JP2006232705A - Anti-polyglutamine disease agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel anti-polyglutamine disease agent (drug). <P>SOLUTION: The anti-polyglutamine disease agent is constituted of geldanamycin or a geldanamycin derivative as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a drug (anti-polyglutamine disease agent) for polyglutamine disease and use thereof.

Spinal and bulbar muscular atrophy (SBMA) is an inherited neurodegenerative disease and was first identified as polyglutamine disease (Non-patent Document 1). Polyglutamine disease is an inherited neurodegenerative disease caused by abnormal extension of CAG repeat sequence, and includes SBMA, Huntington's disease and spinocerebellar degeneration. In SBMA, the polyglutamine chain in the androgen receptor (AR) is abnormally elongated, and this mutant AR (pathological AR) forms or accumulates insoluble inclusion bodies in nerve cells. And is considered to have neurotoxicity (Non-patent Document 2). The number of CAG repeats in AR is abnormally extended to 40 to 62 in SBMA patients, while 5 to 33 in healthy individuals (Non-patent Document 3). Moreover, the phenomenon that the number of CAG repeats is proportional to the onset age and severity of the disease has been confirmed (Non-Patent Document 4). The same phenomenon has been observed in other polyglutamine diseases, and it is considered effective to specifically inactivate or reduce the causative protein in which polyglutamine chains are abnormally elongated in the treatment of polyglutamine diseases. .
The present inventors have already found that the testosterone-dependent nuclear accumulation of mutant AR, which is a pathogenic protein, forms the basis of the disease state using a transgenic mouse which is a model animal of SBMA (Non-patent Document 5). In addition, it has been announced that suppression of testosterone secretion by luteinizing hormone stimulating hormone (LHRH) analogs leads to dramatic therapeutic effects (Non-patent Document 6, Patent Document 1). A clinical trial is currently underway for this drug.

  On the other hand, heat shock protein (HSP) is known as a molecule that inhibits aggregation of mutant proteins. Heat shock protein is a molecular chaperone whose expression is induced by stress such as heat, refolding the shape of an abnormal protein that has undergone structural changes (refolding), not only inhibiting its aggregation, but also a mutant protein in the proteasome It is believed to promote the degradation of The present inventors confirmed in a cultured cell model of bulbar spinal muscular atrophy that suppression of nuclear aggregation of mutant AR by enhancing the expression of Hsp70, which is one of heat shock proteins (Non-Patent Literature). 7) The improvement of neurological symptoms was found by mating a mouse model of bulbar spinal muscular atrophy and a mouse with high Hsp70 expression (Non-patent Document 8). Similar effects have been confirmed in the Drosophila model of bulbospinal muscular atrophy (Non-Patent Document 9) and in the Drosophila and mouse model of spinocerebellar degeneration (Non-Patent Documents 10 and 11). Conceivable.

  In addition, a histone deacetylase inhibitor having an action to improve transcription function has also been confirmed to have a therapeutic effect in this mouse (Non-patent Document 12), and similar results have been reported in a model mouse for Huntington's disease. (Non-patent Documents 13 and 14). In addition, in mouse models of Huntington's disease, therapeutic effects such as congo red and trehalose, which have the effect of inhibiting the aggregation of mutant proteins, and rapamycin, which have the effect of promoting the degradation of mutant proteins, have been reported. It has not reached.

International Publication No. 2004/016083 Pamphlet La Spada AR et al. Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy.Nature. 352, 77-79 (1991). Poletti A et al. The polyglutamine tract of androgen receptor: from functions to dysfunctions in motor neurons. Front Neuroendocrinol. 25, 1-26 (2004). Tanaka F, Doyu M, Ito Y, Matsumoto M, Mitsuma T, Abe K, Aoki M, Itoyama Y, Fischbeck KH, Sobue G et al. Founder effect in spinal and bulbar muscular atrophy (SBMA) .Hum Mol Genet. 5, 1253-1257 (1996). Doyu M, Sobue G, Mukai E, Kachi T, Yasuda T, Mitsuma T, Takahashi A et al. Severity of X-linked recessive bulbospinal neuronopathy correlates with size of the tandem CAG repeat in androgen receptor gene.Ann Neurol. 32, 707 -710 (1992). Katsuno M, Adachi H, Kume A, Li M, Nakagomi Y, Niwa H, Sang C, Kobayashi Y, Doyu M, Sobue G. 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Pharmacokinetics, tissue distribution, and metabolism of 17- (dimethylaminoethylamino) -17-demethoxy geldanamycin (NSC 707545) in CD2F1 mice and Fischer 344 rats.Cancer Chemother Pharmacol. 47, 291-302 (2002 ). Pratt WB & Toft DO.Regulation of signaling protein function and trafficking by the hsp90 / hsp70-based chaperone machinery.Exp Biol Med (Maywood) .228, 111-133 (2003). Neckers L et al. Geldanamycin as a potential anti-cancer agent: its molecular target and biochemical activity.Invest New Drugs. 17, 361-73 (1999). Bonvini PU et al. Ubiquitination and proteasomal degradation of nucleophosmin-anaplastic lymphoma kinase induced by 17-allylamino-demethoxygeldanamycin: role of the co-chaperone carboxyl heat shock protein 70-interacting protein.Cancer Res. 64, 3256-3264 (2004). Stebbins CE et al. Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent.Cell. 89, 239-250 (1997). Kamal A et al. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature.425,407-410 (2003). Fang Y et al. Hsp90 regulates androgen receptor hormone binding affinity in vivo. J Biol Chem. 271,28697-28702 (1996). Solit DB et al. 17-Allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2 / neu and inhibits the growth of prostate cancer xenografts.Clin Cancer Res. 8, 986-993 (2002). Bagatell R et al. Destabilization of steroid receptors by heat shock protein 90-binding drugs: a ligand-independent approach to hormonal therapy of breast cancer.Clin Cancer Res. 7, 2076-2084 (2001). Blagosklonny MV et al. Toretsky J & Neckers L. Geldanamycin selectively destabilizes and conformationally alters mutated p53. Oncogene. 11, 933-939 (1995). Blagosklonny MV et al. Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90.Proc Natl Acad Sci U S A. 93, 8379-8383 (1996). Nagata Y et al. The stabilization mechanism of mutant-type p53 by impaired ubiquitination: the loss of wild-type p53 function and the hsp90 association.Oncogene. 18, 6037-49 (1999). Zou J et al. Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell. 94, 471-480 (1998). Winklhofer KF et al. Geldanamycin restores a defective heat shock response in vivo. J Biol Chem. 276, 45160-45167 (2001). Sittler A, Lurz R, & Wanker EE. Geldanamycin activates a heat shock response and inhibits huntingtin aggregation in a cell culture model of Huntington's disease.Hum Mol Genet. 14, 1307-1315 (2001). Hay DG, Sathasivam K & Bates GP. Progressive decrease in chaperone protein levels in a mouse model of Huntington's disease and induction of stress proteins as a therapeutic approach.Hum Mol Genet. 13. 1389-1405 (2004). Tian ZQ, et al. Synthesis and biological activities of novel 17-aminogeldanamycin derivatives. Bioorg. Med. Chem. 12, 5317-5329 (2004).

  LHRH analog treatment for bulbar spinal muscular atrophy is being studied for clinical application using animal models, but this treatment method is unlikely to be effective for polyglutamine diseases other than spinal and spinal muscular atrophy. On the other hand, drugs such as histone deacetylase inhibitors that have been reported to have therapeutic effects in Huntington's disease model mice have not been established for human safety and are toxic for clinical use in patients. It is considered necessary to evaluate and overcome this. For these reasons, there is an urgent need to search for low toxicity compounds that can be clinically applied. Then, an object of this invention is to provide the novel chemical | medical agent (medicine) with respect to a polyglutamine disease which can meet such a request | requirement.

  Under the above object, the present inventors paid attention to geldanamycin (GA) and its derivatives as drug candidates for polyglutamine disease. GA is the most classic Hsp90 inhibitor, was found as an antifungal agent in 1970 (Non-patent Document 15), and was known to have an excellent antitumor effect (Non-patent Document 16). However, GA was later found to have unacceptable liver toxicity in vivo (Non-patent Document 17), and a search for a less toxic derivative was conducted. As a result, 17-allylamino-17-demethoxygeldanamycin (17-AAG) in which part of the side chain of GA was substituted was found (Non-patent Document 18). Currently, 17-AAG is approaching clinical application as a novel anticancer agent (Non-patent Document 19). Phase I clinical trials are almost complete (Non-Patent Documents 21 to 23), and Phase II has already begun. 17-AAG is an excellent derivative having almost the same pharmacological activity as GA (Non-patent Document 25) while suppressing the side effects seen in GA (Non-patent Document 24). In animal models, it has been confirmed that 17-AAG has good tissue transferability when administered intravenously or intraperitoneally, but on the other hand, its high fat solubility makes oral administration difficult. (Non-patent Document 26). In recent years, 17- (2-dimethylaminoethyl) amino-17-demethoxygeldanamycin (17-DMAG), a further derivative of 17-AAG that can also be administered orally, has been It has been developed (Non-Patent Document 27), and clinical application is being attempted following 17-AAG.

  Hsp90 forms a complex with an Hsp90-dependent client protein together with other molecular chaperones (Hsp70, Hop, p23, p50, etc.), and plays an important role in the stabilization and functional expression of the protein (Non-patent Document 28). ). It has been clarified that administration of a GA derivative inhibits the formation of such an Hsp90 complex, inactivates the function of the protein, and further degrades it in the ubiquitin / proteasome system (Non-patent Documents 29 and 30). So far, it has been clarified that many of the known proteins are Hsp90 client proteins, among which many tumor-related proteins are included (Non-patent Document 28). GA derivatives exert a pharmacological effect by inhibiting the complex formation of these client proteins with Hsp90 (Non-patent Document 31), but the amount of protein in tumor cells is higher than that in normal cells. Since a protein complex is formed, the sensitivity to GA derivatives is very high (Non-patent Document 32). From this, GA derivatives including 17-AAG are said to have a selective antitumor effect, although the mechanism of action impairs the function of a constitutively expressed protein called Hsp90.

This time, the present inventors paid attention to the drug effect of the GA derivative that reduces the expression level of the target protein. As described above, SBMA is a disease in which a pathological protein with an extended polyglutamine chain in AR is abnormally accumulated in nerve cells. We have already demonstrated that hormonal therapy targeting AR is an effective treatment for SBMA. That is, it was shown that by reducing the testosterone which is an AR ligand, AR translocation into the nucleus was inhibited, and formation of inclusion bodies in the nucleus was remarkably suppressed (Non-patent Documents 5 and 6). AR is also a typical Hsp90 client protein, and Hsp90 is essential for its functional expression (Non-patent Document 33). As shown in FIG. 2, the GA derivative causes a change in the formation of the Hsp90-protein complex. As a result, the function of the client protein is deactivated and decomposed in the ubiquitin / proteasome system (Non-patent Document 20). Thus, the phenomenon that the steroid receptors such as AR, which is a client protein, are impaired in function and further promoted by GA derivatives has already been confirmed in cultured cell models and mouse models. (Nonpatent literature 20, 34, 35).
A variant of p53 is a typical Hsp90-dependent client protein. Although expression of this mutant p53 is observed in about 50% of all malignant tumors, it is interesting that the mutant is significantly more sensitive to GA than normal p53 (Non-patent Documents 36 to 38). The mutant pathological protein is structurally unstable and appears to require more Hsp90 for its stabilization. Similarly, if AR is also a mutant with an extended polyglutamine, if the sensitivity to GA derivative is enhanced, the use of GA derivative will increase the expression level of pathological protein more effectively and selectively. It can be reduced in quantity, i.e. an excellent therapy can be provided.

In view of the fact that AR with an abnormally extended polyglutamine chain is also considered to be dependent on Hsp90, the present inventors have mutated AR by administration of an Hsp90 inhibitor by the same mechanism as that of tumor intracellular proteins. We thought it was possible to reduce the quantity. If the expression level of mutant AR (pathological AR) can be suppressed, an excellent therapeutic effect on SBMA can be expected.
On the other hand, Hsp90 inhibitors have a pharmacological action that activates heat shock transcription factor (HSF-1), a transcription factor of heat shock protein, and nonspecifically increases antistress molecular chaperones such as Hsp70 and Hsp40. (Non-patent documents 39 and 40) were also noted. That is, since the Hsp90 inhibitor has one aspect as a molecular chaperone inducer, it was considered that this action can also provide a therapeutic effect on SBMA.
Regarding the possibility of application of GA and its derivatives (especially 17-AAG) to polyglutamine diseases, it has been reported that GA suppresses the inclusion body formation of pathogenic proteins in a cultured cell model of polyglutamine disease (non- Patent Documents 41 and 42), there is no report that the effect of drugs such as GA was examined at the level of individual animals such as mice.
Accordingly, the present inventors have selected 17-AAG, which has been studied for its most pharmacological action and kinetics among GA derivatives, and whose clinical application is imminent, and its therapeutic effect on SBMA has been shown to be a cultured cell model and a pathogenic gene ( Evaluation was performed with a model mouse into which the number of CAG repeats in the AR was 97). As a result, it was surprisingly found that the expression level of pathological AR was significantly reduced by administration of 17-AAG in a cultured cell model. Furthermore, it was confirmed that 17-AAG exerts a dose-dependent significant therapeutic effect in model mice. These results revealed that 17-AAG is actually effective in the treatment of SBMA. In addition, since compounds having a GA basic skeleton (GA derivatives and GA itself) can act by the same mechanism as 17-AAG, these compounds are also considered to be promising candidates for SBMA therapeutics. It was.

  On the other hand, in polyglutamine diseases such as SBMA, it is caused by abnormal extension of CAG repeats in specific genes, the longer the CAG repeat length, the younger the onset age becomes, the more severe the expression, and the expression-promoting phenomenon (through generations) A phenomenon in which the age of onset becomes younger every time), the nerve tissue is selectively damaged, and the aggregation of the mutant protein is observed in the nerve cell and the inclusion body in the nucleus is observed, Many similarities are recognized. The mutant protein accumulated in the nucleus impairs the function of transcriptional regulatory factors such as CBP (c-AMP response element binding protein-binding protein), which is considered to be the main cause of neuronal damage in polyglutamine disease ( Steffan JS, et al. Proc Natl Acad Sci USA 2000; 97: 6763-6768 .; Nucifora FC Jr, et al. Science 2001; 291: 2423-2428.). The above findings obtained from experiments using SBMA model animals, which are one of polyglutamine diseases, can be applied to other polyglutamine diseases from the above-mentioned many common points including disease causes. The nature is extremely high. That is, it is naturally predicted that 17-AAG and the like exert an effective pharmacological action not only on SBMA but also on other polyglutamine diseases. Especially in other polyglutamine diseases, it has been found that Hsp70 and Hsp40 are involved in suppressing the inclusion body formation of the causative protein, and that the causative protein is expected to be a client protein of Hsp90. In view of the above, it is expected that compounds that act by 17-AAG and similar mechanisms have a broad therapeutic effect on polyglutamine diseases.

The present invention has been completed based on the above knowledge and consideration, and provides a medical use of geldanamycin (GA) and its derivatives regarding polyglutamine disease. Specifically, the present invention provides the following configurations.
That is, one form of the present invention is an anti-polyglutamine disease agent containing geldanamycin or a geldanamycin derivative as an active ingredient. The geldanamycin derivative is preferably a compound obtained by replacing the methoxy group at the C-17 position of geldanamycin with an alkylamino group. One of the causes of geldanamycin toxicity is thought to be in the methoxy group at the C-17 position. Therefore, the toxicity of the above compound having no methoxy group is generally reduced.
In one embodiment of the present invention, the geldanamycin derivative comprises the following group of compounds: 17-allylamino-17-demethoxygeldanamycin, 17- (2-dimethylaminoethyl) amino-17-demethoxygeldanamycin, 17- (4- (dimethylamino) butyl) amino-17-demethoxygeldanamycin, 17- (2- (carboxy) ethyl) amino-17-demethoxygeldanamycin, 17- (2- (N-methyl) Ethylamino) ethyl) amino-17-demethoxygeldanamycin, 17- (2- (pyrrolidin-1-yl) ethyl) amino-17-demethoxygeldanamycin, 17- (2- (piperazin-1-yl) ) Ethyl) amino-demethoxygeldanamycin and 17- (4- (dimethylamino) butyl) amino-17-deme It is selected from the group consisting of carboxymethyl geldanamycin.
In a preferred embodiment of the present invention, an anti-polyglutamine disease agent comprising 17-allylamino-17-demethoxygeldanamycin or 17- (2-dimethylaminoethyl) amino-17-demethoxygeldanamycin as an active ingredient is used. Composed.
Furthermore, in another preferred embodiment of the present invention, an anti-polyglutamine disease agent is composed of 17-allylamino-17-demethoxygeldanamycin as an active ingredient, whereas in a preferred embodiment of the present invention, polyglutamine disease is caused by the bulbar spinal cord. Therefore, a drug (medicine) for bulbar spinal muscular atrophy is provided.
Another aspect of the present invention is to use the above compounds (ie, geldanamycin or geldanamycin derivatives, etc.) and to produce geldanamycin or geldanamycin derivatives to produce an anti-polyglutamine disease agent. The present invention relates to medical treatment of polyglutamine disease (prevention and treatment of polyglutamine disease).

The anti-polyglutamine disease agent of the present invention is characterized by containing geldanamycin (also referred to herein as “GA”) or a geldanamycin derivative as an active ingredient. Here, “geldanamycin (GA)” is represented by the following structural formula.
However, R in the formula is CH ₃ O.

Various geldanamycin derivatives have been found. For example, Tian ZQ et al., Bioorg. Med. Chem. 12 (2004) 5317-5329, U.S. Patent No. 5,387,584, U.S. Patent No. 4,261,989, U.S. Patent No. 3,987,035, WO00 / 03737, WO02 / 079167, WO99 / Specific compounds are described in 21552, WO99 / 22761. The entire contents of these documents are incorporated by reference.
Preferred geldanamycin derivatives in the present invention include a compound (17-alkylamino-17-demethoxygeldanamycin) obtained by substituting the alkylamino group for the methoxy group at the C-17 position of geldanamycin. it can. Such a compound is advantageous in terms of toxicity because it does not have a methoxy group at the C-17 position, which is considered to be one of the causes of the toxicity of geldanamycin. Specifically, 17-allylamino-17-demethoxygeldanamycin (17-AAG), 17- (2-dimethylaminoethyl) amino-17-demethoxygeldanamycin (17 -(2-dimethylaminoethyl) amino-17-demethoxygeldanamycin: 17-DMAG), 17- [4- (Dimethylamino) butyl) amino-17-demethoxygeldanamycin (17- [4- (Dimethylamino) butyl] amino- 17-demethoxygeldanamycin), 17- (2- (carboxy) ethyl) amino-17-demethoxygeldanamycin (17- [2- (Carboxy) ethyl] amino-17-demethoxygeldanamycin), 17- (2- (N- Methylethylamino) ethyl) amino-17-demethoxygeldanamycin (17- [2- (N-Methylethylamino) ethyl] amino-17-demethoxygeldanamycin), 17- (2- (pyrrolidin-1-yl) ethyl) amino -17-demethoxy gel Danama Syn (17- [2- (Pyrrolidin-1-yl) ethyl] amino-17-demethoxygeldanamycin), 17- (2- (piperazin-1-yl) ethyl) amino-demethoxygeldanamycin (17- [2- (Piperazin-1-yl) ethyl] amino-17-demethoxygeldanamycin) and 17- (4- (dimethylamino) butyl) amino-17-demethoxygeldanamycin (17- [4- (Dimethylamino) butyl] amino- A compound selected from the group consisting of 17-demethoxygeldanamycin) can be used. In one preferred embodiment of the present invention, among these compounds, 17-allylamino-17-demethoxygeldanamycin (also referred to herein as “17-AAG”) or 17- (2-dimethylaminoethyl) amino-17 -Demethoxygeldanamycin (also referred to herein as "17-DMAG") is used as the active ingredient.
Also, 17- (2- (N-methylethylamino) ethyl) amino-17-demethoxygeldanamycin and 17- (2- (pyrrolidin-1-yl) ethyl) amino-17-demethoxygeldanamycin Has a pharmacological potency equivalent to 17-DMAG and is highly water-soluble, which is preferable in that it can constitute an orally-available drug.
On the other hand, 17- (2- (carboxy) ethyl) amino-17-demethoxygeldanamycin and 17- (4- (dimethyl) are low in cytotoxicity while maintaining the inhibitory action of Hsp90 (high IC50). Amino) butyl) amino-17-demethoxygeldanamycin is a preferred compound. In addition, in the disease (neurodegenerative disease) which this invention makes object, unlike the case where tumorous disease is made object, the one where the cytotoxicity of an active ingredient is low is preferable.

In addition, the anti-polyglutamine disease agent of the present invention is constituted using a derivative of 17-allylamino-17-demethoxygeldanamycin or 17- (2-dimethylaminoethyl) amino-17-demethoxygeldanamycin. Also good.
Here, 17-AAG is represented by the following structural formula.
Here, R in the formula is _{CH 2 = CH-CH 2 -NH} .

Similarly, 17-DMAG is represented by the following structural formula.
However, R in the formula is (CH ₃ ) ₂ NH—CH—CH ₂ —NH.

  Geldanamycin is widely commercially available and is readily available (eg, SIGMA, InvitroGen). On the other hand, a geldanamycin derivative can be prepared according to a known synthesis method. Specifically, 17-alkylamino-17-demethoxygeldanamycin, which is a kind of geldanamycin derivative, can be synthesized, for example, by the following procedure. First, an amine (an alkylamine corresponding to a compound to be synthesized) is added to a geldanamycin solution and stirred for a predetermined time. Then, dilute with ethyl acetate etc. and then wash with bicarbonate water. The obtained organic layer is dried and finally purified by chromatography or the like. For details of the synthesis method, Tian ZQ et al., Bioorg. Med. Chem. 12 (2004) 5317-5329 can be referred to. The document also refers to an alternative method.

  On the other hand, 17-DMAG can be synthesized by the following procedure (see Tian ZQ et al., Bioorg. Med. Chem. 12 (2004) 5317-5329). First, N, N-dimethylethylenediamine is added to a 1,2-dichloroethane solution of geldanamycin and stirred for a predetermined time. Then, it is diluted with ethyl acetate and subsequently washed with bicarbonate water. The obtained organic layer is dried and finally purified by chromatography or the like.

  The compound used as the active ingredient of the present invention may be in the form of a salt. Examples of salts include hydrohalides (specifically hydrofluoride, hydrochloride, hydrobromide, hydroiodide, etc.), inorganic acid salts (specifically sulfate, nitrate) , Perchlorate, phosphate, carbonate, bicarbonate, etc.), organic carboxylates (specifically acetate, trifluoroacetate, oxalate, maleate, tartrate, fumarate) , Citrates, etc.), organic sulfonates (specifically methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, camphorsulfonate, etc.), amino acids Salt (specifically aspartate, glutamate, etc.), quaternary amine salt, alkali metal salt (specifically sodium salt, potassium salt, etc.), alkaline earth metal salt (specifically magnesium salt, calcium) Salt) It can be mentioned.

In the present invention, the term “anti-polyglutamine disease agent” means a drug (medicine) used to suppress the onset of polyglutamine disease, or improves (including partial or complete cure) symptoms of polyglutamine disease. It refers to a drug (medicine) used for the purpose. Therefore, the anti-polyglutamine disease agent of the present invention includes a drug that can be used for the prophylactic treatment of polyglutamine disease and a drug that can be used for the treatment of polyglutamine disease.
“Polyglutamine disease” is a kind of neurodegenerative disease, characterized in that CAG repeat elongation (abnormal elongation) is observed in the coding region of the gene. So far, polyglutamine diseases such as bulbar spinal muscular atrophy, Huntington's disease, spinocerebellar degeneration type 1 (SCA1), spinocerebellar degeneration type 2 (SCA2), Machado-Joseph disease (MJD, SCA3), spinocerebellar degeneration Syndrome type 6 (SCA6), spinocerebellar degeneration type 7 (SCA7), spinocerebellar degeneration type 17 (SCA17), dentate nucleus erythrocytic and pallidal Louis atrophy (DRPLA) have been found. In patients with polyglutamine disease, common symptoms are observed, such as expression promotion (anticipation), variation in the number of CAG repeats (somatic cell mosaic), and mainly nerve tissue is selectively damaged. The agent of the present invention can be used as a prophylactic or therapeutic agent for these polyglutamine diseases, but is particularly preferably applied to bulbospinal muscular atrophy.

The active ingredient of the present invention can be formulated according to a conventional method. When formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, binders, disintegrants, solubilizers, buffers, emulsifiers, suspensions, soothing agents, stabilizers, Preservatives, preservatives, antioxidants, lubricants, colorants, flavoring agents, physiological saline, and the like).
As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. As the binder, polyvinyl pyrrolidone, ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, gum arabic, or the like can be used. As a solubilizer, physiological saline, lactated Ringer's solution, polyoxyethylene hydrogenated castor oil, nicotinamide, and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used. As the antioxidant, ascorbic acid, α-tocophenol and the like can be used. As the lubricant, talc, magnesium stearate, calcium stearate and the like can be used.

The dosage form for formulation is not particularly limited, for example, tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, eye drops, nasal drops, inhalants, and suppositories. Etc. can be prepared.
The drug of the present invention thus formulated can be applied to a subject by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal injection, etc.) depending on the form. The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, Goats, sheep, dogs, cats, chickens, quails, etc.). Suitably, the medicament of the present invention is applied to humans.
The content of the active ingredient (GA or a derivative thereof) in the drug of the present invention generally varies depending on the dosage form, but is, for example, about 0.01% by weight to about 90% by weight so that a desired dose can be achieved.

  In another aspect of the present invention, a method for preventing or treating polyglutamine disease using the above-mentioned drug (hereinafter, these two methods are collectively referred to as “therapeutic methods”) is provided. The therapeutic method of the present invention includes a step of administering a drug containing GA or a derivative thereof as an active ingredient to a subject. The administration route is not particularly limited, and examples thereof include oral, intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, transdermal, transmucosal and the like. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). In addition, oral administration is preferable because administration is easy.

  The dose of the drug varies depending on symptoms, patient age, sex, weight, etc., but those skilled in the art can appropriately set an appropriate dose. For example, for an adult (body weight of about 60 kg), the dose can be set so that the amount of active ingredient per day is about 100 mg to about 10 g, preferably about 150 mg to about 5 g. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In setting the administration schedule, it is possible to consider the patient's medical condition, the duration of the drug effect, and the like.

Materials / reagents and experimental methods in the following examples were as follows unless otherwise specified.
(Androgen receptor expression vector)
A 24-97 CAG full length androgen receptor (AR) construct was cloned into the pPCR3.1 vector (Invitrogen, California, USA).

(Culbus spinal muscular atrophy (SBMA) cultured cell model)
Human neuronal cultured cells (SHSY-5Y) were induced to differentiate with a medium containing 10 μM retinoic acid, and then infected with an expression vector containing a full-length AR having 24 to 97 CAG by the lipofection method. Thus, a cultured cell model was prepared.

(SBMA model mouse (transgenic mouse))
A transgenic mouse (AR-97Q) into which a construct expressing a full-length AR having 97 CAG under the control of a CMV enhancer and a chicken β-actin promoter was used as a model mouse. The outline of the method for producing the transgenic mouse is as follows. First, the NheI site in the pCAGGS vector (Niwa, H., Yamamura, K., and Miyazaki, J. (1991) .Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene 108, 193-199.) After preparation (pCAGGS-NheI), the full-length human AR gene with 97CAG (Kobayashi, Y., Miwa, S., Merry, DE, Kume, A., Mei, L., Doyu, M., and Sobue, G (1998). Biochem. Biophys. Res. Commun. 252, 145-150.) Is subcloned into this pCAGGS-NheI. Next, treatment with SalI-NheI cuts out the AR fragment. This fragment is microinjected into BDF1 fertilized eggs (Japan SLC, Shizuoka, Japan). A fertilized egg that has completed the injection operation is transplanted into the oviduct of a pseudopregnant mouse and bred for a predetermined period to obtain a pup mouse (F0, AR-97Q mouse). These F0 mice were maintained by mating to C57BL / 6J (Japan SLC, Shizuoka, Japan). For details of the production method of AR-97Q, refer to WO2004 / 016083.

(Drug administration)
Administration of 17-AAG to model mice was performed as follows. 17-AAG dissolved in DMSO was administered intraperitoneally. Administration was started from 6 weeks of age and administered 3 times a week until 25 weeks of age. Two groups with different doses (a group with a single dose of 2.5 mg / kg and a group with a single dose of 25 mg / kg) were prepared. The control group (Tg-0) was administered with the solvent DMSO itself.

(Phenotype analysis)
1. Rotarod task was performed every week from the start of drug administration, and motor function was analyzed. The Rotarod task was carried out using Economex Rotarod (Colombus Instruments, USA), as in the previous report (Adachi, H., Kume, A., Li, M., during the light / dark cycle every 12 hours). Nakagomi, Y., Niwa, H., Do, J., Sang, C., Kobayashi, Y., Doyu, M., and Sobue, G. (2001). Hum. Mol. Genet. 10, 1039-1048 .). Trials were performed three times and the time each mouse was able to ride on the rotating bar the longest was recorded. The timer was stopped when the mouse fell from the bar or when 180 seconds were reached, and the time was recorded.
The cage activity, which is the amount of activity per day of a mouse, is measured with an infrared sensor using the AB system (Neuroscience, Japan) by placing the mouse in a transparent cage (16 x 30 x 14 cm). did. Measurements were taken at 10 minute intervals for 24 hours.

2. Create a cultured cell model by transiently forcing human androgen receptor (AR) gene expression vector containing normal length (AR24Q) or abnormally extended polyglutamine chain (AR97Q) in human neuronal cells (SHSY5Y) did. Using this cultured cell model, the pharmacological effects of GA and 17-AAG were evaluated. Administration of the drug (GA, 17-AAG) was performed by adding to the medium after forced expression, and the medium containing the drug (GA, 17-AAG) was replaced once after 24 hours. GA and 17-AAG were dissolved in dimethyl sulfide oxide (DMSO) and administered at concentrations of GA 0.9, 1.8, 3.6, 7.2 nM, 17-AAG 18, 90, 180, and 360 nM, respectively. As a control, a non-administered drug (C) and a DMSO-only drug (DM) were used. After 48 hours of expression, the cells were collected, proteins were extracted, and the expression level of each protein was evaluated by Western blot. The antibodies used are as follows.
Anti-AR antibody: N-20 (Santa Cruzu Biotechnology, Inc.)
Anti-Hsp70 antibody: SPA-810 (Stressgen Biotechnologies corp.)
Anti-Hsp40 antibody: SPA-400 (Stressgen Biotechnologies corp.)
Anti-Hsp90 antibody: SPA-835 (Stressgen Biotechnologies corp.)
Anti-Hop antibody: SRA-1500 (Stressgen Biotechnologies corp.)
Anti-p23 antibody: MA3-414 (Affinity BioReagents Inc.)
Anti-Hsp60 antibody: MS-263 (Neo Markers)

3. An expression vector incorporating the Hsp70 gene was prepared. This and a human AR gene expression vector containing normal length (AR24Q) or abnormally extended polyglutamine chain (AR97Q) were co-expressed in human neuronal culture cells (SHSY-5Y), and the expression level of each AR was determined by Western Evaluated by blot. Moreover, Hsp90α or β was highly expressed in human neuronal culture cells (SHSY-5Y) using an expression vector incorporating the Hsp90α subset or β subset, and the expression level of AR was similarly evaluated by Western blot.
4). For the Western blot of spinal cord and muscle tissues of mice after drug administration, total homogenate of each tissue was used. The anti-AR antibody is H-280 (Santa Cruzu Biotechnology, Inc.), the anti-Hsp70 antibody is SPA-810 (Stressgen Biotechnologies corp.), The anti-Hsp40 antibody is SPA-400 (Stressgen Biotechnologies corp.), And the anti-Hsp90 antibody is SPA. -835 (Stressgen Biotechnologies corp.) Was used and α-tubulin was used as a control protein. In immunostaining of spinal cord and skeletal muscle, 1C2 (CHEMICON International) was used as an antibody that specifically recognizes a protein with abnormally expanded polyglutamine.

  The drug effect of 17-AAG was evaluated in a cultured cell model and a model mouse into which a pathological gene (with 97 CAG repeats in AR) was introduced. It has been confirmed that the model mouse used this time has gender differences in phenotype and exhibits progressive movement disorders (Non-patent Document 5). The evaluation of drug efficacy is weight change, survival rate, Rotarod method (time measurement that can be held without falling on a rod rotating at a constant speed), Cage activity measurement method (measurement of the number of mouse movements for 24 hours) ) As a parameter. The single dose of the drug has been confirmed in previous reports that toxicity has not been a problem with short-term administration (2.5 mg / kg, 25 mg / kg). Was performed three times a week. The pharmacokinetics of 17-AAG in mice has already been studied in detail (Non-patent Document 43). Dimethyl Sulfoxide (DMSO) as a solvent was administered to the control group.

<Pharmacological effects of GA and 17-AAG in cultured cells>
In cultured cell models, the pharmacological effects of GA and 17-AAG were evaluated. As shown in FIGS. 3A and 3B, GA and 17-AAG administration showed an effect of decreasing AR of both repeat numbers in a drug concentration-dependent manner. In the human body, the same effect was observed with the AR of 24 repeats (AR24Q), which is regarded as the normal repeat number, but the AR reduction effect was strongly observed with AR97Q with an abnormally long repeat number. In addition, GA and 17-AAG administration increased Hsp70 and Hsp40 expression, but Hsp90 expression did not change. The expression levels of the other chaperone groups (Hsp60, Hop, p23) did not change.
4A and 4B are graphs showing the average value of the reduction effect of AR24Q and AR97Q in a bar graph after five trials of 17-AAG administration. Comparing these reduction effects in the control group to which only DM was added and the 17-AAG (360nM) administration group, the respective reduction rates were 41.1% (p = 0.0132) for AR24Q and 11.0% (p = 0.0062) for AR97Q there were. Furthermore, when the AR24Q and AR97Q reduction rates were compared, it was found that the AR97Q reduction rate was significantly larger (p = 0.0063), and the pathological AR with abnormally extended polyglutamine was more sensitive to 17-AAG. became. Statistical analysis used the τ test.
Such an AR reduction effect could also be obtained by forcibly expressing Hsp70 by genetic manipulation. However, despite the fact that high expression of Hsp70 almost equivalent to that of drug administration was obtained, the AR reduction effect was inferior to that of drug administration (FIG. 5A). On the other hand, when the two subsets of α and β of Hsp90 were highly expressed, the expression levels of AR24Q and AR97Q were increased in both, and this tendency was strongly observed by AR97Q (FIG. 5B).

<Pharmacological effects of GA and 17-AAG in mouse model>
From the results of the above cultured cell model, it was predicted that the pathological AR in which polyglutamine was extended could be reduced in vivo by 17-AAG. As a GA derivative, 17-AAG, which has already been confirmed to be safe at the individual animal level, was selected, and a drug was administered to a mouse model. Changes in mouse symptoms were evaluated by weight, survival rate, and motor function analysis (Rotarod, cage activity) (FIGS. 6 and 7). The control group is indicated by Tg-0 (DMSO only), and the treatment group (administration group) is indicated by Tg-2.5 (17-AAG: 2.5 mg / kg) and Tg-25 (17-AAG: 25 mg / kg). FIG. 8A is a photograph of a 16-week-old mouse, showing marked muscle atrophy in the treatment group, whereas the treatment group has a physique almost similar to that of a healthy mouse. FIG. 8B is a comparison of mouse stride, but the stride is significantly wider and regular in the treatment group. Thus, the 17-AAG treatment group (administration group) showed a significant therapeutic effect on dose dependency in all analyses, compared to the non-treatment group.
The number of individuals is body weight, survival rate, Tg-0 (n = 27), Tg-2.5 (n = 22), Tg-25 (n = 23) in the Rotarod method, and Tg-0 ( n = 18), Tg-2.5 (n = 17), and Tg-25 (n = 18).

The expression level of the mutant AR (AR97Q) in which the polyglutamine chain was abnormally extended was evaluated in the spinal cord and skeletal muscle of the SBMA mouse model by Western blotting. The expression level of AR97Q was significantly decreased in both the monomer and the protein complex in the stacking gel (FIG. 9A). The high molecular weight AR that accumulates in the stacking gel is considered to indicate an aggregated mutant AR. Although the expression of Hsp70 and Hsp40 increased as in the cultured cell model, the expression level of Hsp90 did not change (FIG. 9A). Comparing the expression level of mutant AR in 5 mice each of treatment group (Tg-0) and non-treatment group (Tg-25), mutation AR is significant in both organs and in protein complex in stacking gel. (FIG. 9B). Statistical analysis used the T test.
In immunostaining using a 1C2 antibody that specifically recognizes abnormally prolonged polyglutamine, the number of 1C2-positive cells decreased in the 17-AAG treatment group (FIG. 10).
The antibodies used for Western blotting and immunostaining are shown below.
Anti-AR antibody: H-280 (Santa Cruzu Biotechnology, Inc.)
Anti-Hsp70 antibody: SPA-810 (Stressgen Biotechnologies corp.)
Anti-Hsp40 antibody: SPA-400 (Stressgen Biotechnologies corp.)
Anti-Hsp90 antibody: SPA-835 (Stressgen Biotechnologies corp.)
Anti-polyglutamine antibody: 1C2 (CHEMICON International)

<Summary>
As described above, administration of 17-AAG to cultured cell models of SBMA and model mice showed a significant therapeutic effect. A pathological AR protein with an extended polyglutamine chain is considered to be more dependent on Hsp90. No obvious side effects were observed in long-term continuous administration to model mice. This time, it was revealed that mutant AR, a pathological protein of SBMA, is a therapeutic target for 17-AAG. On the other hand, if other pathogenic proteins of polyglutamine disease are also Hsp90-dependent, and if the dependence is increased by abnormally extending polyglutamine, it is expected to be a therapeutic target as well. Although GA derivatives such as 17-AAG are being clinically applied as a treatment for malignant tumors, they are promising drugs that can be applied to polyglutamine diseases, which are neurodegenerative diseases.

  The present invention is useful for prevention or treatment of polyglutamine disease. Among the polyglutamine diseases, the present invention can be suitably used particularly for the prevention or treatment of bulbar spinal muscular atrophy.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

FIG. 1 shows geldanamycin (GA) and its derivatives. In 1970, GA was found as an antifungal agent, and thereafter it was found to have an excellent antitumor effect. Several geldanamycin derivatives have been developed for clinical application. 17-AAG is a derivative in which only part of the side chain of GA is substituted. It is an excellent derivative that has succeeded in reducing toxicity in vivo while maintaining the same pharmacological action. 17-DMAG is a further improved derivative of 17-AAG, which can be administered orally by increasing water solubility. FIG. 2 schematically shows the action of Hsp90 and cochaperone. Hsp90 has been shown to express its function in cooperation with several co-chaperones. The Hsp90-client protein complex in which p50 and p23 are bound is the most stable form for functional expression, and conversely, the complex in which Hop and Hsp70 are bound is unstable and becomes the target of the proteasome. GA and other Hsp90 inhibitors exert their pharmacological actions by specifically inhibiting the formation of such Hsp90 complexes. FIG. 3 shows the effects of GA and 17-AAG in a cultured cell model. A, B. The cultured cell model incorporates a human androgen receptor (AR) gene containing a normal length (AR24Q) or abnormally extended polyglutamine chain (AR97Q) into an expression vector and is expressed in cultured human nerve cells (SHSY5Y) It has been made. The pharmacological effects of GA and 17-AAG were evaluated. It is the result of recovering the cells 48 hours after the expression, extracting the protein, and performing Western blotting. GA and 17-AAG were dissolved in dimethyl sulfide oxide (DMSO) and administered at concentrations of GA 0.9, 1.8, 3.6, 7.2 nM, 17-AAG 18, 90, 180, and 360 nM, respectively. As a control, a non-administered drug (C) and a DMSO-only drug (DM) were used. P85 was used as a control protein. FIG. 4 shows the effect of 17-AAG administration on the AR expression level. A. It is a bar graph showing the reduction effect of AR24Q and AR97Q by 17-AAG. B. It is the bar graph which compared the decreasing rate of AR. FIG. 5 shows the effect on the AR expression level caused by high expression of molecular chaperones (Hsp70 and Hsp90) in a cultured cell model. A. The Hsp70 gene is incorporated into an expression vector and co-expressed with a human androgen receptor (AR) gene expression vector containing a normal length (AR24Q) or abnormally extended polyglutamine chain (AR97Q), and the expression level of each AR is expressed. Evaluated by Western blot. B. Two subsets of α and β of Hsp90 were each incorporated into an expression vector for high expression, and the expression level of AR was similarly evaluated. FIG. 6 shows the effect of 17-AAG on the symptoms of SBMA model mice. The 17-AAG dose was 2.5 mg / kg (Tg-2.5) and 25 mg / kg (Tg-25), and the control group (Tg-0) was administered with DMSO as a solvent. The drug was administered intraperitoneally. Administration was started from 6 weeks of age and administered 3 times a week until 25 weeks of age. A. Graphs of body weight up to 25 weeks of age are shown. B. Graph of cumulative survival curve up to 25 weeks of age. Tg-0 is indicated by ◆, Tg-2.5 is indicated by ▲, and Tg-25 is indicated by ●. FIG. 7 shows the effect of 17-AAG on the symptoms of SBMA model mice. The graph of A. rotarod task up to 25 weeks of age is shown. B. Cage activity graph up to 25 weeks of age. FIG. 8 shows the effect of 17-AAG on the symptoms of SBMA model mice. A. Photographs of 16-week-old control group mice (Tg-0) and treatment group mice (Tg-25) are shown. The control group exhibits prominent muscle atrophy of the trunk and limbs, but the mice in the treatment group have a physique close to that of normal mice. B. Shows the footprint of each group at 16 weeks of age. The front legs are colored red and the rear legs are colored blue. FIG. 9 shows the effect of 17-AAG on protein expression and pathological findings in SBMA model mice. A. Western blotting using total homogenate of spinal cord and muscle tissue of control group (Tg-0) and treatment group mouse (Tg-25). Anti-AR antibody was H-280, anti-Hsp70 antibody was SPA-810, and α-tubulin was used as a control protein. B. A graph comparing the amount of mutant AR expressed in the spinal cord and muscle tissue of a control group (Tg-0) and a treatment group mouse (Tg-25). FIG. 10 shows the effect of 17-AAG in SBMA model mice. Immunostaining was performed on spinal cord and skeletal muscle of control group (Tg-0) and treatment group mouse (Tg-25). The antibody that specifically recognizes the protein with abnormally prolonged polyglutamine used 1C2.

Claims (8)

  An anti-polyglutamine disease agent containing geldanamycin or a geldanamycin derivative as an active ingredient.   The anti-polyglutamine disease agent according to claim 1, wherein the geldanamycin derivative is a compound obtained by substituting a methoxy group at the C-17 position of geldanamycin with an alkylamino group. The anti-polyglutamine disease agent according to claim 1, wherein the geldanamycin derivative is a compound selected from the following group:
17-allylamino-17-demethoxygeldanamycin, 17- (2-dimethylaminoethyl) amino-17-demethoxygeldanamycin, 17- (4- (dimethylamino) butyl) amino-17-demethoxygeldana Mycin, 17- (2- (carboxy) ethyl) amino-17-demethoxygeldanamycin, 17- (2- (N-methylethylamino) ethyl) amino-17-demethoxygeldanamycin, 17- (2 -(Pyrrolidin-1-yl) ethyl) amino-17-demethoxygeldanamycin, 17- (2- (piperazin-1-yl) ethyl) amino-demethoxygeldanamycin, and 17- (4- (dimethyl The group consisting of amino) butyl) amino-17-demethoxygeldanamycin.   The anti-polyglutamine disease agent according to claim 1, wherein 17-allylamino-17-demethoxygeldanamycin or 17- (2-dimethylaminoethyl) amino-17-demethoxygeldanamycin is an active ingredient.   The anti-polyglutamine disease agent according to claim 1, wherein 17-allylamino-17-demethoxygeldanamycin is an active ingredient.   The anti-polyglutamine disease agent according to any one of claims 1 to 5, wherein the polyglutamine disease is bulbar spinal muscular atrophy.   Use of the active ingredient according to any one of claims 1 to 5 for producing an anti-polyglutamine disease agent.   A method for preventing or treating polyglutamine disease, comprising a step of administering geldanamycin or a geldanamycin derivative to a subject.