_1-40 and PUT- Aβ_1-40 were calculated using the V_p (μl/g) as a measure of residual plasma volume. Statistical evaluations of PS and V_p were performed using ANONA followed by Bonferroni multiple comparisons.

Putrescine-modified Aβ_1-4o (PUT- Aβmo) exhibited increased permeability at the BBB compared to native Aβi^o. Putrescine modification significantly increased the BBB permeability of PUT- Aβ_1-40 in all brain regions measured, compared to Aβ 1-40, with significant increases in the permeability coefficient x surface area product (PS) ranging from 1.9-fold in the hippocampus and cerebellum to 2.3-fold in the cortex and thalamus

(Table 1). The residual plasma volume (N_p) was increased slightly, but significantly in three of six brain regions (Table 1). This was probably a result of the large increases in the PS values observed for PUT-Aβ_1-40, with some of the peptide crossing the BBB even in the short time used in this experiment for the administration of the second isotope (15 sec).

TABLE 1 PS and Vp of Aβι..« and PUT AEY₄n

Example 2 - Labeling of Amyloid Plaques In Vitro: The next step was to determine if PUT- Aβ_1-4o would bind and label amyloid deposits in AD brain sections in vitro. Aβ_1- o and PUT- Aβ_{1- 0} were first radiolabeled with ¹²⁵I to facilitate detection and

19^ then purified to remove unbound I and unlabeled peptide. Radioiodinated Aβ_1-40 and PUT- Aβi-₄0 were purified using reversed-phase high performance liquid chromatography (RP-HPLC) to remove iodotyrosine and unlabeled peptide in order to achieve very high specific activity. Maggio, J.E. et al., Proc. Natl. Acad. Sci.USA, 89:5462-5466 (1992); and Ghilardi, J.R. et al., NeuroReport 7:2607-2611 (1996). Following radioiodination,

19^ the peptides were dialyzed for 4 hr against 0.2 M Nal to remove unbound I. The peptides were then passed over a C₁₈ preparative cartridge (Sep-Pak Light, Waters Corp.)

19^ to remove more unbound I. The peptide was eluted stepwise with increasing concentrations of acetonitrile (ACN) in 0.05% trifluoroacetic acid (TFA) (10, 20, 40, 80, 100%) ACN, Fisher). The peptides eluted primarily in the 40 and 80% ACN fractions and were reduced by adding 2-mercaptoethanol (2-ME, Bio-Rad). The peptides were concentrated to 0.25 ml with a Speed Vac (Savant). The ¹²⁵I- Aβ_1-40 and ¹²⁵I-PUT- Aβ_1-40 were purified by RP-HPLC (System Gold, Beckman) using a gradient method with a binary solvent system (Buffer A: 0.05% TFA 99.95% water; Buffer B: 80% ACN/0.05%TFA/19.95% water). Each peptide was injected and purified using a 1-hr gradient of 0-100% Buffer B at a rate of 1 ml/min using a small-bore, C₁₈ column (5 μm, 4.6 x 250 mm, #218TP54, Vydac). Chroniatograms of

HPLC elution profiles of unlabeled Aβ_1-40 and PUT- Aβ_1-40 are shown in Figures 1A and IB, respectively. PUT- Aβ_1-4o elutes earlier than Aβι_-40 because the positively charged amine groups of putrescine make PUT- Aβ_1-4o less hydrophobic. One-minute (1 ml) fractions were collected with the detector turned off so as not to quench any of the radioactivity. Aliquots (5 μl) often fractions surrounding the most radioactive fraction were then counted with the gamma counter to identify the fraction with the highest

19^ radioactivity. That fraction containing the purest I-labeled peptide was then concentrated to 0.25 ml with a Speed Vac to remove the ACN and stored at -20°C in the presence of a reducing agent (2 -ME). Aliquots of the RP-HPLC fractions presumed to contain the ¹²⁵I- Aβ_{1- 0} and ¹²⁵I-PUT- Aβ_1-40 were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Only a single band was observed for all fractions of I- Aβ_1-40 and ^{1 5}I-PUT- Aβ_1-40, verifying their purity.

HPLC purified ¹²⁵I- Aβ_1-40 and ¹²⁵I-PUT- Aβ_{1- 0} were incubated with adjacent sections of unfixed AD temporal lobe cortex. Three adjacent sections were incubated with either ¹²⁵I-Aβ 1-40, ¹²⁵I-PUT-Aβ 1-40, or vehicle. Briefly, the sections were first blocked with 0.1% bovine serum albumin (BSA) in 0.05 M Tris HCl/0.9% NaCl (TBS), pH 7.0 for 30 min. The sections were then incubated for 3 hrs with 100 pM I- Aβ_1-40 or ¹²⁵I-PUT-Aβ 1-40, or alone in 250 μl of TBS, pH 7.0 containing 0.1% BSA, 0.6 mg/ml magnesium chloride, 0.04 mg/ml bacitracin, 0.002 mg/ml chymostatin, and 0.004 mg/ml leupeptin. The sections were washed with TBS, pH 7.0 four times and then rinsed briefly with distilled water twice. The sections were allowed to air dry overnight in a box with desiccant at 4°C.

Sections then underwent immunohistochemistry (IH) for amyloid using an anti- Aβ monoclonal mouse antibody (4G8, Senetek). Untreated sections were included as a positive control for antibody staining. After rehydrating with TBS, pH 7.6, the sections were fixed briefly with neutral-buffered, 10% formalin for 3 min. The sections were washed with TBS and then blocked with 1.5% nom al horse serum in TBS for 30 min.

The sections were incubated with the anti-Aβ primary antibody at a dilution of 1 : 1000 in

0.1%) BS A TBS overnight at 4°C. The primary antibody was then visualized using a Vectastain Elite ABC, immunoperoxidase kit with diaminobenzidine (DAB) as a substrate according to the instructions (Vector Laboratories). The sections were allowed to air dry overnight in a box with desiccant at 4°C.

Following immunohistochemistry (IH) to visualize the amyloid deposits, the sections were subjected to film and emulsion autoradiography to detect the presence of the ¹²⁵I- Aβ_{1- 0} or ¹²⁵I-PUT- Aβ_{1- 0}. The sections were exposed to high-resolution autoradiographic film (Hyperfilm MP, Amersham) at -70°C to visualize ¹²⁵I-labeled amyloid deposits. Figures 2A-2B are the film autoradiographs. Note the presence of punctate areas of exposed film located predominantly in the regions corresponding to gray matter. This is similar to the distribution of amyloid deposits seen following IH of the tissue sections. It was possible to overlay the two for direct correspondence under the microscope. A longer duration of exposure was required for ¹²⁵I- Aβ_1-40 (6 days) to achieve equal relative intensity as ¹²⁵I-PUT- Aβ_1-4o (1 day), suggesting that ¹²⁵I-PUT-

19^

Aβ_1-4o may have greater affinity to the amyloid deposits than I- Aβ_{1- 0}. In order to definitively correlate the IH with the iodinated peptide by colocalization, the sections were next dipped in an autoradiographic emulsion (Type NTB-3, Kodak). The slides were dipped in emulsion at 43°C under Safelight illumination in a darkroom. The slides were chilled to solidify the emulsion and then allowed to air dry at room temperature for 3 hrs in a light-proof box. The slides were exposed at 4°C in a light-proof box with desiccant. The slides were developed with Dektol developer

(Kodak) and fixed (Kodak) according to the instructions. The sections were dehydrated with successive changes of ethanol and xylene and then coverslipped with a xylene-based mounting media (CMS).

The presence of the iodinated peptide was revealed by exposed (blackened) silver grains directly on the same tissue previously stained with anti-Aβ antibody. These results are illustrated in the photomicrographs of Figs. 3-5. Figures 3A-3F illustrate the binding of equal amounts of radioactivity of ¹²⁵I- Aβi.₄₀ and ¹²⁵I-PUT- Aβ_1-40 to amyloid deposits in adjacent sections of AD temporal lobe cortex in vitro. All sections were processed for anti-Aβ IH and emulsion autoradiography with an equal exposure time of 6 days. The amyloid deposits appear brown. The presence of iodinated peptide is indicated by increased density of black, exposed silver grains. The binding of both peptides is specific to the amyloid deposits due to the low density of exposed silver grains in the background.

Furthermore, it seems to be selective for the dense-core, neuritic-type plaques on the left than the diffuse plaque located on the right (Figs. 3E, F). Also note that with equal amounts of radioactivity and exposure time there is a higher density of silver grains for ¹²⁵I-PUT-Aβ_1-40j suggesting that the modified peptide has a greater affinity for the neuritic

19^ plaques than I-Aβ_1-4o. Figures 4A-4F illustrate the binding of equal peptide

19^ 19^ concentrations of I- Aβ_1-4o and I-PUT- Aβ_1-4o to amyloid deposits in adjacent sections of AD temporal lobe cortex in vitro. These results are similar to Figures 3A-3F.

1 *1

Based on the result that I-PUT- Aβ_1-40 has a greater affinity for neuritic plaques than ¹²⁵I-Aβ_1-4o, an experiment was performed to determine the effect of putrescine on binding by incubating the peptides in the absence or presence of excess unbound putrescine. If putrescine also binds to amyloid, then one might expect to see decreased binding of iodinated peptide in a competitive manner in the presence of excess unbound putrescine. Figures 5A-5H illustrate the binding of ¹²⁵I- Aβ_1-40 and ¹²⁵I-PUT- Aβ_1-40 to amyloid deposits in AD temporal lobe cortex in vitro in the absence or presence of a 10- fold excess of unbound putrescine. There appears to be no appreciable effect on binding of iodinated peptide in the presence of unbound putrescine, even at 10-fold excess. This suggests that putrescine itself does not bind specifically to amyloid, but may enhance binding by some other mechanism.

In summary, in addition to increased permeability at the BBB, I-PUT- Aβ_1- o also labeled amyloid deposits in vitro with greater affinity than I-Aβ_1-4o, based on shorter exposure times or increased intensity of autoradiography emulsion. There is not a clear explanation for this observation as the addition of ten-fold excess of unbound putrescine did not decrease labeling in a competitive manner. In fact, because amyloid is highly hydrophobic and PUT- Aβ_{1- 0} appears to be relatively less hydrophobic than Aβμ ₄₀, based on a shorter retention time during HPLC, one might expect to observe decreased labeling with ¹²⁵I-PUT-Aβ_1-40. Furthermore, the addition of putrescine to the structure of Aβi-₄₀ could possibly block binding sites and also reduce labeling. The binding of both PUT- Aβ_1-40 and Aβ_1-40, however, appeared to be specific for dense-core, neuritic-type amyloid deposits, since no diffuse deposits were labeled and background labeling was not noticeable.

Example 3 - Labeling of Amyloid Plaques In Vivo: ¹²⁵I-PUT- Aβ_1-40 was then tested for its ability to cross the BBB and label amyloid deposits in vivo following i.v. injection in transgenic mice that express two mutant human proteins associated with familial AD. These mice develop amyloid deposits and behavioral deficits within 12 weeks of age. Hemizygous transgenic mice (Tg2576) expressing mutant human amyloid precursor protein (APP₆₉₅) were mated with a second strain of hemizygous transgenic mice (M146L5.1) expressing mutant human presenilin 1 (PS1). Holcomb, L. et al., Nature Med. 4:97-100 (1998). The animals were genotyped for the expression of both transgenes by a dot blot method using a sample of mouse tail DNA. The mice were housed in a vims-free barrier facility under a 12-hr light/dark cycle, with ad lib access to food and water. All procedures performed were in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. Quantitative histological analyses of amyloid deposition indicate that deposition of neuritic-type plaques occurs at a rapid rate starting around 12 weeks, reaching an amyloid burden of over 3.5%> in cortex and hippocampus in one year.

These APP, PS1 transgenic mice (27 weeks of age) were catheterized in the femoral vein under general anesthesia (sodium pentobarbital, 25 mg/kg, i.p.) and injected with 200 μg of ¹²⁵I- Aβ_1-40 or ¹²⁵I-PUT- Aβ_1-40. One mouse was injected with ¹²⁵I- Aβ_1-40 and two with I-PUT- Aβ 1-40. After four hours, each animal was perfused with PBS and fixed with neutral-buffered, 10% formalin following an overdose with sodium pentobarbital (75 mg/kg, i.p.). After cryoprotecting in 10% and 30%> sucrose in PBS, frozen sections of each brain were cut with a freezing microtome and then processed with anti-Aβ IH and emulsion autoradiography for the presence of radiolabeled amyloid deposits using the same methods described above for the human AD sections. The results are shown in Figure 6. Figure 6A a photomicrograph of a section through the medial septum that exhibits several amyloid deposits radiolabeled with ¹²⁵I- PUT-Aβ_1-4o. This particular section was exposed for 8 weeks, but labeled deposits could be observed after only one week of exposure. Figure 6C illustrates a higher magnification of one of the deposits in which the IH reaction product is visible beneath the exposed silver grains. Figure 6B illustrates the adjacent section that was stained for thioflavin S and confirms the presence and distribution of the same amyloid deposits. Radiolabeled amyloid deposits were also observed in the hippocampus and fimbria/fo ix of each animal. An APP, PS1 transgenic mouse injected i.v. with 200 μg of ¹²⁵I-Aβι_-4o did not exhibit labeling of any parenchymal amyloid deposits, aside from faint pial and some residual vascular labeling, with up to 12 weeks of exposure. These results demonstrate that ¹²⁵I-PUT-Aβ_1-40 is able to cross the BBB and bind to amyloid deposits in vivo following i.v. administration.

In summary, these results indicate that ¹²⁵I-PUT-Aβ_1-4o has increased BBB permeability compared to ¹²⁵I-Aβ_1-40. Also, ¹²⁵I-PUT-Aβi.₄₀ retains its ability to selectively label neuritic plaques in vitro, and with greater affinity than ^I25I-Aβ_1-40. This binding does not seem to be affected by excess unbound putrescine. APP, PS1 transgenic mice exhibit large amounts of amyloid deposition and provide a convenient animal model i n^c 1 to test the ability of I-PUT-Aβ_1-40 to label amyloid deposits in vivo. Furthermore, I- PUT-Aβ_1-40 labels amyloid deposits in the brains of these mice following systemic administration. The success of these experiments supports the development of radiolabeled PUT-Aβ_1-40 as a marker of amyloid deposition for use as a diagnostic tool for AD in humans.

Example 4 - Permeability of Polyamine- and Gd-Modified A3 Peptides: Permeability of Aβ_1-40, PUT-Aβ_1-40, Gd-Aβi^o, or PUT-Gd-Aβ_1-40 at the BBB was determined using an IN. bolus injection technique, as indicated in Example 1. Gadolinium (Gd) modified synthetic human Aβ_1-4o was provided by Dr. Wisniewski, New York University School of Medicine. Putrescine (PUT) modification of synthetic human Aβ_1-40 (with or without Gd) was performed as described in Example 1. Separate aliquots of native (Aβ_1-40), PUT-modified (PUT-Aβ_M0), Gd-modified (Gd-Aβι_-40), and PUT-Gd- modified (PUT-Gd- Aβ^o) peptides were labeled with I and I (Amersham) using a modified chloramine-T procedure. Poduslo, J.F. and Curran, G.L., Molec. Brain Res., 23:157-162 (1994).

PS values were determined in male B6/SJL mice (young adult, 30g) bred on site. All procedures performed were in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. The femoral veins and arteries of pentobarbital-anesthetized mice were catheterized with Intramedic PE50 tubing using a heat-pulling process to exactly match the inside diameter of each femoral artery and femoral vein. Following catheterization, a bolus of 0.9% NaCl containing ¹²⁵I-Aβ_1-40, ¹²⁵I-PUT-Aβ_1-40, ¹²⁵I-Gd- ' ϊ ^ Aβ ι-₄o, or I-PUT-Gd-Aβ_1-4o was injected rapidly into the catheterized femoral vein.

After injection, serial blood samples of 20 μl were collected from the catheterized femoral artery over a 15 minute time course at 0.25, 1, 3, 5, 10, 14, and 15 minutes post-injection.

At 15 seconds before the end of the time course, a bolus of I-labeled protein (100 μCi) was administered intravenously to serve as the residual plasma volume (V_p) indicator for PS calculations. Each 20 μL blood sample was collected using heparinized micro- hematocrit capillary tubes (Fisher) after a 20 μl aliquot of blood comprising the void volume of the catheter was first sampled from the artery and then discarded. Each blood sample was extracted with TCA. Briefly, 80 μl PBS, 100 μl 2% BSA (w/v), and 200 μl 30%) TCA (w/v) were added to each blood sample. Each sample was vigorously vortexed and then centrifuged at 10,000xg for 10 minutes to yield a supernatant and pellet. The supernatant and the pellet were separated, and the radioactivity in both the supernatant and the pellet was measured using a gamma counter. The radioactivity in the pellet was expressed as a percentage of the total radioactivity found in both the pellet and the supernatant.

After collection of the final blood sample, the anesthetized mouse was sacrificed, and the brain and the meninges removed. The brain was then dissected into the cortex, caudate-putamen (neostriatum), hippocampus, thalamus, brain stem, and cerebellum tissues. Tissue samples were lyophilized and dry weights were determined with a microbalance, and converted to respective wet weights with wet weight/dry weight ratios previously determined. The lyophilized tissue samples were then assayed for I and I radioactivity in a two-channel gamma counter (Cobra II, Packard). Radioactivity obtained from the gamma counter was corrected for crossover of ¹³¹I radioactivity into the

1 *1 I channel and background.

The permeability coefficient x surface area products (PS; 10^"6 ml/g/s) for Aβ_1-40 and PUT- Aβ_1-4o were calculated using the V_p (μl/g) as a measure of residual plasma volume. Statistical evaluations of PS and V_p were performed using ANOVA followed by Bonferroni multiple comparisons. PUT-modified Aβι_-4o exhibited increased permeability at the BBB compared to native Aβ_1-40. PUT modification of Aβ_1-4o increased permeability in all brain regions assayed, with increases in PS ranging from 2.2-fold in the hippocampus to 2.5-fold in the brain stem (Table 2). Additionally, Gd-modified PUT-Aβ_1-40 (PUT-Gd-Aβ_1-40) exhibited an increase in permeability at the BBB compared to native PUT-Aβ_1-40. PUT-Gd-Aβ_1- o exliibited increased permeability in all brain regions assayed, with increases in PS ranging from 1.5-fold in the caudate-putamen to 2.0-fold in the brain stem (Table 2). Gd modifications to Aβ_1-40 in the absence of PUT modifications exhibited a non-significant decrease in permeability compared to native Aβ_1-40. TABLE 2

PS of Aβι.40, PUT-Aβi.₄₀, Gd-Aβ_1-40, and PUT-Gd-Aβ_1-40 in B6/SJL Mice

Example 5 - Labeling of Amyloid Plaques with Polyamine- and Gd-Modified

Aβ: AD brain tissue was processed for anti-Aβ immunohistochemistry coupled with emulsion autoradiography for detection of radiolabeled Gd-Aβ_1-4o and PUT-Gd-Aβ_1- o plaques. Sections of the temporal lobe from an AD mouse brain were treated with 1 x 10⁶ cpm of either ¹²⁵I-Gd-Aβ_1-4o or ¹²⁵I-PUT-Gd-Aβ_1-40 for 3 hours at room temperature. A separate section treated with buffer alone under the same conditions was used as a control. After incubation, the treated sections were processed for immunohistochemistry with anti-Aβ antibodies. Following immunohistochemistry, the treated sections were processed for emulsion autoradiography with an exposure time of 1 day ( I-PUT-Gd- Aβ_1-40) or 9 days (¹²⁵I-Gd-Aβ_1-40). ¹²⁵I-PUT-Gd-Aβ_1-40 retained its ability to bind to deposits with higher affinity compared to Gd modification alone (Figures 7A-7H). Additionally, the polyamine modification allowed for a dramatic increase in the affinity for the Aβ binding to plaques, as the exposure time for ¹²⁵I-PUT-Gd-Aβ_1-40 was considerably shorter (1 day) compared to the exposure time for ¹²⁵I-Gd-Aβ_1-4o (9 days). These studies demonstrate that PUT-Gd- Aβ M_O can be used as a probe to target plaques after IN. administration. OTHER EMBODIMENTS

It is understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting extracellular deposits in the brain of a living mammal, said method comprising: a) administering an amount of a polypeptide to said mammal effective to detectably bind to said extracellular deposits, wherein said polypeptide is labeled and polyamine modified, and wherein said polypeptide has specific binding affinity for said extracellular deposits; and b) detecting said polypeptide bound to said extracellular deposits.

2. The method of claim 1, wherein said detecting step comprises diagnostic imaging.

3. The method of claim 2, wherein said diagnostic imaging comprises positron emission tomography, gamma-scintigraphy, single photon emission computerized tomography, magnetic resonance imaging, functional magnetic resonance imaging, or magnetoencephalography.

4. The method of claim 2, wherein said diagnostic imaging comprises magnetic resonance imaging.

5. The method of claim 1, wherein said deposits are β-amyloid plaques.

6. The method of claim 5, wherein said polypeptide is a β-amyloid peptide.

7. The method of claim 6, wherein said β-amyloid peptide is β-amyloid peptide_1-4o.

8. The method of claim 1 , wherein said polypeptide is an antibody having affinity for a β-amyloid peptide.

9. The method of claim 1 , wherein said polypeptide is a prion protein.

10. The method of claim 9, wherein said polypeptide is the protease resistant form of prion protein.

11. The method of claim 1, wherein said polypeptide is an antibody having specific binding affinity for a prion protein.

12. The method of claim 1, wherein said polyamine is selected from the group consisting of putrescine, spermine, and spermidine.

13. The method of claim 12, wherein said polyamine is putrescine.

14. The method of claim 1, wherein said label is a radioisotope.

15. The method of claim 14, wherein said radioisotope is selected from the group consisting of ¹²³1, ¹⁸F, ^mIn, ⁶⁷Ga, and ^99mTc.

16. The method of claim 1 , wherein said label is a contrast agent.

17. The method of claim 16, wherein said contrast agent is Gd, dysprosium, or iron.

18. The method of claim 16, wherein said contrast agent is Gd.

19. The method of claim 16, wherein said contrast agent is radiolabeled.

20. The method of claim 1, wherein said polypeptide is administered intraveneously.

21. Isolated β-amyloid peptide, wherein said β-amyloid peptide is polyamine modified and labeled with a radioisotope or contrast agent suitable for diagnostic imaging.

22. The β-amyloid peptide of claim 21, wherein said β-amyloid peptide is β-amyloid peptide_1-40.

23. The β-amyloid peptide of claim 21 , wherein said polyamine is selected from the group consisting of putrescine, spermine, and spermidine.

24. The β-amyloid peptide of claim 21, wherein said radioisotope is selected from the group consisting of ¹²³1, ¹⁸F, In, ⁶⁷Ga, and ^99raTc.

25. The β-amyloid peptide of claim 21, wherein said contrast agent is selected from the group consisting of Gd, dysprosium, and iron.

26. The β-amyloid peptide of claim 25, wherein said contrast agent is radiolabeled.

27. An isolated antibody having specific binding affinity for a β-amyloid peptide or a prion protein, wherein said antibody is polyamine modified and labeled with a radioisotope or contrast agent suitable for diagnostic imaging.

28. The antibody of claim 27, wherein said β-amyloid peptide is β-amyloid peptidβi.

40-

29. The antibody of claim 27, wherein said prion protein is protease resistant.

30. The antibody of claim 27, wherein said contrast agent is selected from the group consisting of Gd, dysprosium, and iron.

31. The antibody of claim 27, wherein said contrast agent is Gd.