JPWO2006083019A1 - Amyloid β protein aggregation regulator, amyloid β protein abnormality diagnostic agent, and amyloid β protein abnormality diagnostic agent kit - Google Patents

Abstract

It is an object to provide an amyloid β protein aggregation inhibitor. Some or all of the OH groups are substituted with (i) a sulfo group or a salt of a sulfo group, and (ii) a hydrophobic group, and some or all of the remaining OH groups are H, From a sugar chain to which a monosaccharide or two or more monosaccharides are bonded, which can be substituted by a group consisting of NH2, NHR6 and NHR6R7 (R6 and R7 are groups independently selectable from an alkyl group and an acetyl group) It contains a compound represented by a chemical structure to be constituted. In particular, a compound represented by the general formula (1) is desirable. (In Formula (1), R1-5 are each independently selected by the group selected from H, OH, NH2, -NHR6, and -NR6R7; at least one of R1-5 is a sulfo group or A salt of a sulfo group; at least one of R1 to 5 is a hydrophobic group; R6 and R7 are groups independently selectable from an alkyl group and an acetyl group.)

Description

  The present invention relates to an amyloid β protein aggregation regulator, an amyloid β protein abnormality diagnostic agent, and an amyloid β protein abnormality diagnostic kit containing a compound that interacts with amyloid β protein related to Alzheimer-type dementia and the like.

Amyloid β protein is a major component that forms the amyloid core of senile plaques, which is one of the specific pathological changes found in the brain of Alzheimer's dementia patients. It is produced by enzymatic degradation of the penetrating amyloid precursor protein APP (Amyloid Protein Precursor) (for example, Mori, H. et al. (1992) The Journal of Biological Chemistry, Vol. 267, 17082-17086, Lansbury, P. T.). , Jr. (1992) Biochemistry, 31: 6865-6870, Sisodia, SS et al. (1990) Science 248: 492-495, Mullan, M et al. (1993) Trends i. neuroscience see, Vol. 16, 398-403 pages).
From experiments using chemically synthesized amyloid β protein, amyloid β protein has a strong aggregation property (for example, Jarret, JT et al. (1993) Cell 73, 1055-1058, Burdick, D. et al. (1992)). The Journal of Biological Chemistry, Vol. 267, 546-554, Fraser, PE et al. (1992) Biochemistry, Vol. 31, 10716-10723, etc.) It has been reported that direct cytotoxicity and sensitivity to cytotoxicity by excitatory amino acids and the like are increased (for example, Pike, CJ et al. (1993) The Journal of Neuroscience, Vol. 13, 1676-1687. Page, Mattson, MP et al. (1993) Trends in Neuroscience 16: 409-414).
Specifically, the partial peptide β25-35 (amino acid sequence GSNKGAIIGLM) of amyloid β protein exhibits neurotoxicity, and one of the action points of β25-35 is the mitochondrial electron transport system of neurons, It has already been reported that the intensity of cytotoxicity can be determined by measuring the reducing ability of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) reducing ability ( Yankner et al., Science 250, 279-282, 1990; Kaneko et al., Neurochemistry, 32, 148-149, 1993).
Furthermore, senile plaque formation due to deposition of amyloid β protein in the brain is another characteristic pathological change in the brain of Alzheimer's dementia patients, a pathological change that appears earlier than neurogenic changes. (E.g., Seiko, D.J. (1991) Neuron 6: 487-498, Rumbler, B. et al. (1998) The new England of medicine 320: 1446-1452). Etc.)
That is, in Alzheimer-type dementia, aggregation and deposition of amyloid β protein in the brain tissue are triggered, and senile plaques are formed.As a result, neuronal cell death is induced, leading to dementia. is there.
Therefore, a drug that inhibits aggregation and deposition of amyloid β protein is expected to be useful as a therapeutic agent and preventive agent for Alzheimer's dementia. As drugs having such inhibitory activity, rifamycins (WO95 / 11248 pamphlet), hydroquinones (JP-A-8-193026), thionaphthalene derivatives (JP-A-9-95444), pyridine derivatives However, it has not been reported that saccharides have an activity to inhibit aggregation and / or deposition of amyloid β protein.
On the other hand, studies are also being conducted on the mechanism of aggregation of amyloid β protein, which is inherently soluble. In recent years, GMl of acid glycolipids abundant in the brain (Yanagisawa, K et al. Nature Med. Volume 1062-1066 (1995)), sulfated polysaccharide heparin, etc. (Watson, DJ et al. ( (1997) The Journal of Biological Chemistry 272: 31617-31624) has been found to have a function to promote aggregation of amyloid β protein.

The present inventors have predicted that a compound that suppresses peptide aggregation by suppressing the interaction with glycolipids and polysaccharides that cause amyloid β protein aggregation will greatly suppress the formation of Alzheimer amyloid aggregates. It was speculated that such compounds would be effective in suppressing the onset of the disease.
Therefore, as a result of searching for compounds that can suppress the interaction between glycolipids and polysaccharides, which are compounds that promote aggregation of amyloid β protein, and between amyloid β proteins, a promising group of compounds was discovered.
The present invention has been completed in view of the above circumstances, focusing on the interaction between sugar chains and amyloid β protein, producing an artificial sulfated sugar chain, and an inhibitor of amyloid β protein aggregation caused by the artificial sulfated sugar chain Is a problem to be solved.

The present inventors diligently investigated the possibility of a drug that inhibits aggregation and deposition of amyloid β protein, and as a result of mimicking an acidic carbohydrate that interacts with amyloid β protein, artificial sulfate that affects amyloid β protein aggregation I found a chemical sugar. This sugar (chain) was found to have an aggregation inhibitory action by interacting with amyloid β protein, and the present invention was reached.
That is, in the amyloid β protein aggregation regulator of the present invention, part or all of the OH groups are
(I) a sulfo group or a salt of a sulfo group, and
(Ii) a hydrophobic group,
Has been replaced with
Part or all of the remaining OH groups are from H, NH ₂ , —NHR ⁶ and —NR ⁶ R ⁷ (R ⁶ and R ⁷ are groups independently selectable from an alkyl group and an acetyl group). Can be substituted by the group
It contains a compound represented by a chemical structure composed of a sugar chain to which a monosaccharide or two or more monosaccharides are bonded.
In the amyloid β protein aggregation regulator of the present invention, part or all of the OH groups are substituted with —O—φ—NO ₂ (φ is a phenylene group),
Part or all of the remaining OH groups are H, a sulfo group, NH ₂ , NHR ⁶ and NHR ⁶ R ⁷ (R ⁶ and R ⁷ are groups independently selectable from an alkyl group and an acetyl group.) Can be substituted by a group consisting of
It contains a compound represented by a chemical structure composed of a sugar chain to which a monosaccharide or two or more monosaccharides are bonded.
Here, the behavior of this compound with respect to amyloid β protein varies depending on the number of monosaccharides contained and bound in the chemical structure. When the number of monosaccharides in the chemical structure is small, it acts as an amyloid β protein aggregation inhibitor. On the contrary, when the number of monosaccharides is large, it acts as an amyloid β protein aggregation promoter.
The present amyloid β protein aggregation regulator (especially an aggregation inhibitor) suppresses and / or treats the transition to a pathological state against amyloid β protein abnormality such as Alzheimer-type dementia, and amyloid β protein. It is thought that it can be applied to abnormal diagnostic agents. And when acting as an aggregation promoter with respect to amyloid (beta) protein, it is thought that it can mainly be suitably applied mainly to an amyloid (beta) protein abnormality diagnostic agent. Note that the number of monosaccharides that change from an inhibitor to an accelerator cannot be uniquely determined by the type and number of substituents.
Furthermore, the amyloid β protein aggregation regulator of the present invention is characterized by containing a compound represented by the following general formula (1). Among them, as a suitable amyloid β protein aggregation regulator of the present invention, the compound represented by the general formula (1) is a compound represented by the following general formula (2). Although the present compound depends on the type and number of substituents, it mainly tends to exert an action as an amyloid β protein aggregation inhibitor.
(In the formula ^(1), R 1 to 5 is, H, _OH, NH 2, by a group selected from -NHR ^6, and ^-NR 6 ^{R 7,} each independently ^selected; of ^{R 1 to 5} At least one is a sulfo group, a salt of a sulfo group or -O-φ-NO ₂ (φ is a phenylene group); R ⁶ and R ⁷ are groups independently selectable from an alkyl group and an acetyl group. is there.)
In the formula (1), at least one of R ^{1 to 5} is a salt of a sulfo group or a sulfo group, at least one of R ^{1 to 5} is preferably a hydrophobic group.
Here, the hydrophobic group is -A (R ⁸ ) _n (n is 1 or 2; when n = 1, A is —O—, —S—, —NH—; when n = 2, A is —N; R ⁸ is a group that can be independently selected from an alkyl group, a phenyl group, and a naphthyl group, in which part of hydrogen can be substituted with a nitro group, an OH group, a vinyl group, and / or —NHCOCH ₃ . It is desirable.
The amyloid β-amyloid β-protein aggregation controlling agent present invention suitable as a protein aggregation inhibitor, in the compound represented by the general formula (2), said salt of R ¹ is a sulfo group or a sulfo group; wherein R ² And R ³ is an OH group; R ⁴ is —NHCOCH ₃ ; and A is O.
Furthermore, an amyloid β protein abnormality diagnostic agent and diagnostic agent kit comprising these amyloid β protein aggregation regulators as active ingredients can be provided. When acting as an amyloid β protein aggregation promoter, aggregation is rapidly generated by accelerating the aggregation. Therefore, abnormalities in the amyloid β protein (abnormality in quantity or quality) can be detected by observing the generated aggregation. (Abnormal) can be diagnosed. In addition, when acting as an amyloid β protein aggregation inhibitor, it is possible to observe changes in the aggregated amyloid β protein (such as observation under a microscope) or as a probe utilizing the interaction with amyloid β protein. Can be expected.

  The amyloid β protein aggregation regulator of the present invention is a compound derived based on a novel principle, and can control amyloid β protein aggregation based on a new viewpoint.

FIG. 1 is a graph showing the peptide aggregation inhibition effect and its concentration dependency by the amyloid β protein aggregation inhibitor of the present invention in Examples.
FIG. 2 is a graph showing the change over time in the peptide aggregation inhibitory effect of the amyloid β protein aggregation inhibitor of the present invention in Examples.
FIG. 3 is a diagram showing changes in the secondary structure of peptides by the amyloid β protein aggregation inhibitor of the present invention in Examples.
FIG. 4 is a TEM photograph showing changes in peptide aggregation by the amyloid β protein aggregation inhibitor of the present invention in Examples.
FIG. 5 is a graph showing the results of cytotoxicity evaluation in Examples.
FIG. 6 is a graph showing the results of evaluating the relationship between the effect of the amyloid β protein aggregation inhibitor of the present invention and the substituents in Examples.

(Chemical structure)
The amyloid β protein aggregation regulator of the present invention contains a compound represented by a chemical structure composed of a monosaccharide or a sugar chain to which two or more monosaccharides are bound. Some of the OH groups of monosaccharides and sugar chains are substituted. The substituent has either a sulfo group or a salt thereof (hereinafter referred to as “sulfo group or the like”) or a hydrophobic group.
First, one or more OH groups may be substituted with a sulfo group or the like. Examples of the salt of the sulfo group include ordinary salts such as Na and K, and can be freely selected for adjusting the solubility. Substituting one OH group with a sulfo group or the like out of the whole can sufficiently exert its action. In addition, it is preferable to substitute one OH group with a sulfo group or the like for one or two monosaccharide units. The position of substitution with a sulfo group or the like is not limited, but substitution from the 6-position OH group of the monosaccharide or monosaccharide constituting the sugar chain is preferable from the viewpoint of easiness of the synthesis reaction.
One or more OH groups are substituted with hydrophobic groups. Here, the hydrophobic group is used in a general sense and represents a hydrophobic group as a whole. A hydrophobic group having high hydrophobicity is desirable. For example, an aromatic hydrocarbon group such as a phenyl group or a naphthyl group, an aliphatic hydrocarbon group such as an alkyl group, an alkenyl group, or a cycloalkyl group, an alicyclic hydrocarbon group, or a part of the hydrogen that they have. A group substituted with some characteristic group such as a nitro group, an amino group, or an acyl group may be used as necessary (for the purpose of adjusting hydrophobicity or imparting other physiological activity). Substituting one OH group with a hydrophobic group out of the whole can also provide a sufficient effect. In addition, it is preferable to substitute one or two monosaccharide units with a hydrophobic group one by one. The position of substitution with a hydrophobic group is not limited, but substitution of the OH group at the 1-position of a monosaccharide or a monosaccharide constituting a sugar chain is preferable from the viewpoint of ease of synthesis reaction and the like. As the hydrophobic group, —O—φ—NO ₂ (φ is a phenylene group) is particularly desirable. In this case, the nitro group is preferably substituted at the para position.
Furthermore, other OH groups can be substituted with the following characteristic groups as necessary. For example, H, NH ₂ , NHR ⁶ and NHR ⁶ R ⁷ . Here, R ⁶ and R ⁷ are groups independently selectable from an alkyl group and an acetyl group. By substituting these other OH groups with appropriate groups, necessary performance can be imparted. Here, what part of the monosaccharides OH group is substituted by NH ₂ may, glucosamine, as hexosamine such as galactosamine, natural products as N- acetyl form (compound R ⁶ is acetyl SL in NHR ⁶⁾ It is contained in.
Among the compounds described above, particularly preferred compounds are the compounds represented by the aforementioned general formulas (1) and (2). In particular, R ¹ is a sulfo group or a salt of a sulfo group; R ² and R ³ are OH groups; R ⁴ is —NHCOCH ₃ ; and A is O. R ⁸ can be independently selected from an alkyl group, a phenyl group, a naphthyl group, and a group in which part of hydrogen of these groups is substituted with a nitro group, an OH group, a vinyl group, and / or —NHCOCH ₃ .
Furthermore, if the above-mentioned compound can exist as an active ingredient at an action site such as in the body, it can be converted into a prodrug. For example, improvement of absorbability and bioavailability, enhancement of selective migration to target tissues (eg, fat solubilization to improve permeability of blood-brain barrier), inhibition of metabolism and sustained action Examples include prodrug formation for the purpose of improvement. In that case, an appropriate chemical structure is selected according to the use mode described later.
(Usage: Use as an inhibitor of deposition and aggregation by suppressing aggregation of amyloid β protein)
The amyloid β protein aggregation regulator of the present invention is a compound having excellent affinity with amyloid β protein, and can control aggregation of amyloid β protein. For example, in the case of inhibiting or suppressing aggregation, interaction with amyloid β protein can be generated to suppress aggregation. As a result, even if there is an abnormality in the amyloid β protein, aggregation is difficult to be generated, and the generated aggregation can be redissolved, so that relief or cure of symptoms can be expected.
Therefore, the amyloid β protein aggregation regulator (particularly the aggregation inhibitor / inhibitor) of the present invention can be suitably used as an aggregation and / or deposition inhibitor of amyloid β protein. In that case, a pharmaceutically acceptable carrier can be blended.
Examples of the pharmaceutically acceptable carrier in this case include excipients, disintegrants, binders, coating agents, pH adjusters, solubilizers, stabilizers, thickeners, and the like. The amyloid β protein aggregation regulator of the present invention is provided as an oral preparation such as a soft capsule, hard capsule, tablet, granule, powder, suspension, liquid, syrup, injection, suppository or external preparation. it can.
Examples of additives include vegetable oils (eg, corn oil, cottonseed oil, coconut oil, almond oil, peanut oil, olive oil, etc.), oily esters such as medium chain fatty acid glyceride oil, glycerin esters such as mineral oil, tricaprylin, and triacetin. Alcohols such as ethanol and propanol, physiological saline, propylene glycol, polyethylene glycol, petrolatum, cocoa butter, animal fats and oils, cellulose derivatives (crystalline cellulose, hydroxypropylmethylcellulose, methylcellulose), polyvinylpyrrolidone, polyethylene glycol and the like.
(Usage mode: diagnostic agent and diagnostic kit for amyloid β protein aggregation)
The amyloid β protein aggregation regulator of the present invention is a compound having excellent affinity for amyloid β protein. Therefore, when an abnormality occurs in the amyloid β protein in a living body (abnormality in concentration, abnormality in properties, etc.), the abnormality can be detected by causing an interaction with the amyloid β protein. . For example, in the case of the amyloid β protein aggregation regulator of the present invention, which is a compound that promotes aggregation, it is added to a biological sample such as blood or cerebrospinal fluid to produce aggregation when an abnormality occurs in amyloid β protein, The abnormality can be detected promptly.
In addition, when the amyloid β protein aggregation regulator suppresses / inhibits amyloid β protein aggregation, luminescence occurs when the amyloid β protein aggregation regulator interacts with amyloid β protein. As described above, by introducing a fluorescent label or the like into this compound, a simple diagnostic agent and the main constituent of the diagnostic agent for the risk that abnormalities in amyloid β protein occur and amyloid β protein deposition or aggregation occurs. Application as an amyloid β protein aggregation regulator that can be used as a diagnostic kit capable of providing components can be expected.

Examples of the present invention will be shown below to clarify the present invention more specifically. It goes without saying that the present invention is not restricted by the description of such embodiments. In addition to the following examples, in addition to the description in the embodiment of the present invention described above, the present invention is based on the knowledge of those skilled in the art unless it departs from the spirit of the present invention. It should be understood that various changes, modifications, improvements and the like can be made.
(Synthesis of used samples)
(1-1) As an example of the amyloid β protein aggregation regulator of the present invention, the sulfated sugar chain used in this example (para-nitrophenyl 2-acetamido-2-deoxy-6-sulfonate-β-D- Glucopyranoside; the following formula (3)) was synthesized according to the following reaction formula.
1) Synthesis of chloro 2-acetamido-3,4,6-tri-O-acetyl-deoxy-α-D-glucopyranoside Acetylglucosamine (Tokyo Chemical Co., Ltd., 4.90 g, 20.9 mmol) was mixed with chloroacetic acid (Tokyo Kasei). 30 mL) was added, and the reaction solution was stirred for 24 hours. Ice water was added to the reaction solution to stop the reaction, followed by extraction with chloroform, followed by washing twice with saturated NaHCO ₃ water and once with ice water. After drying over magnesium sulfate, the mixture was concentrated and purified by silica gel chromatography (eluent: chloroform / methanol = 50/1 → chloroform only).
The yield was 560 mg, and the yield was 6.9%. _{^{1 H-NMR (CDCl 3,}} 500MHz.): D6.19 (d, J = 4.0,1H, H-1), 5.76 (d, J = 8.5,1H, CONH), 5. 32 (dd, J = 9.10, 1H, H-3), 5.22 (m, 1H, H-4), 4.53 (ddd, J = 3.5, 9.0, 10.5, 1H, H-2), 4.30-4.25 (m, 2H, H-5, H-6ProR), 4.16-4.12 (m, 1H, H-6proS), 2.05 (dd , 12H, Ac).
IR (KBr): 1747 (C = O), 1228 (C-O), 601 (Cl).
2) Synthesis of para-nitrophenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside Chloro 2-acetamido-3,4,6-tri-O-acetyl- Deoxy-α-D-glucopyranoside (560 mg, 1.53 mmol) is dissolved in 5 mL of methylene chloride, 5 mL of 1N NaOH, Bu ₄ NBr (manufactured by Tokyo Chemical Industry Co., Ltd., 490 mg, 1.5 mmol) and p-nitrophenol (Tokyo Chemical Industry Co., Ltd.). Product, 430 mg, 3.1 mmol) was added and stirred for 12 hours. Then, it extracted with ethyl acetate and wash | cleaned by 1N NaOH, a saturated salt solution, and water. Thereafter, the product was purified by silica gel chromatography (eluent: chloroform: methanol = 100: 1).
The yield was 290 mg and the yield was 40%. ¹ H-NMR (CDCl ₃ , 500 MHz): d8.20 (dt, 2H, Ph), 7.07 (dt, 2H, Ph), 5.60 (1H, NHCO), 5.47 (d, 1H, H-1), 5.46 (t, J = 10.5 Hz, 1H, H-3), 5.15 (t, J = 9.5 Hz, 1H, H-4), 4.29 (dd, J = 5.5, 12.0 Hz, 1H, H-6proR), 4.20 (dd, J = 2.0, 5.5, 9.5 Hz, 1H, H-5), 4.10 (ddd, J = 8.5, 8.5, 10.5 Hz, 1H, H-2), 3.94 (ddd, J = 2.0, 5.5, 9.5 Hz, 1H, H-5), 2.08 , 2.067 (sxs, s, 3H, OAc), 1.97 (s, 3H, NHAc). _{IR (KBr): 1745 (C} = O), 1523 (NO 2), 1344 (NO 2), 1228 (C-O).
3) Synthesis of para-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside Para-nitrophenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside (130 mg, 0.29 mmol) was dissolved in 10 mL of methanol, sodium methoxide (manufactured by Tokyo Chemical Industry Co., Ltd., 10 mg, 0.19 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Neutralized with a cation exchange resin (Amberlyst, Organo) and filtered. The filtrate was concentrated and the target compound was obtained by recrystallization.
The yield was 100 mg and the yield was 100%. ¹ H-NMR (D ₂ O, 500 MHz): d8.26 (dt, 2H, Ph), 7.20 (dt, 2H, Ph), 5.32 (d, J = 8.5 Hz, 1H, H— 1), 4,03 (dd, J = 8.5, 10.5 Hz, H1, H-2), 3.96 (dd, J = 2.5, 12.5 Hz, H-1, H-6proS) , 3.80 (dd, J = 5.5, 12.5 Hz, 1H, H-6proR), 3.69 (m, 2H, H-3, H-5), 3.38 (dd, J = 9 .0, 10.0 Hz, 1H, H-4), 2.01 (s, 3H, Ac). IR (KBr): 3307 (OH), 1521 (NO ₂ ), 1346 (NO ₂ ), 1250 (C—O).
4) Synthesis of para-nitrophenyl 2-acetamido-2-deoxy-6-sulfonate-β-D-glucopyranoside 200 mg of para-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside was placed in a 30 mL eggplant flask. Dissolved in 8 mL DMF. After dissolving well, sulfatrioxide trimethylamine complex (Sulfur trioxide-trimethylamine complex) Me ₃ N · SO ₃ 3e. q. (Sigma Aldrich, 240.5 mg) was added dropwise and stirred for 3 hours. Meanwhile, the reaction was followed by TLC (developing solvent: chloroform: methanol = 2: 1). After completion of the reaction, 14 mL of methanol was added, stirred at room temperature for 3 hours, concentrated with an evaporator, and separated and purified with a reverse phase column. Thereafter, 2 mL of a cation exchange resin was added to the obtained compound and stirred for 3 days. Thereafter, the cation exchange resin was filtered off with a glass filter, and the obtained liquid was concentrated and freeze-dried.
The yield was 123.1 mg, and the yield was 47.2%. 1H-NMR (500 MHz, D ₂ O, 30 ° C.) δ 8.02 (m, 2H, H _methyl of phenyl group), 7.00 (m, 2H, _ortho of phenyl group), 5.16 (d, 1H, J = 8.5 Hz, H-1β), 4.27 (dd, 1H, J = 2.0, 11.5 Hz, H-6pros), 4.11 (dd, 1H, J = 5.5) 11.5 Hz, H-6proR), 3.92 (dd, 1H, J = 8.5, 10.5 Hz, H-2), 3.80 (m, 1H, H-5), 3.58 (dd , 1H, J = 10.5, 9.0 Hz, H-3), 3.48 (dd, 1H, J = 9.0, 10.0 Hz, H-4), 2.74 (s, 9H, 3 X Me), 1.89 (s, 3H, Ac).
(1-2) As an example of the amyloid β protein aggregation regulator of the present invention, the following compounds (A) and (B) were synthesized.
The compound (pNP GlcNAc) of the above (A) is obtained as a synthetic intermediate of the aforementioned paranitrophenyl-6-sulfonyl-β-D-acetylglucosamine (pNP 6-Sulfo-GlcNAc). The compound of (B) (Allyl 6-Sulfo-GlcNAc) can be synthesized in the same manner by employing allyl alcohol instead of p-nitrophenol used in the synthesis of the above-described pNP 6-Sulfo-GlcNAc.
(2) Synthesis of Amyloid β1-42 and Amyloid β1-40 Peptide Amyloid β1-42 peptide was synthesized as the amyloid β protein used in the following examples.
Peptide synthesis was performed using a peptide synthesizer (Applied Biosystems) by a solid phase method using Fmoc-amino acid as a raw material. After completion of the synthesis, the peptide was excised from the resin and purified by high performance liquid chromatography using a reverse phase column (C18, Showa Denko). It was confirmed by mass spectrometry (MALDI-TOF-MS, Voyager, Applied Biosystems) that the obtained amyloid β1-42 peptide had the target amino acid sequence. The amyloid β1-42 was lyophilized and used in the following examples. Furthermore, amyloid β1-40 was synthesized in the same manner. Hereinafter, when it is described as “amyloid β protein”, it means amyloid β1-42 unless specifically limited to “amyloid β1-40”.
(3) Test method of amyloid β protein aggregation inhibitory activity The test was performed by a method using a dye (thioflavin T). Thioflavin T (ThT) is reported to bind to the β sheet structure of aggregated proteins such as amyloid β protein and emit new fluorescence (482 nm) that was not shown in the free state (Harry Levine III). 1993, Protein Science 2, 404-410). The intensity of fluorescence is proportional to the degree of aggregation of the bound protein. By measuring the degree of aggregation of the amyloid β protein containing the drug by the intensity of fluorescence of ThT bound thereto, the amyloid β protein aggregation inhibitory activity of the drug can be examined.
Specifically, 5 μL was taken from a solution of amyloid β protein dissolved in 0.02% NH ₄ OH aqueous solution containing pNP 6-Sulfo-GlcNAc (formula (3) above), and this was taken up with 50 mM Gly-NaOH (pH 9. Add to a solution of ThT dissolved at a concentration of 5 μM in 0) and stir. After stirring, the fluorescence of the solution is measured immediately with a spectrotrofluorometer (FP-777 manufactured by JASCO) at an excitation wavelength of 415 nm and a fluorescence wavelength of 482 nm. The measurement was performed while changing the amount of sulfated carbohydrate or over time. If the increase in fluorescence is suppressed as compared with a solution containing only amyloid β protein that does not contain sulfated saccharide, the drug is determined to have amyloid β protein aggregation inhibitory activity.
FIG. 1 shows the results one day after incubating amyloid β protein and pNP 6-Sulfo-GlcNAc. FIG. 1 shows the peptide aggregation-inhibiting effect and its concentration dependence by pNP 6-Sulfo-GlcNAc in this example. After adding 10-1000 μM of pNP 6-Sulfo GlcNAc to a 10 μM amyloid β protein aqueous solution and incubating for one day, ThT was added to measure fluorescence. The bar graph display in the figure represents the concentration of pNP 6-Sulfo GlcNAc, which is 0 μM as a control and the others are aggregation inhibitors. As is clear from FIG. 1, ThT showed strong fluorescence when pNP 6-Sulfo-GlcNAc was not added, whereas when pNP 6-Sulfo-GlcNAc of 10 μM or more was added, the fluorescence intensity was increased. A decrease was observed. From this, it is clear that pNP 6-Sulfo-GlcNAc inhibits aggregation of amyloid β protein. It is known that when heparin is added at 100 μM, the fluorescence intensity becomes 245 (au) after one day. That is, it was found that heparin exerts an aggregating action on amyloid β, and pNP 6-Sulfo-GlcNAc exerts an inhibitory action on aggregation.
As a result of examination under the same conditions except that amyloid β protein was replaced with amyloid β1-40, the fluorescence intensity was 153 (au) in the system in which neither heparin nor pNP 6-Sulfo-GlcNAc was added. When 100 μM heparin was added, the fluorescence intensity was 181 (au), and when 100 μM pNP 6-Sulfo-GlcNAc was added, the fluorescence intensity was 160 (au). That is, it was found that pNP 6-Sulfo-GlcNAc exerts an action of promoting aggregation, although not as much as heparin, with respect to amyloid β1-40.
FIG. 2 shows the results of measuring changes over time from day 1 to day 7 after adding amyloid β protein and 100 μM sulfated carbohydrate and incubating. FIG. 2 shows the change over time in the peptide aggregation inhibitory effect of pNP 6-Sulfo-GlcNAc in this example. The fluorescence value measured by adding 100 μM of pNP 6-Sulfo GlcNAc to 10 μM amyloid β protein aqueous solution, incubating for 7 days, and adding ThT after each elapsed time is shown. Day 0 is one hour later. In the graph, in the graph, ◯ represents only the control amyloid β protein, and □ represents the value after adding 100 μM of the aggregation inhibitor pNP 6-Sulfo GlcNAc. As can be seen from FIG. 2, when no pNP 6-Sulfo-GlcNAc was added, it showed high fluorescence after incubation and was found to be aggregated. On the other hand, it was found that when pNP 6-Sulfo-GlcNAc was added, the fluorescence intensity was low and aggregation was suppressed even after incubation up to 7 days. That is, it was found that aggregation was suppressed over a long period of one week.
(4) Method for analyzing structure of amyloid β protein The structure was measured by circularly polarized dichroism (CD) spectrum. Specifically, 10 μM of a sample solution of amyloid β protein containing pNP 6-Sulfo-GlcNAc was taken, and a CD spectrum from a wavelength of 260 nm to 190 nm was measured with a CD spectrometer (J-725, manufactured by JASCO). In addition, a comparison was made with the addition of natural polysaccharide heparin, which is an aggregation promoter. In the CD spectrum, peaks based on a 218 nm β-sheet structure were compared. Here, the β-sheeted amyloid β protein has a minimum value at 218 nm.
FIG. 3 shows the CD spectrum after 1 day of incubation. As is apparent from FIG. 3, the changes in the 10 μM amyloid β protein solution as it was after adding 100 μM pNP 6-Sulfo GlcNAc and 100 μM heparin after incubation for 1 day were found. When 6-Sulfo GlcNAc was added, the peak at 218 nm was small, but it was found that a strong peak was shown when nothing was added and when the aggregation polysaccharide, heparin, was added. Therefore, it was found that the β sheet property was suppressed in the amyloid β protein solution to which pNP 6-Sulfo-GlcNAc was added.
(5) Observation of aggregation of amyloid β protein The aggregation of amyloid β protein was observed using a transmission electron microscope. Specifically, pNP 6-Sulfo-GlcNAc 100 μM and amyloid β protein 10 μM were incubated for 1 day, copied onto a carbon grid membrane, and negatively stained with a 2% phosphotungstic acid aqueous solution. The form of this sample was observed with a transmission electron microscope (H-800, manufactured by Hitachi, Ltd.).
FIG. 4 shows the results of TEM observation after 1 day of incubation. (A) is obtained by adding heparin to amyloid β protein and incubated, and (b) is obtained by incubating amyloid β protein as it is, and a clear fibrous amyloid aggregate is observed. On the other hand, (c) is obtained by adding pNP 6-Sulfo-GlcNAc as an aggregation inhibitor to the peptide. Only small aggregates are observed, and fibrous amyloid protein aggregates are not observed at all. It was.
(6) Evaluation of cytotoxicity The concentration of amyloid β was divided into three stages, 0 M, 10 ⁻⁸ M, 10 ⁻⁷ M and 10 ⁻⁶ M, and 100 μM heparin, 0.4 mM HCl, 100 mM HEPES, and 0.1 M NaCl. The number of cells after culturing Hela cells for 2 days at 37 ° C. in the presence was counted. Furthermore, the case where 100 μM of pNP 6-Sulfo-GlcNAc was added was also evaluated. And the survival rate which calculated the case where the amyloid (beta) was not added as 100% was computed. The evaluation results are shown in FIG. As apparent from the figure, it was revealed that the toxicity of amyloid β was reduced by adding pNP 6-Sulfo-GlcNAc. In particular, the concentration of amyloid beta is less than 10 -6 ^M (well details 10 -7 ^M or less) in the case of it is revealed that the higher survival rate than without addition of amyloid beta.
(7) Evaluation of Substituents As for pNP GlcNAc (compound (A)) and Allyl 6-Sulfo-GlcNAc (compound (B)), in the same manner as pNP 6-Sulfo-GlcNAc, the amyloid β aggregation inhibitory action was observed in the above (3) amyloid It examined by the method similar to the test method of (beta) protein aggregation inhibitory activity.
100 μM each of pNP GlcNAc, Allyl 6-Sulfo-GlcNAc, and pNP 6-Sulfo GlcNAc was added to 10 μM amyloid β protein aqueous solution and incubated for 1 day, and then ThT was added to measure fluorescence. The results are shown in FIG.
As is clear from FIG. 6, it was revealed that all of pNP GlcNAc, Allyl 6-Sulfo-GlcNAc, and pNP 6-Sulfo GlcNAc exhibit the ability to inhibit aggregation. In particular, it was found that when pNP 6-Sulfo-GlcNAc was added, the highest aggregation inhibition ability was exhibited. Therefore, it was suggested that both the sulfo group and the hydrophobic group (p-nitrophenol group in this evaluation) play an important role in the aggregation inhibition ability.
(8) Evaluation of aggregate dissociation action The action of aggregated amyloid β was examined. In the same manner as the test method of (3) amyloid β protein aggregation inhibitory activity described above, 10 μM amyloid β was agglutinated by allowing it to stand at 37 ° C. for 1 day, as it was and to which 100 μM heparin was added. I let you. Thereafter, 100 μM of pNP 6-Sulfo GlcNAc was added and allowed to stand at 37 ° C. for 1 day, and then the degree of aggregation was evaluated by the same method as the test method for (3) amyloid β protein aggregation inhibitory activity.
As a result, in the system to which heparin was added, the fluorescence intensity of 219 (au) was increased to 184 (au) (84% of the original state) by adding pNP 6-Sulfo GlcNAc. Decreased to. In the case of amyloid β alone, the fluorescence intensity of 183 (au) was reduced to 166 (au) (91% of the original state) by adding pNP 6-Sulfo GlcNAc. did. Therefore, it was revealed that the aggregate can be dissociated even in the case of amyloid β once the aggregation has progressed. That is, it has been found that there is a possibility that aggregation of amyloid β in vivo can be reduced.

Claims (9)

Some or all of the OH groups are
(I) a sulfo group or a salt of a sulfo group, and
(Ii) a hydrophobic group,
Has been replaced with
A part or all of the remaining OH groups are groups composed of H, NH ₂ , NHR ⁶ and NHR ⁶ R ⁷ (R ⁶ and R ⁷ are groups independently selectable from an alkyl group and an acetyl group). Can be replaced by
A amyloid β protein aggregation regulator comprising a compound represented by a chemical structure composed of a sugar chain to which a monosaccharide or two or more monosaccharides are bonded. Some or all of the OH groups are
-O-φ-NO ₂ (φ is a phenylene group),
Part or all of the remaining OH groups are H, a sulfo group, NH ₂ , NHR ⁶ and NHR ⁶ R ⁷ (R ⁶ and R ⁷ are groups independently selectable from an alkyl group and an acetyl group.) Can be substituted by a group consisting of
A amyloid β protein aggregation regulator comprising a compound represented by a chemical structure composed of a sugar chain to which a monosaccharide or two or more monosaccharides are bonded. An amyloid β protein aggregation regulator comprising a compound represented by the following general formula (1):
(In the formula ^(1), R 1 to 5 is, H, _OH, NH 2, by a group selected from -NHR ^6, and ^-NR 6 ^{R 7,} each independently ^selected; of ^{R 1 to 5} At least one is a sulfo group, a salt of a sulfo group or -O-φ-NO ₂ (φ is a phenylene group); R ⁶ and R ⁷ are groups independently selectable from an alkyl group and an acetyl group. is there.) In the formula (1), at least one of R ^{1 to 5} is a sulfo group or a salt of a sulfo group, and at least one of R ^{1 to 5} is a hydrophobic group. The amyloid β protein aggregation regulator described. The hydrophobic group is -A (R ⁸ ) _n (n is 1 or 2; when n = 1, A is —O—, —S—, —NH—; when n = 2, A is —N; R ⁸ is A part of hydrogen is a group which can be independently selected from an alkyl group, a phenyl group and a naphthyl group, which can be substituted with a nitro group, an OH group, a vinyl group and / or —NHCOCH ₃ . Item 5. The amyloid β protein aggregation regulator according to item 1, 3 or 4. The amyloid β protein aggregation regulator according to any one of claims 2 to 5, wherein the compound represented by the general formula (1) is a compound represented by the following general formula (2).
The amyloid β protein aggregation control according to claim 6, wherein R ¹ is a sulfo group or a salt of a sulfo group; R ² and R ³ are OH groups; R ⁴ is -NHCOCH ₃ ; and A is O. Agent. An amyloid β protein abnormality diagnostic agent comprising the amyloid β protein aggregation regulator according to any one of claims 1 to 7 as an active ingredient. An amyloid β protein abnormality diagnostic agent kit comprising the amyloid β protein aggregation regulator according to any one of claims 1 to 7.