WO2007129077A2

WO2007129077A2 - Imaging agent

Info

Publication number: WO2007129077A2
Application number: PCT/GB2007/001669
Authority: WO
Inventors: Brian Austen
Original assignee: St George's Hospital Medical School
Priority date: 2006-05-05
Filing date: 2007-05-04
Publication date: 2007-11-15
Also published as: WO2007129077A3; GB0608960D0; EP2024389A2

Abstract

The invention provides an agent comprising the synthetic amino acid sequence DPhe-DPhe-DVal-DLeu-DLys and an amine or guanidine substituent.

Description

IMAGING AGENT

Field of the Invention

The present invention relates to synthetic peptides capable of recognising and binding to β-amyloid and to the use of the peptides in the diagnosis, monitoring and therapy of Alzheimer's Disease (AD).

Background of the Invention β-amyloid peptide (Aβ) is the major protein component of senile plaques and cerebrovascular amyloid deposits in the brains of AD patients. There is substantial evidence that deposition of Aβ in the form of amyloid fibrils or oligomers is causally linked to the disease. Aβ is toxic to cultured neuronal cells, and this toxicity has been linked to the aggregational and/or conformational status of the peptide.

Under physiological conditions, Aβ readily aggregates into fibrils with a cross-β-sheet conformation. Coincident with the conversion of monomelic Aβ to fibrillar Aβ is a transition from random coil to β-sheet. Several features of Aβ affect the facility of this transition. The Aβ peptide is amphiphilic, with a hydrophilic N- terminus and hydrophobic C-terminus; the length of the latter affects the rate of aggregate formation. In addition, a short hydrophobic stretch at residues 16-20 ( KLVFF) appears to be critical in the formation of fibrillar structure. Inter-molecular interactions of KLVFF are proposed to be involved in the aggregation process.

The penta-peptide KLVFF, containing the 16-20 sequence of full-length Aβ, has been reported to bind to Aβ and disrupt fibril formation (Tjernberg et al., J. Biol. Chem. 271(15):8545-8548, 1996). An octapeptide, QKLVTTAE, with substitutions for the two Phe residues at positions 19 and 20, has also been reported to inhibit fibril formation at a 10- fold molar excess, a result that was attributed to weak interactions between the octapeptide and monomeric Aβ (Hughes et al., Proc. Natl. Acad. Sci. USA 93:2065-2070, 1996). In both cases, fibril inhibition was assessed by electron microscopy. Summary of the Invention

The present inventors have demonstrated that synthetic peptides comprising the amino acid sequence DPhe-DPhe-DVal-DLeu-DLys (FFVLK) are potent inhibitors of β-amyloid fibril formation and toxicity. These inhibitors bind to partially aggregated forms of Aβ, and prevent the formation of full fibrils.

The FFVLK sequence of D-amino acids is the retroinverse of the KLVFF sequence of L-amino acids found in Aβ. The retroinverso configuration gives rise to peptides with increased resistance to proteolytic degradation and turnover in brain, whilst maintaining the amino acid side-chains in the same orientation with respect to each other as in the native sequence. The present inventors have demonstrated that, surprisingly, peptides comprising the retroinverse sequence, FFVLK, are more potent inhibitors of β-amyloid fibril formation than peptides comprising the natural KLVFF sequence.

The FFVLK sequence in the inhibitors of the invention may be extended by addition of a highly basic Arg residue, to actively induce dispersal of the amyloid oligomers. An amine or guanidine substituent may also be present to increase transport across the blood-brain barrier. In addition, the synthetic peptides may be labelled for use as imaging agents. For example, an additional, preferably amino- terminal, substituent such as DOTA may be introduced to provide a ligand for complexation with a contrast agent such as gadolinium ions, to enable MRI imaging of amyloid deposits in patients.

Inhibitors comprising the retroinverse sequence, FFVLK, thus have improved properties as therapeutics over other peptides containing the native sequence KLVFF. When labelled, such peptides are useful in the diagnosis of early or moderate AD and for monitoring therapy of AD.

Accordingly, the present invention provides: an agent comprising the synthetic amino acid sequence DPhe-DPhe-DVal- DLeu-DLys and an amine or guanidine substituent; use of an agent according to the invention in a method of disrupting β- amyloid aggregates; an agent of the invention for use in a method of treating the human or animal body by therapy or in a diagnostic method practised on the human or animal body; use of an agent of the invention in the manufacture of a medicament for the treatment or diagnosis of AD; a method of treating AD, said method comprising administering to a subject in need thereof a therapeutically effective amount of an agent of the invention; - a pharmaceutical composition comprising an agent of the invention and a pharmaceutically acceptable carrier or diluent; use of an agent of the invention in a method of imaging β-amyloid aggregates; a method of imaging β-amyloid aggregates, said method comprising detecting the binding of an agent of the invention to β-amyloid aggregates; and a method of diagnosing AD, said method comprising administering an agent according to the invention to a subject and detecting the presence of β-amyloid aggregates, wherein the presence of β-amyloid aggregates indicates that the subject has AD.

Brief Description of the Figures

Figure 1 illustrates the structure of two exemplary imaging agents of the invention (Imaging Agent 1 and 2). The imaging agents contain three domains: the amyloid-binding domain is the retroinverso sequence in the middle section of the agent; the transport domain is the polyamine in the case of Imaging Agent 1 and hexa-DArginine in the case of Imaging Agent 2 at the C-terminus; and the contrast agent is the gadolinium ion at the N-terminus.

Figure 2 illustrates a molecular model showing how the retroinverse sequence of the central KLVFF sequence of Aβ retains binding to Aβ. Key interactions with the native β-amyloid sequence (e.g. the π-orbital overlaps in phenyl side-chains are enhanced, while the peptide is stabilised against proteolytic degradation by the unnatural D amino acid configuration. The Figure shows an energy-minimised molecular model of residues 15-22 of native Aβ complexed with retroinverso inhibitor rGffvlkGr-NH2 (RI-0MAR2; bottom). Native Aβ residues are shown in mid grey with Glu22 (labelled 88) in dark grey. The inhibitor RI-OM AR2 is shown in light grey with the Arg residues in black. Hydrophobic interactions between Phe residues are enhanced in this structure, compared to structures containing the native sequence. There are ionic bonds between GIu and Arg.

Figure 3 shows that agents containing the retroinverse D-amino acid sequence FFVLK (IR-OMAR2 and Imaging-Frl) are more potent inhibitors of soluble β-amyloid (1-42) aggregate formation than an agent containing the natural sequence KLVFF (KLVFF-amide Fr2), as measured by an ELISA that detects soluble oligomers.

Figure 4 shows that the agent 0MAR2 containing the retroinverse D-amino acid sequence FFVLK is more effective than known inhibitors at inhibiting neuronal cell death induced by aggregated Aβ.

Figure 5 shows that the retroinverse inhibitor rGffvlkGr-NH₂ is more potent at inhibiting beta-amyloid (1-40) aggregation than native sequence inhibitors containing the native KLVFF sequence or the KLVFF sequence with an additional beta-breaking proline (Soto et al (1996) BBRC 226; 2672-680). An ELISA to detect soluble oligomers of Aβ showed that the retroinverso inhibitor (black) is more potent than the native sequence inhibitors (grey and pale grey) at preventing aggregation of Aβ 1-40.

Figure 6 shows that the retroinverse inhibitor rGffvlkGr-NH₂ dissolves fibrillised β-amyloid after 12 days incubation at a concentration of 50μM, whereas native sequence inhibitors have no effect.

Figure 7 shows hippocampal post-mortem sections from an Alzheimer's patient (1, 3 and 5) and a normal male (2, 4 and 6) incubated with biotinylated Imaging Agent 1 (1 and 2) and an adjacent section stained with biotinylated 6E10 antibody to β-amyloid (3 and 4). Control sections incubated in the absence of Imaging Agent or antibody are also shown (5 and 6). Staining was with peroxidase- avidin.

Figure 8 shows hippocampal post-mortem sections from an 80-year old Alzheimer's patient (A, B and D) and a normal 20-year old female (C) incubated with biotinylated Imaging agent 2 containing the retroinverso sequence FFVLK (A, B, C) and an adjacent section stained with biotinylated 6E10 monoclonal antibody to beta-amyloid (D). Staining was with peroxidase-avidin. Amyloid deposits are clear in A, B and D. Figure 9 shows the toxicity of Imaging Agent 1 after incubation for 48hrs with human SHSY-5Y cells with and without aggregrated β-amyloid (50μM). It demonstrates that Imaging Agent 1 is not toxic to these neuronal cells at up to 30μM, and exhibits some protection against amyloid-induced toxicity up to 50μM. Figure 10 shows a measure of binding affinity for biotinylated Imaging

Agents 1 and 2 (referred to as Rl and R2 respectively in the figure) for amyloid beta (1-42 and 1-40). Binding of Imaging Agent 1 to Aβl-40 was non-saturating up to ImM (A) whereas binding of Imaging Agent 2 to Aβl-40 has a kD of about 5 x 10^" ⁷M (B). Saturation of binding to Aβl-42 was observed at 0.1M (C). An expanded view at lower concentrations is shown in (D), from which the Kd was found to be approximately 1x10^"8M.

Figure 11 illustrates the MRI-T2 response in vivo from a 2576 Transgenic APP mouse 2 hours after injection (iv-tail vein) of 0.5mg Imaging Agent 1. Plaques visualise as hypointense patches in the T2-derived image. The plaques indicated by arrows are not seen at 1 hour after intravenous injection.

Figure 12 compares binding to amyloid beta (1-42) preparations by a variety of peptides, including Imaging Agent 2. Peptides are 1. AC-Lys(biotin)-Darg-Gly- Dphe-Dphe-Dval-Dleu-Dlys-Gly-Darg-CONH2 (biotinylated Imaging Agent 1 without the diamine and DOTA-Gd complex); 2. DOTA (Gd)-DOT A-Darg-Gly- Dphe-Dphe-Dval-Dleu-Dlys-Lys(Biotin)-Darg-Darg-Darg-Darg-Darg-Darg-NH2 (biotinylated Imaging Agent 2); 3. Biotin-Lys-Lys-Leu-Val-Phe-Phe- AIa-COOH (natural binding sequence in L conformation); 4 and 5. Darg-Darg-Darg-Darg-Darg- Darg-Lys(biotin)-Gly-Dthr-Dval-Dala-Gly-Darg-NH2 (designed for binding alpha synuclein); 6. (Gd)-DOT A-Darg-Sar-Dval-Dval-Dala-Sar-Darg-Darg-Darg-Darg- Darg-Darg-NH2; 7. Abri peptide; 8. PBS. Imaging Agent 2 (2) shows binding which is higher than that of other agents, and has greater binding to aged, fully fibrillar sample of amyloid beta (1-42) than to fresh partly- fibrillar samples. Detailed Description of the Invention

Agent

The present invention provides an agent that binds to Aβ, and in particular to Aβ aggregates. The agent comprises an amyloid-binding domain which comprises the synthetic peptide sequence DPhe-DPhe-DVal-DLeu-DLys and a transport domain comprising an amine or guanidine. The agent may optionally comprise a detectable label, such as a contrast agent. Labelled agents of the invention are useful in detecting Aβ aggregates and are useful in the diagnosis of AD, particularly mild to moderate AD. Agents of the invention, which need not comprise a detectable label, are also useful in the therapeutic treatment of AD.

Amyloid-binding domain

The amyloid-binding domain in an agent of the invention comprises the synthetic peptide sequence DPhe-DPhe-DVal-DLeu-DLys. The amyloid-binding domain may consist essentially of the sequence DPhe-DPhe-DVal-DLeu-DLys, or may consist essentially of the peptide sequence DPhe-DPhe-DVal-DLeu-DLys.

The synthetic peptide sequence may comprise additional amino acids. The total length of the amyloid-binding domain peptide may be from about 5 to about 30 amino acids, for example, about 6 to about 25, about 7 to about 20, about 8 to about 15, about 9 to about 14 or about 10 to about 12 amino acids.

The additional amino acids present in the amyloid-binding domain may serve to extend the retroinverse Aβ sequence. The total length of the Aβ retroinverse sequence is generally less than 10 amino acids, i.e. the total length of the Aβ retroinverse sequence may be 5, 6, 7, 8 or 9 amino acids in length.

The synthetic peptide sequence may additionally include amino acids which are not part of an Aβ retroinverse sequence. Such amino acids may be present in the D-configuration or the L-confϊguration. Preferred additional amino acids include amino acids that induce dispersal of amyloid oligomers. For example, the additional amino acids may include 1 or more, such as 2, 3, 4, 5 or 6 basic amino acid residues and/or bulky amino acid residues, such as arginine (Arg(R)) or proline (Pro(P)). In one embodiment, the addition of a C- and/or N-terminal DArg residue is preferred. Glycine (GIy(G)) residues may optionally be used as linkers between the retroinverse sequence and the basic and/or bulky amino acid residues. The additional amino acid residues may be present at the N-terminal and/or C-terminal end of the retroinverse peptide sequence. Preferred Amyloid domains of the invention include: - DArg-Gly-DPhe-DPhe-DVal-DLen-DLys-Gly-DArg-Gly;

DArg-Gly-DPhe-DPhe-DVal-DLen-DLys-Gly; DArg-Gly-DPhe-DPhe-DVal-DLeu-DLys-DArg.

The N-terminus and/or the C-terminus of the peptide may be substituted with, for example, an acetyl or amine group. In one preferred embodiment, the peptide comprises an acetyl group at the N-terminus and/or an amide group at the C- terminus.

Transport Domain

The transport domain may comprise or consist of any compound which facilitates transport of the peptide across the blood brain barrier. Preferred transport domains comprise guanidine and/or arginine groups, hi one preferred embodiment, the transport domain comprises a diamine or polyamine. The polyamine typically comprises 3, 4, 5 or 6 amines, preferably 2 or 3 amines. The polyamines may be synthetic or naturally occurring. The polyamine is typically one capable of interacting with the polyamine transporter at the blood brain barrier. Useful naturally occurring polyamines include putrescine, spermidine, 1,3-diaminopropane, norspermidine, spermine, syn-homospermidine, thermine, thermospermine, caldopentamine, homocaldopentamine and canavalmine. hi one preferred embodiment, the polyamine is pentadiamine. In an alternative embodiment, the transport domain may comprise a diguanidine or polyguanidine. The polyguanidine may comprise from 3 to 10 guanidines, for example, 4, 5, 6 or 7 guanidines. In one preferred embodiment, hexaarginine, such as hexa-Darginine, is used as a transport signal. Tetraarginine and pentaarginine may also be used as transport signals. The suitability of a transport signal for inclusion in an agent of the invention may readily be determined by a person skilled in the art. For example, the blood brain barrier permeability of an agent comprising a potential transport signal may be determined in an experimental animal, such as a mouse, by quantifying the permeability co-efficient X surface area (PS) product for each protein. Typically PS is measured after correction for the residual plasma volume (V_p) occupied by the protein in blood vessels in different brain regions following an intravenous bolus injection.

The transport signal may be present at either the N-terminal end or at the C- terminal end of the amyloid-binding peptide, hi one preferred embodiment, the transport signal is present at the C-terminal end.

The amine or guanidine transport signal may be attached to the peptide by any suitable method, for example, by chemical cross-linking. Suitable cross-linkers are well known in the art. One such method is described in Example 2 herein.

Detectable Label

The amyloid-binding peptide of the invention may be labelled to facilitate imaging of a Aβ aggregates. The peptide may, for example, include a detectable label at the C-terminus and/or at the N-terminus. hi one preferred embodiment, the detectable label is present at the N-terminus. The detectable label is typically one which enables the detection of the peptide when bound to Aβ aggregates. The Aβ aggregates may be present in the brain of a living mammal or in a post-mortem brain sample. Useful labels include radiolabels and contrast agents, preferably ones suitable for use in humans.

Suitable radiolabels include ¹⁸F, ¹²³1, ¹¹¹In, ¹³¹I, ^99mTc, ³²P, ¹²⁵1, ³H, ¹⁴C and

RL. Suitable contrast agents include rare earth ions such as gadolinium (Gd), dysprosium and iron. Other examples of such contrast agents include a number of magnetic agents paramagnetic agents and ferromagnetic or superaramagnetic agents, such as particles.

Other labels that may be used include fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography ("PET") scanner, chemiluminescers such as luciferin and enzymatic markers such as peroxidase or phosphatase. Short- range radiation emitters, such as isotopes detectable by short-range detector probes can also be employed. Pep tides of the invention may be labelled using standard techniques. For example, the peptides may be iodinated using l,3,4,6-tetrachloro-3α,6α- diphenylglycouril or chloramine T.

Chelates (e.g., EDTA, DTPA and NTS chelates) can be used to attach (and reduce toxicity) of some paramagnetic substances (e.g., Fe⁺³, Mn⁺², Gd⁺³). Peptides can be labelled with gadolinium ions, for example, by conjugating a low molecular Gd chelate such as l,4,7,10-tetraazacyclododecane-l,4,7-tris(acetic acid-t-butyl ester)- 10-acetic acid (DOTA) or diethylene triamine pentaacetic acid (DTPA) to the peptide. Accordingly, in one embodiment an agent of the invention may comprise the peptide DPhe-DPhe-DVal-DLeu-DLys, a transport signal and a low molecular weight chelate.

In one embodiment of the invention, the detectable label may be one which is suitable for detection by microscopy, such as electron microscopy, confocal microscopy or light microscopy. The detectable label may, for example, be biotin, a fluorescent compound, such as green fluorescent protein, or a peptide tag, such as a his tag, myc or flag.

Imaging Methods

Agents of the invention comprising a detectable label are useful in methods of imaging Aβ aggregates. Accordingly, the present invention provides a method of imaging β-amyloid aggregates, which method comprises detecting the binding of an agent of the invention to Aβ aggregates.

The presence or absence of the Aβ aggregates may be detected in the brain in vivo using any suitable imaging techniques. In such embodiments, the method may further comprise administering an agent of the invention to a subject. The subject is typically a mammal, preferably a human. The subject may be an experimental animal and, in particular, an experimental animal model of AD. Animal models of AD are known in the art and include transgenic mice and transgenic Drosophilia. Suitable imaging techniques 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 a preferred method because the spatial resolution and signal-to-noise ratio provided by MRI (30μm) is suitable for detecting amyloid deposits.

Magnetic Resonance Imaging (MRI) uses NMR to visualise internal features of a living subject, and is useful for prognosis, diagnosis, treatment, and surgery. MRI can be used without radioactive tracer compounds for obvious benefit. Some MRI techniques are summarised in published European patent application EP-A-O 502 814. Generally, the differences related to relaxation time constants Tl and T2 of water protons in different environments is used to generate an image. However, these differences can be insufficient to provide sharp high resolution images. The differences in these relation time constants are enhanced by contrast agents.

The presence or absence of the Aβ aggregates may also be detected in vitro, for example, in experiments designed to identify agents that inhibit Aβ aggregates. Agents of the invention may also be used to detect Aβ aggregates in brain sections from experimental animals or in post-mortem brain sections from a human subject. In such embodiments, the imaging method may be microscopy, such as electron microscopy, confocal microscopy or light microscopy.

Agents of the invention may be used in methods of diagnosing AD. hi one preferred embodiment, agents of the invention are useful in diagnosing mild or moderate AD. Diagnosis of AD in the mild or moderate stage is currently difficult because it relies on complex psychiatric profiling. Use of a labelled agent of the invention as an MRI-imaging agent will allow a decisive diagnosis to be made at early stages of the disease, when protective therapy can be instituted before widespread destruction of the brain has occurred. As a number of therapeutics are coming through trials for the purpose of ridding the brain of amyloid deposits, imaging (in particular MRI) using an imaging agent of the invention will provide a way of tracking the effectiveness of therapy.

In one embodiment, the invention provides a method for diagnosing AD in a subject, the method comprising determining the presence or absence of Aβ aggregates, wherein the presence of Aβ aggregates indicates that the subject has AD. The absence of Aβ aggregates indicates that the subject does not have AD. The images obtained from a subject may be compared to images taken from control subjects who do not have AD and/or to images from other subjects known to have AD in order to reach or confirm a diagnosis.

A method of diagnosing AD of the invention typically comprises administering a detectably labelled agent of the invention to a subject; imaging the brain of said subject to detect any of said agent bound to Aβ aggregates; and determining the presence or absence of Aβ aggregates. An agent of the invention is administered to a subject in need of diagnosis in an amount sufficient to bind to any Aβ aggregates and be detected by imaging techniques, such as MRI. The invention also provides methods for monitoring the status of AD in a subject. The methods may, thus, be used to determine disease progression. For example, the methods may be used to monitor growth of amyloid plaques in the brain of a subject. The method may also be used to monitor the effectiveness of therapy and/or to evaluate the efficacy of new AD treatments. A subject may be tested on a regular basis, for example monthly, six monthly or yearly, to monitor disease progression within the subject.

Thus, in a further embodiment, the present invention provides a method for monitoring AD in a subject, the method comprising determining the presence or absence of Aβ aggregates in the brain of the subject by detecting binding of an agent of the invention to the Aβ aggregates. The images are typically compared to one or more image taken from the same subject at an earlier time point.

The number and/or size of Aβ aggregates present in the brain of a subject correlates with disease progression. An increase in the number and/or size of Aβ aggregates indicates a progression of AD. Conversely, a decrease in the number or size of Aβ aggregates indicates disease regression. Where no change is observed in the number and/or size of Aβ aggregates, the disease is in a steady state. Where the monitoring method is determine the efficacy of a treatment for AD, maintenance of a steady state or a decrease in the number or size of Aβ aggregates typically indicates that the treatment is successful. Levels of Aβ aggregates may be compared to standards to determine AD status. Therapeutic Methods

The invention provides a method of treating AD by administering a therapeutically effective amount of an agent of the invention to a subject in need thereof. A subject in need in treatment is typically a subject having AD. The subject is generally human, but any other animal such as another mammal, for example a rodent, such as a rat or mouse. In one embodiment, the subject may be an animal which serves as a model for AD. Animal models of AD are well known in the art and include transgenic mice, such as the APPSW transgenic mouse (2576) model of AD, developed in 1996 by Karen Hsiao, University of Minnesota. In a method of treating AD, a therapeutically effective amount of the agent is administered to an individual in need thereof. A therapeutically effective amount is an amount effective to alleviate or attenuate one or more symptom of AD, when administered to treat AD. When administered as a prophylactic treatment of AD, a therapeutically effective amount is an amount effective to prevent the onset of one or more symptom of AD. Typically, a prophylactically effective amount is an amount that delays or inhibits the formation of plaques.

Formulation and Administration

The formulation of any of the agent will depend upon factors such as the nature of the agent and the condition to be treated. Any such agent may be administered or delivered in a variety of dosage forms. It may be administered or delivered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion or inhalation techniques. The agent may also be administered or delivered as suppositories. A physician will be able to determine the required route of administration or delivery for each particular patient.

The agent may be administered directly to the site of a plaque, typically by injection into a blood vessel supplying the brain or into the brain itself. Typically the agent is formulated with a pharmaceutically acceptable carrier or diluent. The invention provides a pharmaceutical composition comprising an agent of the invention and a pharmaceutically effective diluent or carrier. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about 0.1mg/kg to lOmg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Kits The invention also provides kits for carrying out the diagnostic and monitoring methods of the invention. The kit may comprise an imaging agent of the invention and means for administering the imaging agent to a subject. Means for administering the agent may comprise or consist of a sterile syringe. Instructions for using the kit to monitor or diagnose AD may also be included. The following Examples illustrate the invention.

Example 1 : Retroinverse inhibitor of β-amyloid aggregation

The peptide Acetyl-rGffvlkr-NH₂ (RI-Omar2), containing the retro-inverse recognition sequence in β-amyloid, where residues in lower case are amino acid residues in D-configuration, was synthesised as follows.

The sequence was assembled on Fmoc PAL-PEG-Polystyrene resin (Applied Biosystems) by coupling, in succession, D-Arg, D-Lys, D-Leu, D-VaI, D-Phe,D- Phe,Gly,D-Arg, with PyBOP and HATU by the methods described in El-Agnaf O, Sheridan J, Goodwin H & Austen BM (2000) Improved Solid-Phase syntheses of Amyloid Proteins associated with neurodegenerative Diseases. Peptide and Protein Letters 7; 1-8. The N-terminus was acetylated by treatment with acetylimidazole 4- fold molar excess. The target peptide was cleaved and deprotected in TFA, triisopropylsilane (5% by vol) and water (5% by vol), triturated in ether, and purified by HPLC on a column of Dynamax C8, in 0.1% TFA with an acetonitrile gradient. The purified peptide was characterised by Maldi (Krauts Axima CFR) Mass spectrometry analysis (MH=I 121).

Example 2: Retro-inverse inhibitor/imaging agent: Imaging Agent 1

The peptide (l,4,7,10-tetraazacyclododecane-l,4,7-tris(acetic acid-t-butyl ester)- 10-acetic acid) (DOTA)-rGffvlkGrG-pentadiamine, incorporating the DOTA ligand for complexing gadolinium at the N-terminus of the retro-inverse recognition sequence, and a pentadiamine at the C-terminus to enable passage across the blood- brain barrier (Imaging Agent 1; Figure 1) was synthesised as follows.

The synthetic procedure was begun with a reductive amination of formyl polystyrene resin that leads to a polyamine substitution on the COOH-terminus of a peptide. 4- {4-Formyl-3-methoxyphenoxy} butyric acid (FMBP) NovaGel TM resin (0.55 mequivs/g)(Nova Biochem) was suspended in dichloroethane (8ml) and trimethylortho formate (8ml) under helium agitation. Mono-1-Butoxycarbonyl, 1,5- diminopentane (Tosyl salt) (Nova Biochem) (Ig) was added along with sodium acetoxy borohydride (0.6g) and dimethyl formamide (6ml). The mixture was stirred overnight, and the resin washed with excess dimethyl formamide, followed by 10%(v/v) diisopropylethylamine in dimethylformamide, and then dimethylformamide alone. Fmoc-Glycine (1.2g) and O-(7-Azabenzotriazol-l-yl) 1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU) (1.5g) was added and stirring continued overnight. Mass spectrometry (spec) analysis of a small portion of the resin-complex gave a compound of mass 385, the same as expected from FmocGly-(α-tBoc- diamino-pentane.

After Fmoc deprotection with 20% piperidine, the following residues were coupled from the C-terminus to the N-terminus, using double coupling with HATU for each position, as described by El-Agnaf O, Sheridan J, Goodwin H & Austen BM (2000) Improved Solid-Phase syntheses of Amyloid Proteins associated with neurodegenerative Diseases. Peptide and Protein Letters 7; 1-8: Fmoc-D-Arg(Pbf), Fmoc-Gly, Fmoc D-Lys(tBoc), Fmoc-D-Leu, Fmoc-D-Val, Fmoc-D-Phe, Fmoc-D- Phe, Fmoc-Gly, Fmoc-D-Arg(Pbf) (in 4-fold molar excess), all from Nova-Biochem, and finally DOTA (l,4,7,10-tetraazacyclododecane-l,4,7-tris(acetic acid-t-butyl ester)- 10-acetic acid) (Macrocyclics Inc.) (in 3-fold molar excess).

The modified peptide was released from the resin and deprotected with trifluoroacetic acid, triisopropyl silane (5%) and water (5%), triturated with ether, and purified on a column of Phenomenix C4 in 0.1% TFA with an acetonitrile gradient to produce the target compound DOTA-rGffvlkGrG-pentadiamine (mass spec gave MH=I 597 calc 1595). The purified peptide (44mg in 5ml water) was incubated with 4.84ml of gadolinium trichloride (25mM), pH adjusted to 7.0 with NaOH, and the gadolinium complex isolated by reverse-phase HPLC on Phenomenix C4 as above to give the required Imaging Agent (MH=1753; CaIc 1755).

Example 3: Retro-inverse inhibitor/imaging agent: Imaging Agent 2

The peptide (l,4,7,10-tetraazacyclododecane-l,4,7-tris(acetic acid-t-butyl ester)- 10-acetic acid) (DOTA)-rGffVlkGG-πτπτ, incorporating the DOTA ligand for complexing gadolinium at the N-terminus of the retro-inverse recognition sequence, and a hexa-DArginine sequence at the C-terminus to enable transfer across the blood-brain barrier was synthesised as follows.

Fmoc-P AL-PEG-PS resin (Applied Biosystems Cat No. GEN913383)) 0.2mmoles per g) was suspended in dimethyl foramide and loaded into a MELLGEN 9050 Peptide Synthesiser. After Fmoc deprotection with 20% piperidine, the following residues were coupled from the C-terminus to the N-terminus, using HATU for each position (as described by El- Agnaf O, Sheridan J, Goodwin H &

Austen BM (2000) Improved Solid-Phase syntheses of Amyloid Proteins associated with neurodegenerative Diseases" Peptide and Protein Letters 7 1-8): Six successive residues of Fmoc-D-Arg(Pbf), two residues of Fmoc-Gly, Fmoc D-Lys(Boc), Fmoc- D-Leu, Fmoc-D-Val, two residues of Fmoc-D-Phe, one of each of Fmoc-Gly, Fmoc- D-Arg(Pbf) (in 4-fold molar excess), all from Nova-Biochem, and finally t-butyl

DOTA (l,4,7,10-tetraazacyclododecane-l,4,7-tris(acetic acid-t-butyl ester)- 10-acetic acid) (Macrocyclics Inc.) (triple coupled in 2-fold molar excess).

The modified peptide was released from the resin and deprotected with a mixture of trifluoroacetic acid (90%), triisopropyl silane (5%) and water (5%), triturated with ether, and purified on a column of Phenomenix C4 in 0.1% TFA with an acetonitrile gradient to produce the target compound DOTA-rGffvlkGGrrrrrr- NH2 (lower case represents amino acids in the D configuration) (mass spec gave MH=2237 calc 2239). The purified peptide (56mg in 5ml water) was incubated with 4 ml of gadolinium trichloride (25mM) (4 fold molar excess), pH adjusted to 7.0 with NaOH, and the gadolinium complex isolated by reverse-phase HPLC on Phenomenix C4 as above, to give the required Imaging Agent (MH=2397; Calc 2396.5). Example 4: Inhibition of β-amyloid oligomer formation

Imaging Agent 1 and RI-Omar2 were assessed for inhibition of β-amyloid (1- 42) oligomer formation using the previously described ELISA assay (Sian et al, BioChem. J. 349(l):299-308, 2000). RI-Omar2 was also assessed for inhibition of β-amyloid (1-40) oligomer formation using the same assay.

The ELISA plate was coated with 50 μL/well anti-NT A4 antibody diluted in filtered PBS (pH 7.4) to 1 μg/mL. Anti-NT A4 is an affinity-purified rabbit antibody to the ten N-terminal residues of human Aβ. The plate was sealed and incubated overnight at 4°C. Antibody solution was discarded and the plate was washed three times with 100 μL/well blocking buffer (PBS [pH 7.4]), containing 0.05% v/v Tween-20 and 1% w/v gelatin). The plate was incubated for 1 hour with 100 μL/well blocking buffer.

Following incubation at 37⁰C for 15 hours, Aβ 1-42 (Figure 3) or A Aβ 1 -40 (Figure 5) peptides incubated with equimolar concentrations of inhibitor peptides at the concentrations shows in Figure 3 or 5, were diluted to 20 μg/mL in blocking buffer. Blocking buffer was removed from the wells and 50 μL/well of the peptide solutions were added to each well and incubated for 2 hours. The plate was washed with blocking buffer four times and blotted dry. After washing, 100 μL/well of the second antibody (biotinylated anti-NT A4, 1 μg/mL in blocking buffer) was added and incubated for 1.5 hours. The wells were washed three times with 100 μL/well blocking buffer. The plate was incubated for 1 hour with Extravidin-linked Horse-Radish Peroxidase (HRP) (1:1000 in PBS), 100 μL/well. After washing three times with 100 μL/well blocking buffer and once with 100 μL/well PBS, colour was allowed to develop with the addition of 100 μL/well TMB (Europa Biosciences, Cambridge, UK) at room temperature. This reaction was terminated after sufficient colour development with 25 μL of 2 M H₂SO₄ (approximately 5 minutes). The absorbance at 450 nm was read using a Multiskan microtitre plate reader.

The ELISA assay demonstrated that both Imaging Agent 1 and RI-0mar2 are highly potent at inhibiting Aβ(l -42) oligomer formation when compared to the non- reversed recognition sequence in L-configuration, KLVFF-amide (Figure 3). The ability of RI-Omar 2 (Acetyl-rGffvlkr) to inhibit Aβ(l-40) oligomer formation (monitored using the ELISA assay) was also compared to two inhibitors in clinical trials, Leu-Pro-Phe-Phe-Asp (J26) (Soto et al, Nature Med. 4:822, 1998) and Ac-Gln-Lys-Leu-Val-Phe-Phe-NH2 (AcetylAmide; Tjernberg et al, J. Biol. Chem. 271 : 8545-8548, 1996). The ELISA assay demonstrated that RI-0MAR2 is a more potent inhibitor of Aβ oligomer formation then the two known inhibitors (Figure 5).

Example 5: Inhibition of neuronal cell death The retroinverse inhibitor, RI-0mar2 was also compared in its activity in inhibiting neuronal cell death (by a dye-binding MTT assay) induced by aggregated β-amyloid 1-40 with the inhibitors Leu-Pro-Phe-Phe-Asp (J26) (Soto et al, Nature Med. 4:822, 1998) and Ac-Gln-Lys-Leu-Val-Phe-Phe-NH2 (AcetylAmide; Tjernberg et al, J. Biol. Chem. 271:8545-8548, 1996). For the MTT assay, SH-S Y5 Y cells were plated and the peptides and inhibitors were added. The peptide, Aβl-40 or Aβl-42, was diluted to the appropriate concentration with the addition of inhibitor (in OPTI-MEM). Solutions of inhibitor (at various concentrations) without peptide and solutions of peptide alone were also made in OPTI-MEM (these acted as controls). 100 μL/well of these solutions were added to the cells. Following incubation for 48 hours with the peptides and inhibitors, the solutions were removed and the cells were washed with 100 μL/well PBS. 100 μl of MTT (0.6mg/ml in OPTI-MEM) was added to each well. Plates were incubated for 4.5 hours at 37°C. 50 μl was removed from each well and replaced with 150 μl of lysis buffer (20% w/v SDS and 50% v/v N, N- dimethylformamide, pH 4.7). Plates were incubated for a further 24 hours at 37°C. Absorbance readings were taken at 570nm using the micro titre plate reader. Results were expressed as % MTT reduction compared to a 100% signal from untreated cells.

RI-Omar2 was found to be more protective than both Leu-Pro-Phe-Phe-Asp and Ac-Gln-Lys-Leu-Val-Phe-Phe-NH2, which are known to be in clinical trials (Figure 4). Example 6; Ability to dissolve β-amyloid in plaques

The ability of the retroinverse peptide RI-0mar2 to dissolve fibrillised β- amyloid in plaques was shown by the disappearance of fibrils, seen by Negative Stain Electron Microscopy (EM), after long periods of incubation (12 days) (Figure 6).

For the Negative Stain Electron Microscopy (EM), suspensions of 100 μM Aβ 1-40 fibrils were incubated with inhibitor peptides at 50μM in Tris for 12 days at 37°C, spotted onto 400 mesh copper formvar/carbon-coated EM grids (Agar Scientific, UK) and air dried for 10 minutes. Centrifuged uranyl acetate was spotted onto the same grids, air dried for 10 minutes, after which excess was removed using filter paper (Whatman). Grids were then examined using a JEOL-1010 transmission electron microscope.

Rl-Omar 2 was more potent than normal L-peptides KLVFF-amide over the same time period of incubation. Thus, retroinverse sequences are more potent than peptide inhibitors based on the recognition sequence in the natural L-configuration, and therefore possess superior properties as therapeutic agents to target AD.

Example 7; Imaging of AD plaques with Imaging Aeent 1 The binding of a biotinylated derivative of Imaging Agent 1, to senile plaques was investigated in 7μm sections of hippocampus from post-mortem sections from AD patients.

Imaging Agent 1 with gadolinium complexed was resynthesized as described in Example 2 but replacing GIy^"2 with e-Biotin-Lys. Sections of human brain taken from an AD patient and a normal 74-year old male were incubated with biotinylated Imaging Agent 1 (10 mg/ml; sections 1 and 2), biotinylated antibody 6E10 to β- amyloid (sections 3 and 4) or biotin (control sections 5 and 6) for 2 hours and developed with peroxidase-avidin (1-500) in 3% goat serum in PBS for 1 hour. The results are shown in Figure 7. The AD brain section incubated with Imaging Agent 1 (Section 1) was similar in appearance to the AD brain section incubated with biotinylated antibody 6E10 to β-amyloid (section 3), indicating that Imaging Agent 1 binds to fibrillised β-amyloid within plaques (see Figure 7). Example 8: Imaging of AD plaques with Imaging Agent 2

The binding of a biotinylated derivative of Imaging Agent 2 to senile plaques was investigated in 7μm sections of hippocampus from post-mortem sections from AD patients.

Imaging Agent 2 with gadolinium complexed was resynthesised as described in Example 3 but replacing GIy² with eBiotin-Lys. Sections of human brain taken from an 80-year old AD patient (A, B and D) and a normal 20-year old female (C) were incubated with biotinylated Imaging Agent 2 (lOμg/ml; sections A, B and C) or biotinylated antibody 6E10 to β-amyloid (section D) for 2 hours and developed with peroxidase-avidin (1-5000) with 1% gelatine and 0.5% gelatine and 0.5% Tween in PBS for 1 hours. Colour was produced with broad-spectrum DAB reagent (Zymed labs), and slides were counter-stained with haematoxylin, washed with water, and visualised under a Zeiss microscope (x20 lens). The results are shown in Figure 8. The DAB staining in the grey matter of

AD brain section incubated with Imaging Agent 2 (section A) was similar in appearance to the AD brain section incubated with biotinylated antibody 6E10 to β- amyloid within plaques (see Figure 8). Section B showed the Imaging Agent 2 binding to vascular deposits, whereas no binding of Imaging Agent 2 was observed in a section from a 20-year old control (image C).

Example 9: Binding to Amyloid Fibrils

The binding of Imaging Agent 1 to amyloid fibrils was assessed by Malditof mass spectrometry. A suspension of fibrils formed by incubation of Aβ 1-40 at pH7.6 were spotted onto a Maldi plate, followed by 5μl of a solution of Imaging Agent 1 (lμg/ml) diluted in PBS. Attachment of Imaging Agent 1 to the fibrils was shown by the appearance of the agent (M = 1763) in the mass spectrometer. Binding of Imaging Agent 1 was observed at concentrations as low as lOfg/ml, whereas a control peptide (HLAG) of similar molecular weight (M=2141) did not bind at lng/ml.

Example 10: Toxicity of Imaging Agent 1 to neuronal cells The toxicity of Imaging Agent 1 was investigated by monitoring survival of SHSY-5Y neuronal cells after incubation with the agent. The cells were incubated for 48 hours with and without aggregated β-amyloid (50μM) and survival was measured using a dye-binding MTT assay. Figure 9 shows that as little as 1 μM of the agent actually protected cells against amyloid toxicity (more living cells) and that up to 30μM agent was not toxic to the cells. At 50μM, the agent exhibits some protection against amyloid-induced toxicity.

Example 11 : Binding affinity of Imaging Agent to Amyloid beta To find the binding affinity of Imaging Agent 1 and 2 to amyloid beta the agents,_with gadolinium complexed, were resynthesized as described in Example 2 but replacing GIy^"2 (Imaging Agent 1) and GIy⁸ (Imaging Agent 2) with e-Biotin- Lys. Binding affinity of the agent to amyloid was obtained from a plate assay in which Amyloid beta fibrils (fibrillised by incubation at 37⁰C for 4 days) were attached to a 96-well plate then incubated for 2 hours at 37⁰C with each imaging agent diluted into 0.5% tween and 1% fish gelatine. Detection was by avidin- peroxidase.

Binding of Imaging Agent 1 to amyloid beta (1-40) was non-saturated up to ImM (A). Figure 10 shows that binding of Imaging Agent 2 to amyloid beta (1-40) has a Kd of about 5x10^"7M (B). Saturation of binding of Imaging Agent 2 to 1-42 was observed at 0.ImM in (C). An expanded view at lower concentration is shown in (D), from which the Kd was found to be approximately Ix 10^"8M.

Binding affinity of Imaging Agent 2 and a slightly modified version of Imaging Agent 1 to amyloid beta was also tested in comparison to other agents (Figure 12). lOOpmo I/well of each agent was added to wells containing amyloid beta (1-42) aggregates (from dmso plus pbs original) at 200pmol/well as follows: 1. AC-Lys(biotin)-Darg-Gly-Dphe-Dphe-Dval-Dleu-Dlys-Gly-Darg-CONH2 (biotinylated Imaging Agent 1 without the diamine and DOTA-Gd complex); 2. DOTA (Gd)-DOTA-Darg-Gly-Dphe-Dphe-Dval-Dleu-Dlys-Lys(Biotin)-Darg-Darg- Darg-Darg-Darg-Darg-NH2 (biotinylated Imaging Agent 2); 3. Biotin-Lys-Lys-Leu- Val-Phe-Phe- AIa-COOH (natural binding sequence in L conformation); 4 and 5. Darg-Darg-Darg-Darg-Darg-Darg-Lys(biotin)-Gly-Dthr-Dval-Dala-Gly-Darg-NH2 (designed for binding alpha synuclein); 6.(Gd)-DOTA-Darg-Sar-Dval-Dval-Dala- Sar-Darg-Darg-Darg-Darg-Darg-Darg-NH2; 7. Abri peptide; 8. PBS.

Imaging Agent 2 bound strongly with selective discrimination for aged samples of amyloid, whereas Imaging Agent 1 bound weakly. The hexa-Arg brain penetration sequence (present in Imaging Agent 1 and not in Imaging Agent 2) appears to increase affinity for the FFVLK sequence to amyloid beta, possibility as a kosmotrope, increasing surface tension and hydrophobic intereactions involved in binding to amyloid beta (Gibson TJ and Marphy RM (2005) Biochemistry 44; 8898- 8907).

Example 12: Imaging plaques in vivo

The potential for imaging plaques in Alzheimer's patients has been assessed in a 12-month old APP transgenic mouse (2576) model of AD, developed in 1996 by Karen Hsiao, University of Minnesota. Imaging Agent 1 (0.5mg) in 0.5 ml of PBS was injected intravenously into the tail vein of an anaesthetised 2576 mouse. Tl and T2 images of the brain were taken at intervals over 4 hours. 2 hours after injection, dark spots corresponding to expected β-amyloid plaques were seen in the cortex of the mouse, particularly in CAl of the hippocampus (Figure 11). No dark spots were seen either 1 hour or 4 hours after injection.

These preliminary results suggest that Imaging Agent 1, and compounds of similar structure, will be useful both for the MRI diagnosis of AD in humans, and for monitoring the appearance or disappearance of amyloid plaques, thought to be causative of neuronal dysfunction in AD.

Claims

I . An agent comprising the synthetic amino acid sequence DPhe-DPhe- DVal-DLeu-DLys and an amine or guanidine substituent.

2. An agent according to claim 1, which further comprises a basic amino acid residue.

3. An agent according to claim 2, wherein the basic amino acid residue is D-Arg.

4. An agent according to claim 2 or 3, wherein the basic amino acid residue is linked to said amino acid sequence via a GIy residue.

5. An agent according to any one of the preceding claims comprising the amino acid sequence DArg-Gly-DPhe-DPhe-DVal-DLeu-DLys-Gly.

6. An agent according to any one of the preceding claims comprising the amino acid sequence DArg-Gly-DPhe-DPhe-DVal-DLeu-DLys-Gly- DArg-Gly.

7. An agent according to any one of claims 1 to 4 comprising the amino acid sequence DArg-Gly-DPhe-DPhe-DVal-DLeu-DLys-DArg.

8. An agent according to any one of the preceding claims, wherein the amine or guanidine substituent is present at the C terminus.

9. An agent according to any one of the preceding claims, wherein the amine substituent is a diamine or polyamine.

10. An agent according to claim 8, wherein the amine substituent is pentadiamine.

I I. An agent according to any one of claims 1-7, wherein the guanidine substituent is hexa-DArginine.

12. An agent according to any one of the preceding claims further comprising a detectable label.

13. An agent according to claim 12, wherein the detectable label is a contrast agent.

14. An agent according to claim 12, wherein the contrast agent comprises gadolinium ions.

15. An agent according to any one of the preceding claims further comprising a substituent capable of complexing to an imaging agent.

16. An agent according to claim 15, wherein the agent capable of complexing to an imaging agent is DOTA.

17. An agent according to any one of claims 12 to 16, wherein the imaging agent and/or the substituent capable of complexing to an imaging agent is present at the N terminus.

18. Use of an agent according to any one of claims 1 to 17 in a method of disrupting β-amyloid aggregates.

19. An agent according to any one of claims 1 to 17 for use in a method of treating the human or animal body by therapy or in a diagnostic method practised on the human or animal body.

20. An agent according to any one of claims 1 to 17 for use in a method of treating or diagnosing AD.

21. Use of an agent according to any one of claims 1 to 17 in the manufacture of a medicament for the treatment or diagnosis of AD.

22. A method of treating AD, said method comprising administering to a subject in need thereof a therapeutically effective amount of an agent according to any one of claims 1 to 17.

23. A pharmaceutical composition comprising an agent according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier or diluent.

24. Use of an agent according to any one of claims 12 to 17 in a method of imaging β-amyloid aggregates.

25. A method of imaging β-amyloid aggregates, said method comprising detecting the binding of an agent according to any one of claims 1 to 17 to β-amyloid aggregates.

26. A method according to claim 25, wherein the β-amyloid aggregates are present in a human or animal subject.

27. A method according to claim 26, further comprising administering an agent according to any one of claims 1 to 17 to said subject.

28. A method according to any one of claims 25 to 27, wherein said agent is detected by magnetic resonance imaging (MRI).

29. A method of diagnosing AD, said method comprising administering an agent according to any one of claims 1 to 27 to a subject and detecting the presence of β-amyloid aggregates, wherein the presence of β-amyloid aggregates indicates that the subject has AD.

30. A method of monitoring AD, said method comprising administering an agent according to any one of claims 1 to 17 to a subject and detecting the presence of β-amyloid aggregates.

31. A method according to claim 30, wherein said subject is undergoing therapy to treat AD and said method is for monitoring the effectiveness of said therapy.

32. A kit for imaging Aβ aggregates, said kit comprising a detectably labelled agent of the invention and means for administering the agent to a subject.