WO2004055201A2

WO2004055201A2 - Cholesterol 24-hydroxylase (cyp46) as therapeutic target for the treatment of alzheimer's disease

Info

Publication number: WO2004055201A2
Application number: PCT/EP2003/013760
Authority: WO
Inventors: Yan-Hong Liu; Gerhard König; Stefan Golz; Martin GÜLDENAGEL
Original assignee: Bayer Healthcare Ag
Priority date: 2002-12-13
Filing date: 2003-12-05
Publication date: 2004-07-01
Also published as: WO2004055201A3; AU2003288231A1; AU2003288231A8

Abstract

The invention relates to the novel association of cholesterol 24-hydroxylase (CYP46) to Alzheimer's disease. The invention also relates to novel methods of screening for therapeutic agents for the treatment of the disease and relates to pharmaceutical compositions for the treatment of Alzheimer's disease comprising regulators of Cholesterol 24-hydroxylase (CYP46) or activity.

Description

Cholesterol 24-hydroxylase (CYP46) as therapeutic target for the treatment of Alzheimer's disease

Technical Field of the Invention

The invention relates to human Cholesterol 24-Hydroxylase (CYP46) which is associated with Alzheimer's disease. The invention provides assays for the identification of compounds useful in the treatment or prevention of Alzheimer's disease. The invention also features compounds which bind to and/or activate or inhibit the activity of cholesterol 24-hydroxylase (CYP46) as well as pharmaceutical compositions comprising such compounds.

Background of the Invention:

Alzheimer's disease (AD) is the most prevalent form of all neurodegenerative disorders. Approximately 100,000 victims die and 360,000 new cases of Alzheimer's disease are diagnosed each year. To date, there is no effective treatment for Alzheimer's disease. Research has suggested a number of possible approaches to treatment, such as Cholinergic strategies: Acetylcholinesterase inhibitors (e.g., Tacrine, Cognex, or Exelon) and Ml muscarinic receptor agonists; Neurotrophic factors (e.g., Nerve growth factor); Inhibitors of oxidation (e.g., vitamin E); Metal chelating agents; Immunotropic drugs; Non-narcotic analgesics (e.g., Ibuprofen); Inhibitors of beta-A4 aggregation; Estrogen, etc. But so far, none of these approaches has been clearly demonstrated to cause a significant improvement in the majority of Alzheimer's disease sufferers. Thus, a long felt and high medical need exists for new drugs with a novel mode of action for the treatment of Alzheimer's Disease.

Alzheimer's disease is the most common cause of dementia in the aged population. Epidemiological studies show that high blood cholesterol levels correlate with a higher risk of developing AD [M. Kivipelto et al., Neurology 56: 1683-1689, 2001]; and indeed, patients treated with cholesterol-lowering statins become protected against the disease [H. Jicl et al., Lancet 356:1627-1631, 2000; B. Wolozin et al., Arch. Neurol. 57: 1439-1443, 2000].

The brain is the most cholesterol-rich organ in the human body and disturbances in cholesterol metabolism may lead to severe neurological disorders. Thus, the regulation of central cholesterol turnover is crucial for a proper brain function. The presence of the blood-brain barrier (BBB) impedes the direct transport of cholesterol out of the central nervous system (CNS). A small amount of cholesterol is transported from the brain to the cerebrospinal fluid (CSF) via an apolipoprotein E (APOE)-dependent mechanism [Pitas et al., Biochim Biophys Acta 917 (1): 148- 161, 1987]. The enzymatic conversion of CNS cholesterol to 24S-hydroxycholesterol, which readily crosses the BBB, is the major pathway for the elimination of brain cholesterol and the maintenance of brain cholesterol homeostasis [Lutjohann et al., Proc Natl Acad Sci USA 93 (18 ): 9799-9804. 1996; Bjόrkhem et al, J Biol Chem. 272 (48): 30178-30184.1997; Bjorkhem et al, J Lipid Res. 39 (8): 1594-1600. 1998; Meaney et al, J Lipid Res. 42 (1): 70-78. 2001]. The enzyme mediating this conversion was recently identified as cholesterol 24-hydroxylase, representing a new cytochrome P450 subfamily (CYP46) [Lund et al, Proc Natl Acad Sci USA 96 (13 ): 7238- 7243.1999].

The current invention provides a new mechanism for the treatment of Alzheimer disease, namely, through regulation of cholesterol 24-hydroxylase activity to maintain the brain cholesterol homeostasis.

Brief Description of the Figures

Ez

Fig 1. Shows the relative expression of cholesterol 24-hydroxycholesterol (CYP46) in different brain regions of aged wildtype and tg2576 mice. _A

Fig. 2

Fig. 2 shows the neurotoxicity of 24-hydroxycholesterol measured by lactate dehydrogenase (LDH) release. LDH measurement is a colorimetric assay for the quantification of cell death and cell lysis, based on the measurement of (LDH) activity released from the cytosol of damaged cells into the supernatant.

Fig. 2 a): LDFf measured in medium of primary mouse cortical neurons after 24 hours incubation with 25μM cholesterol, 24-OH cholesterol, 25-OH cholesterol, and 10 mM camptothecin (as positive control).

Fig. 2 b): LDH measured in medium of primary mouse cortical neurons after 48 hours incubation with 25μM cholesterol, 24-OH cholesterol, 25-OH cholesterol, and 10 mM camptothecin (as positive control, incubated for 24 hours).

Fig-1

Fig 3 shows the Caspase 3 activity induced by oxycholesterols. Fig. 3 a) shows the Caspase-3 activity measured in primary mouse cortical neurons after 24 hours incubation with 25 μM 24-OH cholesterol, 25-OH cholesterol, cholesterol, and 10 mM camptothecin (positive control).

Fig 3 b) despicts the Caspase-3 activity measured in primary mouse cortical neurons after 48 hours incubation with 25 μM 24-OH cholesterol, 25-OH cholesterol, cholesterol, and 10 mM camptothecin (positive control, incubated for 24 hours)

Fig. 4

Fig. 4 shows the nucleic acid sequence of human cholesterol 24-hydroxylase, CYP46 (SEQ ID NO: 1 , Ace. No. NM_00068). The ORF is depicted in bold and is underlined.

Fig. 5

Fig. 5 shows the amino acid sequence of human cholesterol 24-hydroxylase, CYP46 (SEQ LD NO: 2).

Fig. 6

Fig. 6 shows the sequence of the human CYP46 forward primer (SEQ ID NO: 3).

Fig. 7

Fig. 7 shows the sequence of the human CYP46 probe (SEQ ID NO: 4).

Fig. 8 shows the sequence of the human CYP46 reverse primer (SEQ ID NO: 5).

Fig. 9

Fig. 9 shows the sequence of the mouse CYP46 forward primer (SEQ ID NO: 6).

Fig- 10

Fig. 10 shows the sequence of the mouse CYP46 probe (SEQ ID NO: 7).

Fig- 11

Fig. 11 shows the sequence of the mouse CYP46 reverse primer (SEQ ID NO: 8). Fig. 12

Fig. 12 shows the sequence of the mouse forward β-actin primer (SEQ ID NO: 9).

Fig. 13

Fig. 13 shows the sequence of the mouse β-actin probe (SEQ ID NO: 10).

Fig. 14

Fig. 14 shows the sequence of the mouse β-actin reverse primer (SEQ LD NO: 11).

Fig. 15

Fig. 15 shows the nucleic acid sequence of mouse cholesterol 24-hydroxylase, CYP46 (SEQ ID NO: 12, Ace. No. NM_010010)

Detailed Description of the Invention

It was previously shown that the concentration of 24-hydroxycholesterol in plasma of Alzheimer patients was higher than that of healthy subjects [Lϋtjohann et al, J. Lipid Res*. 41: 195-198, 2000]. Cholesterol 24-hydroxylase (CYP46) catalysing the 24-hydroxylation of cholesterol was mainly detected in the brain (Lund et al.,1999), where it is almost exclusively located to neuronal structures in the brain. An abnormal induction of this enzyme in glial cell was observed in patients with Alzheimer's disease [Bogdanovic N et al. Neuroscience Letters 314: 45-48. 2001].

Interestingly, when analysing the expression profile using the TaqMan (quantitative real-time RT- PCR) technique, we found that the expression of cholesterol 24-hydroxylase (CYP46) is dramatically decreased in the postmortem brain of Alzheimer's disease patients. Similarly, we observed a decrease of cholesterol 24-hydroxylase (CYP46) expression in the cortex and hippocampus of aged transgenic mice (tg2576) which are recognised as^' Alzheimer's disease models (table 1 and fig. 1).

24-hydroxycholesterol, the brain cholesterol elimination product, is the major oxysterol synthesised in the brain. It appears to play a major role in cholesterol homeostasis. It was also described that pathologically increased levels of 24-hydroxycholesterol result in neuronal cell death [Kδlsch et al. Brain Res. 818: 171-175, 1999], thus 24-hydroxycholesterol may be a risk factor in neurodegenerative disorders. Here, in primary cortical neuron cultures, we found that 24-hydroxycholesterol exerts an neurotoxic effect on the cells. For example, administration of 24-hydroxycholesterol increased the LDH release of primary neurons which indicates decreased cell viability. This neurotoxic effect was also found not to be caspase-3 mediated apoptosis in contrast to the proapoptotic effect of 24- hydroxycholesterol on the neuroblastoma cell line SH-SY5Y (figs. 2 and 3).

Thus, in Alzheimer's Disease patients, or in patient with other neurodegenerative disorders with low brain levels of CYP46 or CYP46 activity, increasing the CYP46 activity might result in ameliorating the symptoms of Alzheimers disease or of the neurodegenerative disorder. On the other hand, in Alzheimer's disease patients or in patients with other neurodegenerative disorders with high levels of CYP46, inhibiting the CYP46 activity may result in ameliorating the symptoms. Other neurodegenerative disorders are, but are not limited to Parkinson's Disease, Niemann Pick type C disease, or amyotrophic lateral sclerosis (ALS).

This invention relates to the use of human cholesterol 24-hydroxylase (CYP46) as a therapeutical target for the treatment or prevention of neurodegenerative disorders, preferably Alzheimer's disease.

An object of the invention is a method of screening for therapeutic agents useful in the treatment and/or prophylaxis of neurodegenerative disorders, preferably Alzheimer's disease in a mammal comprising the steps of

i) contacting a test compound with a CYP46 polypeptide,

ii) detect binding of said test compound to said CYP46 polypeptide,

wherein those compounds are selected as potential therapeutic agents which bind to the CYP46 polypeptide.

Another object of the invention is a method of screening for therapeutic agents useful in the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a mammal comprising the steps of

i) determining the activity of a CYP46 polypeptide at a certain concentration of a test compound, ii) determining the activity of said polypeptide in the absence or at a different concentration of said test compound,

wherein those compounds are selected as potential therapeutic agents, for which the activity of a CYP46 polypeptide as determined in i) and ii) is significantly different.

A further object of the invention is a method of screening for therapeutic agents useful in the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a mammal comprising the steps of

i) determining the activity of a CYP46 polypeptide at a certain concentration or certain concentrations of a test compound,

ii) determining the activity of a CYP46 polypeptide at the presence of a compound known to be a regulator of a CYP46 polypeptide,

wherein those compounds are selected as potential therapeutic agents, which show an effect on the activity of a CYP46 polypeptide as determined in i), and step ii) serves as a control.

Another object of the invention is the method of any of the screening methods mentioned above, wherein the step of contacting is in or at the surface of a cell.

Another object of the invention is the method of any of the screening methods mentioned above, wherein the cell is in vitro.

Another object of the invention is the method of any of the screening methods mentioned above, wherein the step of contacting is in a cell-free system.

A further object of the invention is the method of any of the screening methods mentioned above, wherein the polypeptide is coupled to a detectable label.

Another object of the invention is the method of any of the screening methods mentioned above, wherein the compound is coupled to a detectable label.

Another object of the invention is the method of any of the screening methods mentioned above, wherein the test compound displaces a ligand which is first bound to the polypeptide.

A further object of the invention is the method of any of the screening methods mentioned above, wherein the polypeptide is attached to a solid support. Another object of the invention is the method of any of claims the screening methods mentioned above, wherein the compound is attached to a solid support.

In a further embodiment, the invention provides a method of screening for therapeutic agents useful in the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a mammal comprising the steps of

i) contacting a test compound with a CYP46 polynucleotide,

ii) detect binding of said test compound to said CYP46 polynucleotide.

Another object of the invention is the method mentioned above, wherein the nucleic acid molecule is RNA.

Another object of the invention is the method mentioned above, wherein the contacting step is in or at the surface of a cell.

A further object of the invention is the method described above, wherein the contacting step is in a cell-free system.

Another object of the invention is the method described above, wherein polynucleotide is coupled to a detectable label.

Another object of the invention is the method described above, wherein the test compound is coupled to a detectable label.

Furthermore, the invention provides a method of diagnosing Alzheimer's disease in a mammal comprising the steps of

i) determining the amount of a CYP46 polynucleotide in a sample taken from said mammal,

ii) determining the amount of CYP46 polynucleotide in healthy and/or diseased mammals.

A further object of the invention is a pharmaceutical composition for the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a mammal comprising a therapeutic agent which binds to a CYP46 polypeptide. Another object of the invention is a pharmaceutical composition for the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a mammal comprising a therapeutic agent which regulates the activity of a CYP46 polypeptide.

Furthermore, the invention provides a pharmaceutical composition for the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a mammal comprising a therapeutic agent which regulates the activity of a CYP46 polypeptide, wherein said therapeutic agent is

i) a small molecule,

ii) an RNA molecule,

iii) an antisense oligonucleotide,

iv) a polypeptide,

vi) an antibody, or

vii) a ribozyme.

Another object of the invention is a pharmaceutical composition for the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a' mammal comprising a CYP46 polynucleotide.

Another object of the invention is a pharmaceutical composition for the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a mammal comprising a CYP46 polypeptide.

Another object of the invention is the use of regulators of a CYP46 for the preparation of a pharmaceutical composition for the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a mammal.

Another object of the invention is a method for the preparation of a pharmaceutical composition useful for the treatment and/or prophylaxis of neurodegenerative disorders, preferably of Alzheimer's disease in a mammal comprising the steps of

i) identifying a regulator of CYP46, ii) determining whether said regulator ameliorates the symptoms of Alzheimer's disease in a mammal; and

iii) combining of said regulator with an acceptable pharmaceutical carrier.

Another object of the invention is the use of a regulator of CYP46 for the regulation of CYP46 activity in a mammal having Alzheimer's disease.

Test Compounds

Suitable test compounds for use in the screening assays of the invention can be obtained from any suitable source, e.g., conventional compound libraries. The test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. Examples of methods for the synthesis of molecular libraries can be found in the art. Libraries of compounds may be presented in solution or on beads, bacteria, spores, plasmids or phage.

Screening / Screening Assays

The invention provides methods (also referred to herein as "screening assays") for identifying compounds which can be used for the treatment of Alzheimer's Disease. The methods entail the identification of candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other molecules) which bind to CYP46 and/or have a stimulatory or inhibitory effect on the biological activity of CYP46 or its expression and then determining which of these compounds have an effect on symptoms of Alzheimer's Disease in an in vivo assay.

Candidate or test compounds or agents which bind to CYP46 and/or have a stimulatory or inhibitory effect on the activity or the expression of CYP46 are identified either in assays that employ cells which express CYP46 (cell-based assays) or in assays with isolated CYP46 (cell-free assays). The various assays can employ a variety of variants of CYP46 (e.g., full-length CYP46, a biologically active fragment of CYP46, or a fusion protein which includes all or a portion of CYP46). Moreover, CYP46 can be derived from any suitable mammalian species (e.g., human CYP46, rat CYP46 or murine CYP46). The assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known CYP46 ligand to CYP46. The assay can also be an activity assay entailing direct or indirect measurement of the activity of CYP46. The assay can also be an expression assay entailing direct or indirect measurement of the expreβsion of CYP46 mRNA or CYP46 protein. The various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms of Alzheimer's Disease.

In various embodiments of the invention it is necessary to clone the CYP46 nucleotide or parts thereof and/or to express and/or purify the CYP46 polypeptide or parts thereof. This is done by methods well known to a person skilled in the art.

The present invention includes biochemical, cell free assays that allow the identification of inhibitors and agonists of CYP46 suitable as lead structures for pharmacological drug development. Such assays involve contacting a form of CYP46 (e.g., full-length CYP46, a biologically active fragment of CYP46, or a fusion protein comprising all or a portion of CYP46) with a test compound and determining the ability of the test compound to act as an antagonist or an agonist of the enzymatic activity of CYP46.

In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of CYP46. Such assays can employ full-length CYP46, a biologically active fragment of CYP46, or a fusion protein which includes all or a portion of CYP46. As described in greater detail below, the test compound can be obtained by any suitable means, e.g., from conventional compound libraries.

Determining the ability of the test compound to modulate the activity of CYP46 can be accomplished, for example, by determining the ability of CYP46 to bind to or interact with a target molecule. The target molecule can be a molecule with which CYP46 binds or interacts with in nature.

In various embodiments of the above assay methods of the present invention, it may be desirable to immobilize CYP46 (or a CYP46 target molecule) to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to CYP46, or interaction of CYP46 with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro- centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto gluta- thione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized rnicrotitre plates, which are then combined with the test compound or the test compound and either the non- adsorbed target protein or CYP46, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or rnicrotitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of CYP46 can be determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either CYP46 or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin-coated plates (Pierce Chemical). Alternatively, antibodies reactive with CYP46 or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with CYP46 or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with CYP46 or target molecule.

In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding CYP46 specifically compete with a testcompound for binding CYP46. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with CYP46.

Gene Expression Assays

The screening assay can also involve monitoring the expression of CYP46. For example, regulators of expression of CYP46 can be identified in a method in which a cell is contacted with a candidate compound and the expression of CYP46 protein or rnRNA in the cell is determined. The level of expression of CYP46 protein or mRNA the presence of the candidate compound is compared to the level of expression of CYP46 protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of CYP46 based on this comparison. For example, when expression of CYP46 protein or mRNA is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of CYP46 protein or mRNA expression. Alternatively, when expression of CYP46 protein or mRNA is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of CYP46 protein or mRNA expression. The level of CYP46 protein or mRNA expression in the cells can be determined by methods well known in the art.

Binding Assays

For binding assays, the test compound is preferably a small molecule which binds to and occupies the active site of CYP46 polypeptide, thereby making the ligand binding site inaccessible to substrate such that normal biological activity is prevented.

In binding assays, either the test compound or the CYP46 polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to CYP46 polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product. Alternatively, binding of a test compound to a CYP46 polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a CYP46 polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and CYP46.

Determining the ability of a test compound to bind to CYP46 also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules. In yet another aspect of the invention, a CYP46-like polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay, to identify other proteins which bind to or interact with CYP46 and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding CYP46 can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein- dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g. , LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with CYP46.

Functional Assays

Test compounds can be tested for the ability to increase or decrease CYP46 activity of a CYP46 polypeptide. The CYP46 activity can be measured, for example, using methods described in the specific examples, below. CYP46 activity can be measured after contacting either a purified CYP46 or an intact cell with a test compound. A test compound which decreases CYP46 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing CYP46 activity. A test compound which increases CYP46 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing CYP46 activity.

Regulators

Regulators as used herein, refer to compounds that affect the activity of CYP46 in vivo and/or in vitro. Regulators can be agonists and antagonists of CYP46 polypeptide and can be compounds that exhert their effect on the CYP46 activity via the enzymatic activity, expression, post- translational modifications or by other means. Agonists of CYP46 are molecules which, when bound to CYP46, increase or prolong the activity of CYP46. Agonists of CYP46 include proteins, nucleic acids, carbohydrates, small molecules, or any other molecule which activate CYP46. Antagonists of CYP46 are molecules which, when bound to CYP46, decrease the amount or the duration of the activity of CYP46. Antagonists include proteins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the activity of CYP46.

The term "modulate", as it appears herein, refers to a change in the activity of CYP46 polypeptide. For example, modulation may cause an increase or a decrease in enzymatic activity, binding characteristics, or any other biological, functional, or immunological properties of CYP46.

As used herein, the terms "specific binding" or "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope "A" the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.

Applications

The present invention provides for prophylactic, therapeutic and / or diagnostic methods for Alzheimer's Disease.

The regulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of CYP46. An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or any small molecule. In one embodiment, the agent stimulates one or more of the biological activities of CYP46. Examples of such stimulatory agents include the active CYP46 and nucleic acid molecules encoding a portion of CYP46. These regulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject).

Pharmaceutical Compositions

The nucleic acid molecules, polypeptides, and antibodies of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

An additional embodiment of the invention relates to the administration of a pharmaceutical composition containing CYP46 in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of CYP46, antibodies to CYP46, and mimetics, agonists, antagonists, or inhibitors of CYP46. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatine capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and- used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation^' and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. For pharmaceutical compositions which include an antagonist of CYP46 activity, a compound which reduces expression of CYP46, or a compound which reduces expression or activity of a protein in the CYP46 signaling pathway or any combination thereof, the instructions for administration will specify use of the composition for Alzheimer's Disease. For pharmaceutical compositions which include an agonist of CYP46 activity, or a compound which increases expression of CYP46 the instructions for administration will specify use of the composition for Alzheimer's disease.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases CYP46 activity relative to CYP46 activity which occurs in the absence of the therapeutically effective dose. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅o/ED₅₀. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts can vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intra- cellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun", and DEAE- or calcium phosphate-mediated transfection.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. Examples;

Example 1: Expression profiling

Expression profiling of human cholesterol 24-hydroxylase (CYP46) :

Total cellular RNA was isolated from cells by one of two standard methods: 1) guanidine isothiocyanate/Cesium chloride density gradient centrifugation [Kellogg, (1990)]; or with the Tri- Reagent protocol according to the manufacturer's specifications (Molecular Research Center, Inc., Cincinatti, Ohio). Total RNA prepared by the Tri-reagent protocol was treated with DNAse I to remove genomic DNA contamination.

For relative quantitation of the mRNA distribution of cholesterol 24-hydroxylase (CYP46), total RNA from each cell or tissue source was first reverse transcribed. 85 μg of total RNA was reverse transcribed using 1 μmole random hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000 U RnaseQut (Invitrogen, Groningen, Netherlands) in a final volume of 680 μl. The first strand synthesis buffer and Omniscript reverse transcriptase (2 u/μl) were from (Qiagen, Hilden, Germany). The reaction was incubated at 37°C for 90 minutes and cooled on ice. The volume was adjusted to 6800 μl with water, yielding a final concentration of 12.5 ng/μl of starting RNA.

For relative quantitation of the distribution of cholesterol 24-hydroxylase (CYP46) mRNA in cells and tissues the Applied Biosystems 7900HT Sequence Detecton system was used according to the manufacturer's specifications and protocols. PCR reactions were set up to quantitate cholesterol 24-hydroxylase (CYP46) and the housekeeping genes HPRT (hypoxanthine phosphoribosyl- transferase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-actin, and others. Forward and reverse primers and probes for human cholesterol 24-hydroxylase (CYP46) were designed using the Perkin Elmer ABI Primer ExpressTM software and were synthesized by TibMolBiol (Berlin, Germany). The human cholesterol 24-hydroxylase (CYP46) forward primer sequence was: Primerl (SE Q ID NO: 3). The human cholesterol 24-hydroxylase (CYP46) reverse primer sequence was Primer2^' (SEQ ID NO: 5). Probel (SEQ LD NO: 4), labelled with FAM (carboxy- fluorescein succi imidyl ester) as the reporter dye and TAMRA (carboxytetramethylrhodamine) as the quencher, is used as a probe for the human cholesterol 24-hydroxylase (CYP46). The following reagents were prepared in a total of 25 μl : lx TaqMan buffer A, 5.5 mM MgC12, 200 nM of dATP, dCTP, dGTP, and dUTP, 0.025 U/μl AmpliTaq GoldTM, 0.01 U/ μl AmpErase and Probel (SEQ ID NO: 4), human cholesterol 24-hydroxylase (CYP46) forward and reverse primers 'J each at 200 nM, 200nM human cholesterol 24-hydroxylase (CYP46) .FAJVI/TAMRA-labelled probe, and 5 μl of template cDNA. Thermal cycling parameters were 2 min at 50°C, followed by 10 min at 95°C, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for l min.

5 Calculation of corrected CT values

The CT (threshold cycle) value is calculated as described in the "Quantitative determination of nucleic acids" section. The CF-value (factor for threshold cycle correction) is calculated as follows .

1. PCR reactions were set up to quantitate the housekeeping genes (HKG) for each cDNA 0 sample.

2. CTHKG-values (threshold cycle for housekeeping gene) were calculated as described in the "Quantitative determination of nucleic acids" section.

3. CTHKG-mean values (CT mean value of all HKG tested on one cDNAs) of all HKG for each cDNA are calculated (n = number of HKG):

5 CTHKG-n-mean value = (CTHKG1 -value + CTHKG2-value +... + CTHKG-n-value) / n

4. CTpannel mean value (CT mean value of all HKG in all tested cDNAs) =

(CTHKGl-mean value + CTHKG2-mean value +...+ CTHKG-y-mean value) / y (y = number of cDNAs)

5. CFcDNA-n (correction factor for cDNA n) = CTpannel-mean value - CTHKG-n-mean 0 value

6. CTcDNA-n (CT value of the tested gene for the cDNA n) + CFcDNA-n (correction factor for cDNA n) = CTcor-cDNA-n (corrected CT value for a gene on cDNA n)

Calculation of relative expression

Definition : highest CTcor-cDNA-n ≠ 40 is defined as CTcor-cDNA [high] 5 Relative Expression = 2(CTcor-cDNA[high] - CTcor-cDNA-n) Tissues

Expression of human CYP46 was investigated in the following (tissue) samples:

Fetal heart, heart, pericardium, heart atrium (right), heart atrium (left), heart ventricle (left), interventricular septum, fetal aorta, aorta, aorta sclerotic, artery, coronary artery, coronary artery sclerotic, vein, coronary artery smooth muscle primary cells, HUVEC cells, fetal brain, brain, alzheimer brain, cerebellum, cerebellum (right), cerebellum (left), cerebral cortex, alzheimer cerebral cortex, frontal lobe, alzheimer brain frontal lobe, occipital lobe, parietal lobe, temporal lobe, precentral gyrus, postcentral gyrus, tonsilla cerebelli, vermis cerebelli, pons, substantia nigra, cerebral meninges, cerebral peduncles, corpus callosum, hippocampus, thalamus, dorsal root ganglia, spinal cord, neuroblastoma SK-N-MC cells, neuroblastoma SH-SY5Y cells, neuroblastoma IMR32 cells, glial tumor H4 cells, glial tumor H4 cells + APP, HEK CNS, HEK CNS + APP, retina, leukocytes (peripheral blood), Jurkat (T-cells), bone marrow, erythrocytes, lymphnode, thymus, thrombocytes, bone marrow stromal cells, bone marrow CD71+ cells, bone marrow CD33+ cells, bone marrow CD34+ cells, bone marrow CD15+ cells, cord blood CD71+ cells, spleen, spleen liver cirrhosis, fetal lung, fetal lung fibroblast IMR-90 cells, lung, lung right upper lobe, lung right mid lobe, lung right lower lobe, lung tumor, lung COPD, trachea, prostata, prostate BPH, bladder, ureter,, penis, corpus cavernosum, fetal kidney, kidney, kidney tumor, HEK 293 cells, adrenal gland, thyroid, thyroid tumor, pancreas, pancreas liver cirrhosis, esophagus, esophagus tumor, stomach, stomach tumor, colon, colon tumor, small intestine, ileum, ileum tumor, ileum chronic inflammation, rectum, salivary gland, fetal liver, liver, liver liver cirrhosis, liver tumor, HEP G2 cells, skeletal muscle, adipose, skin, cervix, testis, HeLa cells (cervix tumor), placenta, uterus, uterus tumor, ovary, ovary tumor, breast, breast tumor, MDA MB 231 cells (breast tumor), mammary gland

Expression profile

The results of the the mRNA-quantification (expression profiling) is shown in Table 1. Table 1: Relative expression of cholesterol 24-hydroxylase in Various human tissues.

TISSUE Rel. Expression

fetal heart 80 heart 64 pericardium 38 heart atrium (right) 128 heart atrium (left) 333 heart ventricle (left) 28 interventricular septum 246 fetal aorta 4 aorta 32 aorta sclerotic 51 artery ^• 80 coronary artery 372 coronary artery sclerotic 265 vein 32 coronary artery smooth muscle primary 25 cells

HUVEC cells 39 fetal brain 282 brain 2180 alzheimer brain 750 cerebellum 39 cerebellum (right) 109 cerebellum (left) 175 cerebral cortex 1235 alzheimer cerebral cortex 588 frontal lobe 512 TISSUE Rel.

Expression alzheimer brain frontal 657 lobe occipital lobe 1499 parietal lobe 1585 temporal lobe 1563 precentral gyrus 1024 postcentral gyrus 46 tonsilla cerebelli 201 vermis cerebelli 140 pons 197 substantia nigra 605 cerebral meninges 53 cerebral peduncles 345 corpus callosum 190 hippocampus 1201 thalamus 455 dorsal root ganglia 160 spinal cord 168 neuroblastoma SK-N-MC cells 38 neuroblastoma SH-SY5Y cells 18 neuroblastoma IMR32 cells . 84 glial tumor H4 cells 32 glial tumor H4 cells + APP 12

HEK CNS 85

HEK CNS + APP 79 retina 96 leukocytes (peripheral blood) 9

Jurkat (T-cells) 7 bone marrow 1 erythrocytes 35 lymphnode 37 thymus 50 TISSUE Rel.

Expression thrombocytes 21 bone marrow stromal cells 8 bone marrow CD71+ cells 31 bone marrow CD33+ cells 446 bone marrow CD34+ cells 28 bone marrow CD 15+ cells 36 cord blood CD71+ cells 48 spleen 10 spleen liver cirrhosis 7 fetal lung 53 fetal lung fibroblast IMR-90 cells 34 lung 1 lung right upper lobe 148 lung right mid lobe 107 lung right lower lobe 151 lung tumor ^{' '" • '} 77 lung COPD 114 trachea 26 prostata 20 prostate BPH 13 bladder 4 ureter 78 penis - 75 corpus cavernosum 13 fetal kidney 145 kidney 47 kidney tumor 19

HEK 293 cells 29 adrenal gland 29 thyroid 22 thyroid tumor 20 pancreas 50 TISSUE Rel.

Expression pancreas liver cirrhosis 38 esophagus 33 esophagus tumor 18 stomach 7 stomach tumor 89 colon 4 colon tumor 19 small intestine 14 ileum 52 ileum tumor 31 ileum chronic 79 inflammation rectum 428 salivary gland 5 fetal liver 8 liver 4 liver liver cirrhosis 72 liver tumor 23

HEP G2 cells 4 skeletal muscle 27 adipose 20 skin 91 cervix 9 testis 53

HeLa cells (cervix tumor) 1 placenta 4 uterus 29 uterus tumor 17 ovary 44 ovary tumor 541 breast 92 breast tumor 40 TISSUE Rel.

Expression

MDA MB 231 cells (breast tumor) 22 mammary gland 12

Expression profiling of mouse cholesterol 24-hydroxylase (CYP46)

The expression profile was also investigated in aged tg2576 mice (older than 20 months) in hippocampus, frontal cortex, and temporal cortex.

Tissue and RNA preparation

A total of 6 Tg2576 transgenic mice and 6 wildtype littermate controls (>20 months) were sacrificed by cervical dislocation. After removing their brains, hippocampus, frontal cortex and temporal cortex were dissected from the rest of the brain and deeply frozen in liquid nitrogen. Total RNA from mouse brain was isolated by homogenizing frozen tissue using the QBiogene FastPrep 120 System and FastRNA purification kit (Carlsbad, CA, USA) according to instructions provided by the manufacturer. Total RNA isolation was performed with the Tri-Reagent protocol according to the manufacturer's specifications (Molecular Research Center, Lie, Cincinatti, Ohio).

cDNA Synthesis

cDNA from each tissue source was synthesized using the ThermoScript RT-PCR system (Invitrogen, Groningen, the Netherlands): 2 μg of total RNA together with 50 ng/μl random hexamer primers were used for cDNA synthesis following the manufacturer's instructions.

Semi-quantitative real time PCR

For relative quantification of CYP46 cDNA in mouse brain the 7700HT Sequence Detection system (Applied Biosystems, Foster City, CA, USA) was used according to the manufacturer's specifications and protocols. PCR reactions were set up to quantitate the mouse housekeeping gene β-actin and the CYP46 gene using sets of gene specific upstream and downstream primers in combination with probes labeled with carboxyfluorescein succinimidyl ester (FAM) which was quenched by carboxytetramethylrhodamine (TAMRA) at the 3' end of the oligonucleotide. Sequences of appropriate oligonucleotides used for quantification PCR were obtained using the Perkin Elmer ABI Primer Express software and synthesized by TibMolBiol (Berlin, Germany): The mouse cholesterol 24-hydroxyϊase (CYP46) forward primer sequence was: primer3 (SEQ ID NO: 6); The reverse primer sequence was: primer 4 (SEQ ID NO: 8); Probe2 (SEQ ID NO: 7). β- actin forward primer sequence was : primer5 (SEQ ID NO: 9); β-actin reverse primer sequence was primerό (SEQ ID NO: 11); β-actin probe sequence was: Probe 3 (SEQ ID NO: 10). The final amplification mix consisted of lx TaqMan universal master mix (Applied Biosystems), 5 μl of template cDNA as well as of primers and probes each at 200 nM. Thermal cycling parameters were 2 min at 50°C, followed by 10 min at 95° C, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extending at 60° C for 1 min.

Data Evaluation

1. Calibration of cDNA-level by β-actin PCR: Since cDNA preparations differ in their total amount of cDNA, it is neccessary to calibrate individual cDNA preparations on a specific housekeeping gene (e.g. β-actin) of which each cell contains nearly the same level. In this experiment, a β-actin PCR was used for calibration and estimation of a correction factor (CF)j which reflects the aberration of the individual cDNA content (measured as CT^;) compared to the mean cDNA content of all cDNA preparations (measured as C _mn aii)- Thus, the CF allows a compensation of different cDNA contents.

The β-actin content was double-estimated for each cDNA preparation during the same PCR run (CT_b CT₂). If the difference between both CT values (CT_D;_ff) was ≥ 1 or < -1, data were rejected; values in-between -1 and 1 were taken as mean value (individual cDNA: CT_mn i, all cDNAs: CTmn all)-

• CT_Di_ff: the difference between 2 individual measurements of the same cDNA during the same Taqman run:

CT_Diff= CTι+ CT. [rejected if CT_Diff > 1 or < -1]

• CT_mn j: the mean of 2 individual measurements of the same cDNA during the same Taqman run:

CT_mni = (CT₁+ CT₂)/ 2

• CT_{mn aI1}: the mean of all CT_mn i of all cDNAs • CFp: correction factor of an individual cDNA to adjust CT_mn values of β-actin measurements (assumed that β-actin is expressed at the same level in each tissue):

^»-*' ^— *-' J- nin i - 1 mn all

2. Estimation of the relative expression of CYP46: For estimation of the relative expression of CYP46 cDNA, a C _mn i was estimated as described above. A corrected CT (CT_00r) value was calculated by subtracting the CF from the CT_mn i- The maximum CT_cor value (CT_max) of all cDNAs of one tissue (WT and Tg2576) was used to get a relation between an individual CT_cor and the maximum value of the tissue; the difference between an individual CT_cor and the CT_maχ was expressed in ACT. Since target amplification during PCR occurs logarithmically, the final relative expression was calculated as 2^ΛCT.

• CT_cor= CT_mn i (target gene) - CF

• CT_max: maximum CT_cor value (≠40) of cDNAs of a certain tissue (WT and Tg2576)

• ACT: CT_max corrected by β-actin correction factor

ΔCT = CT_raax - CF

• relative expression: expression level of a target gene of a specific cDNA preparation relative to the cDNA preparation with CT_maχ.: rel Exp = 2^ΔCT

3. Statistical analysis: Extreme values of relative expression data of cDNAs within one tissue group (WT or Tg2576) were excluded using the Dixon's test at p<0.05 (Dixon, 1950, 1951, .

Dixon, WJ. (1950). Analyses of extreme values. Annals of Mathematical Statistics, 21, 488-506; Dixon, WJ.(1951). Ratios involving extreme values. Annals of Mathematical Statistics, 22, 68-78.). Statistical significance of relative expression data between different genotypes (WT and Tg2576) of one tissue was determined using a student's t-test.

Relative Expression of cholesterol 24-hydroxylase (CYP46) in the brain of aged wildtype and tg 2576 mice are shown in Fig 1. Example 2: Neurotoxicity of 24-Hydroxycholesterol

SH-SY5Y neuroblastoma cell line and primary mouse cortex neurons were utilized to determine the neurotoxicity effect of 24-hydroxycholesterol.

SH-SY5Y Cells are cultured under standard conditions (medium: DMEM/F12 nut mix plus Non- essential amino acids, Glutamax and 10% Fetal Bovine serum). For experiments, cells are plated at a density of 400,000 cells per well in 6- ell plates on day 1, and treated on day 3 with 24- hydroxycholesterol, 25-hydroxycholesterol and free cholesterol for 24 hours and 48 hours respectively.

Our data showed that 24-hydroxycholesterol significantly increased the LDH release, decreased the mitochondria potential and induced DNA laddering. These data are consistent with the previous data described in the literature [H. Kδlsch, et al; Brain Res. 818: 171-175, 1999].

Cortical neuronal cultures were established from brains of embryonic day 16 to day 18 fetal transgenic (APP-SL) mice. The dissected brain cortexes were cooled on ice. Separation of cortical neurons were done according to a standard protocol with Papain Dissociation system (Worthington Biochemical Corporation, Lakewood, New Jeysey 08701, USA). The dispersed cells were collected by centrifugation and plated at ~3 x 106 cells/well on 6-well cell culture plates (coated with poly-D-lysine lOOμg/ml for 20 minutes) in B27/Neurobasal media (GTBCO/BRL, Gaithersburg, MD). Neurons were allowed to mature for up to 7 days in culture before they were used for experiments. After 7 days, medium was replace with 1 ml Neurobasal medium containing various substances: 24-hydroxycholesterol, 25-hydroxycholesterol, free cholesterol, and camptothecin (as possitive control for apoptosis. The cells were incubated at 37°C for 24 hours or 48 hours. To determine the neurotoxicity of 24-hydroxycholesterol, the culture medium was used for the LDH release and cells were used for caspase-3 activity assay. Statistical analysis was done to compare each group to the group treated with free cholesterol: ***P<0.001.

Neurotoxicity of 24-hydroxycholesterol measured by LDH release

LDH measurement is a colorimetric assay for the quantification of cell death and cell lysis, based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the supernatant. Assays were done following the protocol provided by the manufacturer (Cytotoxicity Detection Kit, Roche, No. 1644793) Fi .2

a) LDH measured in medium of primary mouse cortical neurons after 24 hours incubation with 25μM cholesterol, 24-OH cholesterol, 25-OH cholesterol, and 10 mM camptothecin (as positive control)

b) LDH measured in medium of primary mouse cortical neurons after 48 hours incubation with 25μM cholesterol, 24-OH cholesterol, 25-OH cholesterol, and 10 mM camptothecin (as positive control, incubated for 24 hours)

Caspase 3 activity induced by oxycholesterols:

The activation of caspase 3 plays a key role during the apoptotic process. Once caspase 3 has been activated, there is no way back to normal viability, the program for cell death is irreversibly activated. The Caspase 3 Activity Assay provides a specific, sensitive method for analyzing this early apoptotic event. The Assays were performed according to the manufacturer's specification (Caspase 3 Fluorometric Assay Kit, Sigma No. CASP-3-F).

Fig. 3

a) Caspase-3 activity measured in primary mouse cortical neurons after 24 hours incubation with 25μM 24-OH cholesterol, 25-OH cholesterol, cholesterol, and 10 mM camptothecin (positive control)

b) Caspase-3 activity measured in primary mouse cortical neurons after 48 hours incubation with 25 μM 24-OH cholesterol, 25-OH cholesterol, cholesterol, and 10 mM camptothecin (positive control, incubated for 24 hours)

Example 3: Drug Screening

Cell lysates of Chinese hamster ovarian cells stably expressing cholesterol 24-hydroxylase (CYP46) are used for drug screening. Cell lysates are prepared using a polytron set at 10,000 rpm, with three bursts of 3s each with 30-s intervals between bursts. Incubations are performed at 37°C with 140 μg of cell lysate protein in 50 mM potassium phosphate buffer, pH7.4, containing 5mM NACPH. [4~^l4C]Cholesterol is added in 4 μl of 45% (w/v) 2-hydroxypropylcyclodextrin in water to a final concentration of 5 μM. Compounds are added at various end concentrations. The total volu e of the incubation is adjusted to 200 μl. After 2 hours, reactions are extracted with chloroform/methanol (2:1, v/v) and analyzed by thin layer chromatography.

^■ For High Throughput-Screening, another format that provides higher throughput is used. For example, the decrease in NADPH that occurs during the hydroxylation of cholesterol can be determined by measuring the absorbance at 3430 nm.

Example 4: Animal experiments:

Compounds are tested in elderly mice and rats, or in Alzheimer's disease models (e.g APP-SL mice, APP-SL x PSl mice etc). Compounds are administered intraperitoneally or orally, levels of 24-hydroxycholesterol in serum and CSF are determined at various time points. Changes of cognition activity are assessed in T-maze, Passive Avoidance, Morris water maze, Cone field, 8- Arm radial maze, Social Recognition and Object Recognition tasks. These are the recognized behavioural tests to assess various congnition functions: where T-maze is mainly for working memory, Passive Avoidance and Morris water maze mainly for reference memory, whereas cone field and 8-arm radial maze are for both working memory and reference memory. Social recognition and Object Recognition are tests to measure the capability of rats/mice to differentiate the novel individual (or object) from the familiar ones. The neuronal survival in these animal brains are further examined by immunohistochemistry.

References:

Bjδrkhem et al, J Biol Chem. 272 (48): 30178-30184.1997

Bjδrkhem et al, J Lipid Res. 39 (8): 1594-1600. 1998

Bogdanovic N et al. Neuroscience Letters 314: 45-48. 2001

Dixon, WJ. Annals of Mathematical Statistics, 21, 488-506, 1950

Dixon, W.J. Ratios involving extreme values. Annals of Mathematical Statistics, 22, 68-78 1951

JickH. etal. Lancet 356:1627-1631, 2000;

Kellogg, Analytical Biochemistry, 189: 202-208. 1990

Kivipelto M. et al. Neurology 56: 1683-1689, 2001

Kolsch et al. Brain Res. 818: 171-175, 1999

Lund et al, Proc Natl Acad Sci USA 96 (13 ): 7238-7243.1999

Lutjohann et al, J. Lipid Res. 41 : 195-198, 2000

Lϋtjohann et al, Proc Natl Acad Sci USA 93 (18 ): 9799-9804. 1996

Meaney et al, J Lipid Res.42 (1): 70-78. 2001

Pitas et al, Biochim Biophys Acta 917 (1): 148-161, 1987

Wolozin B. et al., Arch. Neurol. 57: 1439-1443, 2000

Claims

Claims:

1. A method of screening for therapeutic agents useful in the treatment and/or prophylaxis of Alzheimer's disease in a mammal comprising the steps of

i) contacting a test compound with a CYP46 polypeptide,

ii) detect binding of said test compound to said CYP46 polypeptide,

2. A method of screening for therapeutic agents useful in the treatment and/or prophylaxis of Alzheimer's disease in a mammal comprising the steps of

i) determining the activity of a CYP46 polypeptide at a certain concentration of a test compound,

ii) determining the activity of said polypeptide in the absence or at a different concentration of said test compound,

3. A method of screening for therapeutic agents useful in the treatment and/or prophylaxis of Alzheimer's disease in a mammal comprising the steps of

4. A method of screening for therapeutic agents useful in the treatment and/or prophylaxis of

Alzheimer's disease in a mammal comprising the steps of i) contacting a test compound with a CYP46 polynucleotide,

ii) detect binding of said test compound to said CYP46 polynucleotide,

wherein those compounds are selected as potential therapeutic agents which bind to the CYP46 polynucleotide.

5.- A method of diagnosing Alzheimer' s disease in a mammal comprising the steps of

6. A pharmaceutical composition for the treatment and/or prophylaxis of Alzheimer's disease in a mammal comprising a therapeutic agent which binds to a CYP46 polypeptide.

7. A pharmaceutical composition for the treatment and/or prophylaxis of Alzheimer' s disease in a mammal comprising a therapeutic agent which regulates the activity of a CYP46 polypeptide.

8. A pharmaceutical composition for the treatment and/or prophylaxis of Alzheimer ' s disease in a mammal comprising a CYP46 polynucleotide.

9. A pharmaceutical composition for the treatment and/or prophylaxis of Alzheimer's disease in a mammal comprising a CYP46 polypeptide.

10. Use of regulators of a CYP46 for the preparation of a pharmaceutical composition for the treatment and/or prophylaxis of Alzheimer's disease in a mammal.

11. Method for the preparation of a pharmaceutical composition useful for the treatment and/or prophylaxis of Alzheimer's disease in a mammal comprising the steps of

i) identifying a regulator of CYP46,

ii) determining whether said regulator ameliorates the symptoms of Alzheimer's disease in a mammal; and iii) combining of said regulator with an acceptable pharmaceutical carrier,

iv) combining of said regulator with an acceptable pharmaceutical carrier.

1/12

Fi .l

cm WT

Hippocampus frontal Cortex temporal Cortex

2/12-

Fig.2a

3/12

Fig. 2b

^■ 4/12 -

Fig. 3a

5/12.

Fig.3b

6/12 ^■

Fig. 4:

SEQ ID NO: 1

ATGAGCCCCGGGCTGCTGCTGCTCGGCAGCGCCGTCCTGCTCGC

CTTCGGCCTCTGCTGCACCTTCGTGCACCGCGCTCGCAGCCGCT

ACGAGCACATCCCCGGGCCGCCGCGGCCCAGTTTCCTTCTAGGA

CACCTCCCCTGCTTTTGGAAAAAGGATGAGGTTGGTGGCCGTGT

GCTCCAAGATGTGTTTTTGGATTGGGCTAAGAAGTATGGACCTG

TTGTGCGGGTCAACGTCTTCCACAAAACCTCAGTCATCGTCACGA

GTCCTGAGTCGGTTAAGAAGTTCCTGATGTCAACCAAGTACAAC

AAGGACTCCAAGATGTACCGTGCGCTCCAGACTGTGTTTGGTGA

GAGACTCTTCGGCCAAGGCTTGGTGTCCGAATGCAACTATGAGC

GCTGGCACAAGCAGCGGAGAGTCATAGACCTGGCCTTCAGCCGG

AGCTCCTTGGTTAGCTTAATGGAAACATTCAACGAGAAGGCTGA

GCAGCTGGTGGAGATTCTAGAAGCCAAGGCAGATGGGCAGACCC

CAGTGTCCATGCAGGACATGCTGACCTACACCGCCATGGACATC

CTGGCCAAGGCAGCTTTTGGGATGGAGACCAGTATGCTGCTGGG

TGCCCAGAAGCCTCTGTCCCAGGCAGTGAAACTTATGTTGGAGG

GAATCACTGCGTCCCGCAACACTCTGGCAAAGTTCCTGCCAGGG

AAGAGGAAGCAGCTCCGGGAGGTCCGGGAGAGCATTCGCTTCCT

GCGCCAGGTGGGCAGGGACTGGGTCCAGCGCCGCCGGGAAGCC

CTGAAGAGGGGCGAGGAGGTTCCTGCCGACATCCTCACACAGAT

TCTGAAAGCTGAAGAGGGAGCCCAGGACGACGAGGGTCTGCTG

GACAACTTCGTCACCTTCTTCATTGCTGGTCACGAGACCTCTGCC

AACCACTTGGCGTTCACAGTGATGGAGCTGTCTCGCCAGCCAGA 7/12

GATCGTGGCAAGGGTGCAGGCCGAGGTGGATGAGGTCATTGGTT

CTAAGAGGTACCTGGATTTCGAGGACCTGGGGAGACTGCAGTAC

CTGTCCCAGGTCCTCAAAGAGTCGCTGAGGCTGTACCCACCAGC

ATGGGGCACCTTTCGCCTGCTGGAAGAGGAGACCTTGATTGATG

GGGTCAGAGTCCCCGGCAACACCCCGCTCTTGTTCAGCACCTAT

GTCATGGGGCGGATGGACACATACTTTGAGGACCCGCTGACTTT

CAACCCCGATCGCTTCGGCCCTGGAGCACCCAAGCCACGGTTCA

CCTACTTCCCCTTCTCCCTGGGCCACCGCTCCTGCATCGGGCAG

CAGTTTGCTCAGATGGAGGTGAAGGTGGTCATGGCAAAGCTGCT

GCAGAGGCTGGAGTTCCGGCTGGTGCCCGGGCAGCGCTTCGGG

CTGCAGGAGCAGGCCACACTCAAGCCACTGGACCCCGTGCTGTG

CACCCTGCGGCCCCGCGGCTGGCAGCCCGCACCCCCACCACCCC

CCTGCTGAGGGGGCCTCCAGGCAGGACGAGACTCCTCGGGCAAGGG

CCGTGCCCGCCCACCTCTGCTGCCCACGGCCACCCACCCTTCTCCCCT

GCCCCGTCCCCTGGGCCACCCTTCACGCTGGCTTCCAGCGGGCCCTCT

GCCGACCGCCTGCTTCACACCCCTCAGCGCTCCCTGTCGCCTGCGGA

CTCCATGGCCCTTCCTGGACTGGCCCTTGCCCAACTCCCAGCCACCAC

CACTGTCCCTACCACTGAGCCCTTGCACAGGCCACTTGCTCAGACGA

GACACCCTAACTCTTGCTCACTCCCTAAAGCCCTCTTCAGGGGTCACC

TCCTCCAAGAAGCCCTCCTTGCCACCCCCCGCCGGCAGGGGCCCCTC

CTCTGTGCTCCCTCGGTCACCTGTGCTACCTCTAACACCACACTGACC

ACACTGTATCGTGAGTGTCCGTTGACGTGACCAATTGCCCTGCCAGG

CTGTCAGCGCCTCAAGGGTAGGGTCTGCGTGTGATTTGTCTCTGAGCC

CCCTGTGCCCACCCAGGGCCCGGCACAGAGTCGATGCTCAATAAATG

TGTGTTGACTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAA 8/12

Fig. 5:

SEQ ID NO: 2

MSPGLLLLGSAVLLAFGLCCTFVHRARSRYEHIPGPPPvPSFLLGHLPCFW

KKDEVGGRVLQDVFLDWAKKYGPVVRVNVFHKTSVIVTSPESVK FLM

STKYNKDSKMYRALQTVFGEP .FGQGLVSECISn ER HKQRRVIDLAFS

RSSLVSLMETFNEKAEQLVEILEAKADGQTPVSMQDMLTYTAMDILAK

AAFGMETSMLLGAQKPLSQAVKLMLEGITASRNTLAKFLPGKRKQLRE

VRESI^LRQVGRDWVQRRREALKRGEEVPADILTQILKAEEGAQDDEG

LLDNFVTFFIAGHETSANHLAFTVMELSRQPEIVARLQAEVDEVIGSKRY

LDFEDLGRLQYLSQVLKESLRLYPPAWGTFRLLEEETLIDGVRVPGNTPL

LFSTYVMGRMDTYFEDPLTFNPDRFGPGAPKPRFTYFPFSLGHRSCIGQQ

FAQMEVKVVMAKLLQRLEFRLVPGQRFGLQEQATLKPLDPVLCTLRPR

GWQPAPPPPPC.

Fig. 6:

SEQ ID NO: 3

GAGCAGCTGGTGGAGATTCTAGA 9/12

Fig. 7:

SEQ ID NO: 4

CAAGGCAGATGGGCAGACCCCAG

Fig. 8;

SEQ ID NO: 5

TCAGCATGTCCTGCATGGA

Fig. 9:

SEQ ID NO: 6

TGCAGTATCTGTCGCAGGTC

Fig. 10:

SEQ ID NO: 7

AGTCTCTGAGGCTGTACCCGCCAG 10/12

Fig. 11:

SEQ ID NO: 8

TAGGTGCTGAACAGGAGAGG

Fig. 12:

SEQ ID NO: 9

CGTTGACATCCGTAAAGACCT

Fig. 13:

SEQ ID NO: 10

CAACACAGTGCTGTCTGGTGGTACCA

Fig. 14:

SEQ ID NO: 11

CAGCAATGCCTGGGTACAT - 11/12

Fig. 15:

SEQ ID NO: 12

CGCAGCGCTGACAGCTAGTCGGTCGCAGCCTCCGGCCCCCTCTGCAC

CGGCGCCGACCACGAGCCATGAGCCCCGGGCTGCTGCTGCTCGGCAG

CGCCGTCCTGCTCGCCTTCGGCCTCTGCTGCACCTTCGTGCATCGCGC

TCGCAGCCGCTATGAGCACATCCCCGGGCCGCCGCGGCCCAGCTTCC

TTCTTGGACATCTCCCCTACTTTTGGAAAAAGGACGAAGATTGTGGC

CGTGTGCTCCAAGATGTGTTTCTGGATTGGGCTAAGAAGTATGGTCCT

GTTGTAAGAGTCAATGTCTTCTACAAGACCTCAGTCATTGTCACGAGT

CCGGAGTCAGTCAAGAAGTTCCTGATGTCCACCAAGTACAACAAGGA

CTCCAAGATGTACCGCGCGCTTCAGACTGTGTTTGGGGAGAGACTGT

TTGGCCAGGGCTTGGTGTCTGAATGTGACTATGGGCGCTGGTACAAG

CAGAGGAAAGTCATGGACTTGGCCTTCAGCCGCAGCTCCTTGGTCAG

CCTGATGGAGACGTTCAACGAGAAGGCGGAGCAGCTGGTGGAAATC

CTAGAAGCTAAGGCAGATGGACAGACACCCGTGTCCATGCAGGACA

TGCTGACCTGTGCCACCATCGACATCCTGGCCAAGGCAGCTTTTGGG

ATGGAGACCAGTATGTTACTGGGTGCCCAGAAACCGCTGTCCCAGGC

AGTGAAGGTCATGCTGGAGGGCATCAGTGCATCCCGTAACACCCTGG

CAAAGTTCATGCCAGGGAAGAGAAAGCAGCTTCGGGAGATCCGCGA

GAGCATCCGTCTGCTGCGCCAGGTGGGGAAGGATTGGGTGCAGCGCC

GCCGCGAAGCCCTGAAGAGGGGCGAGGACATGCCGGCTGACATCCT

CACGCAGATCCTCAAAGCTGAAGAGGGAGCTCAGGACGATGAGGTT

CTGCTGGACAACTTTGTCACCTTCTTCATTGCGGGTCATGAGACTTCT

GCCAACCATCTGGCATTCACAGTGATGGAGTTGTCTCGCCAGCCAGA 12/12 -

GATTGTGGCAAGGCTGCAGGCCGAGGTGGATGAGGTTGTCGGTTCCA

AGAGGCACCTGGACTACGAGGATCTGGGGAGACTGCAGTATCTGTCG

CAGGTCCTCAAAGAGTCTCTGAGGCTGTACCCGCCAGCGTGGGGCAC

CTTTCGCCTGCTGGAGGAGGAGACCTTGATTGATGGGGTCAGAGTCC

CTGGCAATACTCCTCTCCTGTTCAGCACCTACGTCATGGGGAGAATG

GACACCTACTTTGAAGACCCATTGACTTTCAACCCTGACCGCTTCGGC

CCTGGAGCGCCCAAGCCACGGTTCACTTACTTTCCCTTTTCCCTGGGC

CACCGGTCCTGCATCGGCCAACAGTTTGCTCAGATGGAGGTGAAAGT

TGTCATGGCCAAGCTGCTTCAGAGGATCGAGTTCCGGCTAGTGCCTG

GGCAGCGCTTTGGGCTGCAAGAGCAGGCTACGCTCAAGCCATTGGAC

CCCGTGCTGTGCACCCTGAGGCCCCGGGGCTGGCAGCCTGCACCCCC

ACCCCCACCCTGCTGAGCCCTGGGTAAGGTCAGACTCCTTGGACCAG

GGCCTCATTGGCCCCCATTACTGCCCATGGCCACCCACCGGCACCCC

TGACCCTTCTGCCACCTTCCACGATGGCTCCTGGCGAGCCCTGTCCCC

TCGCCTGCTTCACACCCTCAGTGCTTCCTGTCATCGCTGGCTTTTCAG

CTCTTTCTGGTGGGCCCTGCCTGACTCCCAGCTATCACCACAACTACC

ATCGTCTCTCCCAAACCTCCGAACTTCTGCACTGGCCACACCCTCAAA

AGAGACACCCTAACTCTTGCTCACTCCCTAAACTCTTCAAGTGACATG

TCCCCCAGGAAGCCCTGTTTGCTTCTCACAGCCAGGCCAGGGTCTCTC

CTCTGTGCTCCTACCTCTAACACACTGACCACACTGTATTGTGAATGT

TTGCTCATGTGACCAGCTGCCCTGCCAGGCTGTGAGGGCCACAGGGC

AGGGTCTGTGTGCACTTTGCCTCTGAGGCCCCTATTGCTCACTAGGGT

CTGGAACAGAGTCGATGCCCAATAAACGTGTGTTGACTGCAAAAAAA

AAAAAAAAAAAAAAAAAAAAAA