HK1029937B - Use of phanquinone for the treatment of alzheimer's disease - Google Patents

Description

Use of ubiquinones for the treatment of alzheimer's disease

Technical Field

The present invention relates to the use of known compounds for the manufacture of a pharmaceutical composition for the treatment or prevention of alzheimer's disease. The invention also relates to pharmaceutical compositions for use in such treatment or prevention. The invention also relates to methods of treating or preventing Alzheimer's pain.

Background

Alzheimer's Disease (AD), a leading cause of dementia in adults in industrialized societies, is a degenerative brain disease that is clinically characterized by progressive loss of memory, confusion, dementia, and ultimately death. The histopathological features of alzheimer's disease are the presence of two brain lesions in the neocerebral cortex, particularly the hippocampus: namely neurofibrillary tangles (NFTs) of helical filaments (PHF) paired in neurons and nerve (senile) plaques in the extracellular space. The formation of age spots is associated with the appearance of symptoms and signs of disease, including memory loss. After the formation of senile plaques, neurofibrillary tangles are generated in the neuron body. The formation of neurofibrillary tangles is associated with a worsening of memory deficits and a worsening of other symptoms of dementia.

The major component of age spots is amyloid deposits. Cataracts are a disease that often occurs concurrently with alzheimer's disease. It can occur in the eye and is also caused by amyloid deposits. Thus, the methods of treatment of patients suffering from alzheimer's disease disclosed in the present specification and claims may also be used to treat cataracts. Thus, as used in the specification and claims, the term Alzheimer's disease also includes cataracts.

The major component of amyloid deposits is a polypeptide referred to herein as a β (beta amyloid). Normally, A.beta.is cerebrospinal fluidA soluble component at a concentration of about 3-5 nm. In the mature state, A.beta.can have 39-43 amino acids, typically 40 amino acids, and is derived as a proteolytic cleavage product from a cell surface protein known as the Amyloid Precursor Protein (APP) (Kang et al, 1987, Nature)325：733-736)。

A number of studies have shown that A.beta.has toxicity in vitro when added directly to neuronal cell cultures (Yankner BA, Duffy LK, Kirschner DA, Science 1990, 250 (4978): 279-jar 282; Koh JY, Yang LL, Cotman CW, Brain Res 1990, 533 (2): 315-jar 320; and Pike CJ, Burdick D, Walencz AJ, Glabe CG, Cotman CW, J.Neurosci 1993, 13 (4): 1676-jar 1687).

The neurotoxicity of a β was found to lie in the peptide sequence between amino acid residues 25 and 35 (a β (25-35)). A.beta.25-35 was as effective as full-length A.beta.1-40 in inducing neuronal cell death (Yankner BA, Duffy LK, Kirschner DA, Science 1990, 250 (4978): 279 282). The normal function of A.beta.is not known at present, but it is likely to form cation selective channels across cell membranes (Kawahara M. et al, 1997, biophysical journal 73/1, 67-75).

Precipitation of synthetic A β has been shown to be caused by a variety of environmental factors including low pH, high salt concentrations, and the presence of metals such as zinc, copper, and mercury (Bush, A.I. et al, 1995, Science 268: 1921-1923). It has been reported that A.beta.itself can exhibit high affinity (K) at a molar ratio of 1: 1 (Zn: A.beta.)_D107nM) binds specifically and saturably to zinc (Bush, a.i. et al, 1994, j.biol.chem.269: 12152-12158). This binding occurs at physiological concentrations of zinc (Bush, A.I. et al, 1994, Science 265: 1464-.

It is hypothesized that removal of amyloid deposits from patients with alzheimer's disease may alleviate the symptoms of alzheimer's disease. Accordingly, there have been many efforts to prepare such drugs because methods for treating alzheimer's disease are urgently needed.

International patent application publication No. WO 93/10459 discloses a method of treating alzheimer's disease by administering a zinc binding agent. As preferred compounds phytic acid, deferoxamine, sodium citrate, EDTA, 1, 2-diethyl-3-hydroxy-pyridin-4-one and 1-hydroxyethyl-3-hydroxy-2-methyl-pyridin-4-one are described.

German patent application No. DE 3932338 discloses the use of aluminium chelators such as 8-hydroxy-quinoline for the treatment of Alzheimer's disease.

Us patent No. 5,373,021 discloses the use of disulfiram and its salts and analogues. According to this patent, the disclosed compounds can be used to reduce nerve damage caused by alzheimer's disease.

International patent application publication No. WO 98/06403 discloses the use of clioquinol in the manufacture of a pharmaceutical composition for the treatment of alzheimer's disease.

The compounds currently known for the treatment of alzheimer's disease have several drawbacks, thus preventing their widespread use. Most of these compounds are unable to cross the blood brain barrier and therefore cannot easily reach the area of amyloid deposition. Disulfiram, which is permeable to the blood-brain barrier, has the disadvantage that when mixed with ethanol by the patient, it can cause serious side effects including headache, nausea, vomiting, sweating, thirst, weakness and hypotension. Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) also permeates the blood brain barrier and has a history of liability because it has side effects causing subacute myelooptic neuropathy (SMON).

Until now, ubiquinone (4, 7-phenanthroline-5, 6-dione) has been used for the treatment of various diseases, such as amebiasis. Ubiquinone is sold by CIBA-GEIGY under the trademark ENTOBEX. In contrast to clioquinol, no side effects were detected when the ubiquinone was used in the normal dosage range.

In the past, pharmaceutical formulations of anti-amoebae drugs containing clioquinol and ubiquinone have been sold by CIBA-GEIGY under the trademark Mexafor. However, when one realized that clioquinol could cause SMON, the formulation was stopped from being marketed.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a new use of known compounds for the treatment or prevention of alzheimer's disease. It is another object of the invention to provide an enhanced effect for the treatment of alzheimer's disease. Another object is to avoid any harmful side effects in the treatment or prevention of alzheimer's disease.

According to the present invention we provide the use of ubiquinone in the manufacture of a pharmaceutical composition for the treatment or prevention of alzheimer's disease.

The ubiquinone may be administered in an amount effective to treat or prevent Alzheimer's disease. The ubiquinone is preferably administered 1-3 times daily in an amount of 5mg to 250mg, and most preferably in an amount of 10mg to 50 mg.

In one embodiment of the present invention, a compound or a mixture of compounds selected from the group consisting of: antioxidant, acetylcholine enhancer, trace metals, prosthetic group and clioquinol.

The invention also relates to a pharmaceutical composition comprising a ubiquinone and a compound or a mixture of compounds selected from the group consisting of: an antioxidant, an acetylcholine enhancer, a trace metal, a prosthetic group and clioquinol, provided that when clioquinol is selected, at least one further compound is selected from the above compounds.

Preferably, the antioxidant is vitamin C, vitamin E, Q10, or a combination thereof. Preferably the acetylcholine enhancer is a μ l agonist or anticholinesterase inhibitor. Preferred anticholinesterase inhibitors are tacrine (trade mark: Cognex) and donepezil (trade mark: Aricept). Preferably, the prosthetic group is vitamin B₁₂。

In a preferred embodiment of the invention, a combination of ubiquinone and chloroiodine is usedHydroxyquine for the manufacture of a pharmaceutical composition for the treatment or prevention of Alzheimer's disease. Preference is given to using ubiquinone, clioquinol and vitamin B₁₂To produce the pharmaceutical composition.

The pharmaceutical composition may be formulated for oral, parenteral or transdermal administration. Furthermore, the pharmaceutical formulation may be formulated as a single pharmaceutical composition or as two or more separate dosage forms for sequential or substantially simultaneous administration.

The invention also relates to a method of treating a patient having or suspected of having alzheimer's disease comprising administering to said patient an effective amount of a ubiquinone to treat or prevent alzheimer's disease.

Brief description of the drawings

FIG. 1 depicts the effect of ubiquinone on A β (25-35) dose-response in PC12 cells.

FIG. 2 depicts the effect of ubiquinone on A β (25-35) induced toxicity in PC12 cells.

FIG. 3 depicts the pair of Zn by the use of ubiquinone and clioquinol²⁺Induced aggregation of a β.

FIG. 4 depicts clioquinol and ubiquinone vs Cu²⁺Induced aggregation of a β.

FIG. 5 depicts the NMR spectra of the three solutions. Solution a contains vitamin B₁₂(hydroxycobalamin) at a concentration of 2.6 mM. Solution B contains 2.6mM vitamin B₁₂And 10mM glucuronide clioquinol (molar ratio about 1: 4). Solution c contained 10mM of the glucuronide clioquinol.

Fig. 6 depicts the right half of fig. 5 expanded to facilitate comparison of the location of resonance.

Fig. 7 depicts the left half of the fig. 5 expansion to facilitate comparison of the location of resonance.

Detailed Description

There is a need in the market to introduce effective drugs for the treatment or prevention of alzheimer's disease. According to the present invention, we propose the known drug ubiquinone.

In the examples it is shown that a β (25-35) in PC12 cells has a significant effect on the redox activity of the cells. In the method used in example 2, the redox activity of cells exposed to 1 μ M A β (25-35) was reduced to 60% of the normal level (see fig. 2, open circles). However, when the cells treated with A.beta. (25-35) were treated with not less than 1. mu.g/ml of ubiquinone, the normal redox activity could be restored. This suggests that when ubiquinone is administered to a mammal such as a human, it can alleviate the adverse effects of a β.

While we do not intend to limit the present invention to any particular mechanism of action, we presently believe that the antioxidant effect of ubiquinone prevents the effect of a β on cellular redox activity.

The antioxidant effect of the ubiquinone can be enhanced by co-administering an additional antioxidant such as vitamin C, vitamin E, vitamin Q10, or a combination thereof.

The presence of senile plaques in the extracellular space may prevent or inhibit the transmission of impulses in the cholinergic nervous system. The function of the nervous system depends on acetylcholine transmitters. Thus, compounds that enhance acetylcholine levels, such as compounds that prevent or inhibit the normal hydrolysis of acetylcholine by acetylcholinesterase, so-called anticholinesterase inhibitors, can be administered with the ubiquinone to enhance the effect. Preferred anticholinesterase inhibitors are tacrine and donepezil.

In the studies reported in the examples, it has been shown that ubiquinones can reduce Cu deposition²⁺And Zn²⁺Induced aggregation of A β (1-40).

The mechanism of action of the present invention set forth below is not intended to limit the invention to that mechanism. Applicants currently believe that ubiquinone and A β can competitively chelate with zinc, copper and other heavy metals. Ubiquinone has the ability to cross the blood brain barrier. When the heavy metal ions are captured in the extracellular space, the ubiquinone can enter the blood and be eliminated from the body. Due to aggregation of a β or a salt thereof, heavy metal ions are in equilibrium with free a β and heavy metal ions, so as long as aggregated a β (i.e., amyloid) is present, ubiquinone can continue to capture and transport free heavy metal ions into the blood. Free a β can cross the blood brain barrier by passive diffusion rather than by degradation by proteolytic enzymes normally present in the extracellular space. Thus, free a β or denatured portions thereof can potentially damage neuronal cells before being digested to a non-toxic extent. Meanwhile, due to its ability to counteract the reduction of redox activity normally produced by a β, ubiquinone can prevent or inhibit the potential cytotoxic effects of a β.

Reduction of aggregation by ubiquinone on Cu²⁺50-60% of Zn²⁺The content was 10%. It can be further demonstrated that clioquinol has the opposite trend. The clioquinol can convert Zn into²⁺Induced Ass (1-40) aggregation is reduced by more than 60%, while Cu is reduced²⁺Induced aggregation was reduced by about 30%. In other words, ubiquinone lowers Cu²⁺The best induced A beta (1-40) aggregation, while clioquinol reduces Zn²⁺The induced aggregation is best.

Due to Cu²⁺And Zn²⁺Naturally occurring in patients suffering from or suspected of suffering from alzheimer's disease, there is a need to reduce or resolubilize both ion-induced aggregates. Therefore, it is preferable to administer clioquinol before, together with or after the administration of the ubiquinone.

However, administration of clioquinol is also problematic, as clioquinol causes side effects of SMON. Prior studies (see WO 98/06403) have shown that clioquinol may be active against vitamin B₁₂Has an effect.

Clioquinol is known to be excreted by the kidney as a glucuronide or sulfonate derivative (Kotaki H. et al: Enterophaatic circulation of cyclic in the rate, J. Pharmacobiodyn.1984.6 months; 7 (6): 420-5 and Jurmima M. et al: Metabolism of 14C-iodochlorodihydroquinoleine and the rate, J. Pharmacobiodyn.1984.3 months; 7 (3): 164-70), as compound (5-chloro-7-iodo-quinolyl-2 ', 3 ', 4 ' -tri-O-acetyl-glucopyranoside) methyl uronate. For simplicity, this compound will be referred to hereinafter as clioquinol glucuronide.

The in vivo detoxification of hydrophobic substances such as clioquinol occurs mainly in the liver. Thus, it is believed that clearance of clioquinol occurs as follows: the chloroiodohydroxyquine is first converted to chloroiodohydroxyquine glucuronide in the liver. After the formation of clioquinol glucuronide, the water soluble clioquinol glucuronide is secreted into the bile. Bile enters the intestine, where most of the clioquinol glucuronide is excreted in the feces. And a portion of the clioquinol glucuronide is resorbed from the intestine into the blood. The chloroiodohydroxyquine glucuronide in the blood is filtered in the kidney and excreted in the final urine.

Treatment of mice with clioquinol and subsequent administration of vitamin B with a radioisotope₁₂([⁵⁷Co]Cyanocobalamin), which was shown in international patent application publication No. WO 98/06403 to be vitamin B in the brain and liver of clioquinol-treated mice₁₂The concentration of vitamin B in the kidneys of these mice remained at normal levels, compared to normal levels₁₂The concentration of the isotope decreases. Furthermore, these findings suggest that the kidney is a target organ, in which chloroiodoquinol-dependent metabolism occurs.

For studying chloroiodohydroxyquine glucuronide and vitamin B₁₂With possible interactions between them, we designed an assay in which chloroiodohydroxyquine glucuronide and vitamin B were tested₁₂Mixing in water. By using¹The mixture was analyzed by H NMR.¹H NMR spectra (see FIGS. 5-7) showed a portion of vitamin B₁₂Shifted in the two resonances of clioquinol glucuronide (corresponding to the quinoline moiety)The same phenomenon is also observed. The applicants therefore believe that similar results are expected to be obtained using free clioquinol, but that clioquinol cannot be detected by NMR when dissolved in aqueous solution.

The results show that the chloroiodohydroxyquine glucuronide and the vitamin B₁₂The hydrophobic interaction between the two may be due to vitamin B₁₂The benzimidazole moiety of (a) and the quinoline moiety of clioquinol glucuronide.

General vitamin B₁₂Is actively reabsorbed from the kidney plasma after filtration. In this way the body can recover most of the vitamin B₁₂Otherwise it will be lost in the urine. Recently, vitamin B has been demonstrated₁₂Resorption of (D) is mediated by the action of the membrane protein megalin (Moestrup S.K. et al Proc. Natl. Acad. Sci.1996; 93 (16): 8612-7). The meglin pair has been shown to consist of vitamin B₁₂The binding of the complex formed with the transporter cobalamin transporter has a strong affinity.

Vitamin B based on the novel finding reported here₁₂Can be combined with chloroiodohydroxyquine glucuronide, and is considered as vitamin B₁₂Having bound to clioquinol glucuronide, it is unable to bind to megalin protein and/or cobalamin transporter. Thus, vitamin B could not be performed₁₂So that sufficient amounts of neovitamin B are provided to the body after a period of time if not through the normal dietary route₁₂If so, the body will suffer from vitamin B₁₂Deficiency syndrome.

Because the human body can not synthesize vitamin B₁₂So that human vitamin B₁₂The only source of (a) is food. Thus, it is clear that diets low in meat and/or microorganisms can cause vitamin B₁₂And (4) lack. If too low vitamin B is provided to the human₁₂Diet at a content such that vitamin B is prevented by competitive binding of clioquinol to megalin₁₂The administration of clioquinol will worsen the condition. SMON is observed only in Japan and Japanese's diet is mainly composed of vegetables and cereals (in particularIs rice) composition (Kromhout D et al: "FoodConsumption pattern in the 1960s in seven counts", am.J.Clin.Nutr.49: 889-894, 1989) explain the reason why SMON diseases are restricted to japan.

In order to combat the side effects of the administration of ubiquinone and/or clioquinol, trace amounts of metals and/or prosthetic groups need to be co-administered. Preferred prosthetic groups are vitamin B₁₂(cyanocobalamin).

The compositions according to the invention, the use of the compositions according to the invention and the process according to the invention are illustrated by the following non-limiting examples: 1) patients diagnosed with any clinical stage of alzheimer's disease, 2) prevention of alzheimer's disease in patients with early or prodromal symptoms or signs, and 3) delay the appearance or progression or worsening of alzheimer's disease symptoms and signs. Thus, the methods and compositions of the invention may be used, for example, to treat alzheimer's disease, ameliorate or alleviate the symptoms and signs of alzheimer's disease, improve any pathology or laboratory outcome of alzheimer's disease, delay the progression of alzheimer's disease, delay the appearance of any symptoms and signs of alzheimer's disease, prevent the onset of alzheimer's disease, and prevent the appearance of any symptoms and signs of alzheimer's or parkinson's disease.

The subject or patient may be an animal, such as a mammal, but is preferably a human, and may be a fetus, a neonate or an adult.

The optimal in vivo dosage of ubiquinone to resolubilize a β can be determined by a physician according to routine experimentation. An example of such an assay is monitoring the amount of soluble a β in cerebrospinal fluid (CSF) (WO 93/10459, 5 months and 27 days 1993, University of Melbourne). Starting from a relatively low dose (10-25 mg/day), the physician can monitor the amount of dissolved a β in the CSF of the patient. If the dissolved a β, which is an indicator of zinc-a β aggregate resolubilization, is not increased in response to administration of ubiquinone, the dosage of ubiquinone may be increased until an increase in dissolved a β is observed. Another example is monitoring clinical signs and symptoms of disease through clinical, behavioral, and psychometric observations and determinations.

Before administration of a drug to a human, the efficacy of the drug is best demonstrated in an animal model. Any animal model of Alzheimer's disease known in the art may be used.

The pharmaceutical compositions according to the invention preferably contain one or more pharmaceutically acceptable carriers and active ingredients. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. In a preferred embodiment, the ubiquinone and optionally additional active ingredients in the pharmaceutical composition are purified.

It will be appreciated that the amount of ubiquinone and optionally additional active ingredient required for such treatment or prevention will vary with the following factors: the route of administration, the disease to be treated, the physical condition, age, medical history of the subject, and galenic forms of the pharmaceutical composition, and the like. When treating a patient diagnosed with Alzheimer's disease, the amount of ubiquinone is preferably an amount effective to increase the solubility of A β -aggregates in the cerebrospinal fluid of the patient.

Generally, an appropriate therapeutically effective amount of the ubiquinone in the pharmaceutical composition is, for example, 5 to 250mg, preferably 10 to 50 mg. The appropriate amount of a compound selected from the group consisting of antioxidants, μ l agonists, anticholinesterase inhibitors, trace metals, prosthetic groups and clioquinol or a mixture of these compounds in the pharmaceutical composition is for example 5 μ g to 250mg, preferably 0.5 to 1 mg. If chloroiodohydroxyquine and vitamin B are selected₁₂The amount of clioquinol is preferably an amount effective for the treatment or prevention of Alzheimer's disease, and vitamin B₁₂The amount of (a) is preferably an effective amount to inhibit the deleterious side effects of administration of clioquinol. Clioquinol and vitamin B₁₂The amounts are preferably 5mg to 250mg, most preferably 10mg to 50mg and 5. mu.g to 2mg, most preferably 0.5mg to 1mg, respectively.

Actually administered ubiquinone and optionally additional active ingredients such as clioquinol and vitamin B₁₂The amount of (c) can be determined by the monitoring physician. If the pharmaceutical composition contains in addition to the ubiquinoneThe additional active ingredients may then be administered simultaneously in the same composition or in different compositions for substantially simultaneous but separate or sequential administration.

Therapeutic formulations include those suitable for parenteral (including intramuscular and intravenous), oral, rectal or transdermal administration, although oral administration is the preferred route. Thus, the pharmaceutical composition may be formulated as tablets, pills, syrups, capsules, suppositories, preparations for transdermal use, powders (in particular, lyophilized powders for intravenous injection, reconstituted with a carrier), and the like.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. The carrier in the pharmaceutical composition may include binders such as microcrystalline cellulose, polyvinylpyrrolidone (povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; disintegrating agents such as alginic acid, corn starch, etc.; lubricants or surfactants such as magnesium stearate or sodium lauryl sulfate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; and/or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Therapeutic preparations suitable for oral administration, such as tablets and pills, may be obtained by tabletting or molding, optionally containing one or more accessory ingredients. Compressed tablets are prepared by mixing the ingredients and compressing the mixture into tablets of a certain size on a suitable apparatus. Prior to mixing, the ubiquinone may be mixed with a binder, lubricant, inert diluent, and/or disintegrant and the other components optionally present additionally mixed with a diluent, lubricant, and/or surfactant.

In a preferred embodiment, free-flowing ubiquinone powder is mixed with a binder (e.g., microcrystalline cellulose) and a surfactant (e.g., sodium lauryl sulfate) until a uniform mixture is obtained. Subsequently, another binder, such as polyvinylpyrrolidone, is transferred to the mixture with stirring. After achieving uniform distribution, vitamin B is added under constant stirring₁₂An aqueous solution of (a). Make itThe mixture is passed through a granulation screen and dried by dehydration and then compressed into tablets on standard tabletting equipment.

In a second preferred embodiment, a free-flowing powder of ubiquinone is combined with a surfactant and/or emulsifier such as Sapamine^(N- (4' -stearamidophenyl) -trimethylammonium methylsulfate) and lactose monohydrate were mixed until a homogeneous distribution of the various components was achieved. A second preparation containing a disintegrant such as corn starch is added to the ubiquinone mixture under continuous stirring. This second preparation was prepared by adding an excess of boiling water to corn starch suspended in cold water. The final mixture is granulated and dried as above and mixed with corn starch and magnesium stearate and finally compressed into tablets on standard equipment.

The tablets may be coated or uncoated. The uncoated tablets may be scored tablets. Coated tablets may be coated with sugar, shellac, film or other enteric coating agents.

Therapeutic formulations suitable for parenteral administration include sterile solutions or suspensions of the active ingredient. Aqueous or oily vehicles may be used. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Formulations for parenteral administration also include lyophilized powders comprising ubiquinone and optionally additional active ingredients, which can be reconstituted by dissolving in a pharmaceutically acceptable carrier, such as an aqueous solution of carboxymethylcellulose and sodium lauryl sulfate, in which the active ingredients are dissolved.

When the pharmaceutical composition is a capsule, it may contain a liquid carrier such as a fatty oil, e.g. cocoa butter.

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, extended release formulations and the like. The compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides.

In another embodiment, a controlled release system may be used to deliver the therapeutic compound. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC crit. Ref. biomed. Eng.14: 201 (1987); Buchwald et al, Surgery 88: 507 (1980); Saudek et al N.Engl. J. Med.321: 574 (1989)). In another embodiment, polymeric materials may be used (see Medical applications of Controlled Release, Langer and Wise (eds.), CRC Pres, Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.Macromol.Sci.Rev.Macromol.Chem.23: 61 (1983); also see Levy et al, Science 228: 190 (1985); During et al, Ann.neurol.25: 351 (1989); Howard et al, J.Neurosurg.105: 1989)). In another embodiment, a controlled release system may be placed near the target of the therapeutic agent, i.e., the central nervous system, such that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of controlled Release, supra, Vol.2, pp.115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249: 1527-.

In one embodiment of the pharmaceutical composition of the present invention, the ubiquinone and the additional active ingredient are formulated as separate pharmaceutical formulations. For example, one formulation may contain ubiquinone and clioquinol, while another formulation may contain vitamin B₁₂. The two formulations may be administered simultaneously or sequentially. For example, a formulation containing both ubiquinone and clioquinol may be administered first, followed by administration of vitamin B within one day, one week or one month of the administration of clioquinol and ubiquinone₁₂. If the two formulations are administered sequentially, it is preferred that the formulation containing ubiquinone and clioquinol is administered for 1-3 weeks followed by a 1-4 week washout period during which the formulation containing the vitamin is administeredElement B₁₂But not a formulation containing clioquinol. After the washout period, the treatment is repeated.

The invention also provides a kit or kit comprising one or more containers containing one or more of the components of the pharmaceutical compositions of the invention. Such containers optionally carry a notice described by a regulatory agency regulating the production, use or sale of a pharmaceutical or biological product, which notice reflects the approval of the agency to produce, use or sale of a human medication.

Other features and advantages of the present invention will become apparent from the following description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention.

Examples

Example 1

Effect of ubiquinone on A β (25-35) dose-response in PC12 cells

Ap (25-35) was supplied by bachem (ch) or sigma (usa) and was dissolved in Phosphate Buffered Saline (PBS) at pH7.4 2 hours prior to use. The neurotoxicity of A.beta.lies in the sequence between residues 25 and 35 of amino acids (A.beta. (25-35)), and decapeptides containing this region were as effective in inducing neuronal cell death as full-length A.beta. (1-40) (Yankner, DuffyLK, Kirschner DA: neurophotophic and neuropathic effects of amyloid. beta. -protein: reverse by tachykinin in neuropopeptides. science 1990; 250 (4978): 279-282).

Rat PC12 pheochromocytoma cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) in 5% CO₂Growth in a humidity-adjusting incubator: 1% penicillin-streptomycin, 5% fetal bovine serum and 10% horse serum.

PC12 cells were seeded in 96-well microtiter plates containing 100. mu.l of the appropriate medium. After 24 hours, a β (25-35) peptide at the indicated concentration was added alone or together with the indicated concentration of ubiquinone. Incubation was continued for 24 hours. After incubation, MTT reduction was determined using a commonly available assay according to the manufacturer's instructions (boehringer mannheim). The measurement obtained using the vehicle alone was defined as 100%.

MTT is a substrate for intracellular and plasma membrane (plasma membrane) oxidoreductases and is widely used to measure the reduction of cellular redox activity. The reduction of redox activity of this cell was found to be an early indicator of A.beta.mediated cell death (Shearman MS, Ragan CI, Iversen LL: Inhibition of PC12 cell redox is a specific, early indicator of the mechanism of beta-amyloid-mediated cell death, Proc. Natl.Acad. Sci.USA 1994; 91 (4): 1470-.

To examine the effect of ubiquinone on A β (25-35) -induced cytotoxicity of PC12, PC12 cells were contacted with A β (25-35) peptide at a concentration of 0-10 μ M. In the absence of ubiquinone (i.e., vehicle only), A β (25-35) produces a dose-dependent inhibition of MTT reduction (FIG. 1, filled circle). A β (25-35) concentration as low as 0.01 μ M produces a significant reduction, and MTT reduction is reduced by a maximum of about 50% at A β (25-35) concentrations of 0.1 μ M or above.

In the presence of 10. mu.g of ubiquinone per ml, the toxic effects of A.beta. (25-35) were virtually eliminated (FIG. 1, open circles). The presence of ubiquinone can completely combat the toxic effects of a β even at a β concentration as high as 1 μ M. Only at the highest concentration of a β (10 μ M) does a β have a slight inhibitory effect on MTT reduction. However, the inhibition was moderate, being only about 10% compared to the absence of the ubiquinone (about 50%).

Example 2

Effect of ubiquinone on A β (25-35) -induced toxicity in PC12 cells

The same materials and methods as in example 1 were used in this study, except that PC12 cells were contacted with a fixed concentration of A β of 1. mu.M, and the concentration of ubiquinone was varied between 0-10. mu.g/ml. In the absence of ubiquinone, A β (25-35) produced about 40% inhibition of MTT reduction (figure, open circle). The inhibitory effect was maintained with up to 0.1. mu.g of ubiquinone per ml. Increasing the concentration of ubiquinone above 0.1. mu.g/ml produced a significant reduction in the toxic effects of A.beta. (25-35). At concentrations of 0.1. mu.g/ml and above, the toxic effects of A.beta. (25-35) were completely eliminated.

Example 3

Ubiquinone and clioquinol pair Zn²⁺And Cu²⁺Effect of induced Ass aggregation

Prior to each assay, a 5mg/ml stock of A β (1-40) (supplied by Bachem (CH)) was freshly prepared by dissolving the lyophilized peptide in 0.01M HCl followed by a 1: 1 dilution with 0.01M NaOH to give a neutral pH. Each aliquot of A.beta. (1-40) was diluted to 100. mu.M with PBS (pH7.4) and incubated at room temperature for 24 hours in a total volume of 30. mu.l. In the co-incubation assay, the indicated concentrations of metal ions and/or aliquots of test compound are added. Test compound was added to a final molar concentration of 10. mu.g/ml.

Amyloid formation was quantified by thioflavin T fluorescence assay. Thioflavin binds specifically to amyloid, which shifts its emission spectrum, while the fluorescence signal is proportional to the amount of amyloid formation. After incubation, A β (1-40) peptide was added to PBS (pH6.0) and 3 μ M thioflavin T in a final volume of 1 ml. Fluorescence was detected with a Fluoroscan II fluorometer (Molecular devices, UK) at 454nm excitation and 482nm emission. A time scan of fluorescence was performed and the background fluorescence of 3 μ M thioflavin T was subtracted after the decay reached a plateau (about 5 minutes) and the average of the three values was calculated. During the co-incubation assay, the fluorescence of the individual test compounds is measured. Samples were run in triplicate. The mean ± SD of a typical experiment is shown in the figures (fig. 3 and 4).

The ability of ubiquinone and clioquinol to prevent aggregation of A β (1-40) into amyloid structures was determined. Clioquinol is known as a compound for the treatment of alzheimer's disease (see international publication No. WO 98/06403) and was originally tested in this assay for comparison.

Whether the two compounds are paired or not was investigatedMetal ion catalyzed Ass aggregation, particularly by Zn²⁺And Cu²⁺The resulting aggregation has an effect. The test results are shown in fig. 3 and 4.

As can be seen from FIGS. 3 and 4, Cu is relative to spontaneous aggregation²⁺And Zn²⁺Aggregation of a β into amyloid structures may be increased to a lesser extent. In the presence of ubiquinone and clioquinol at a concentration of 10 μ g/ml, the metal ion-induced Α β aggregation was significantly reduced.

Cu in the presence of test concentrations of ubiquinone at 10. mu.g/ml²⁺The induced aggregation can be reduced by 50-60%, while Zn²⁺Induced aggregation was only moderately inhibited by about 10%. Surprisingly, clioquinol showed the opposite trend. Clioquinol p-Zn²⁺Induced Ass (1-40) aggregation is reduced by over 60%, while for Cu²⁺Catalytic aggregation was reduced by about 30%. Therefore, the pharmaceutical composition containing the ubiquinone and clioquinol has a wider use than that containing only one compound.

Example 4

In this example, a clioquinol metabolite was synthesized.

Clioquinol is known to be excreted by the kidney as a glucuronide derivative of clioquinol (Kotaki H., et al: "Enterophastic circulation of clinical in the rat", J.Pharmacobiodyn.1984, 6 months; 7 (6): 420-5 and Jurmima M., et al: "Metabolism of 14C-iodochlorohydroline in the dog and at", J.Pharmacobiodyn.1984, 3 months; 7 (3): 164-70). It is envisaged that conversion of clioquinol to the corresponding glucuronide occurs in the liver. After the chloroiodohydroxyquine glucuronide is formed in the liver, it is eventually transferred to the kidney for excretion with the urine.

The glucuronide derivative methyl ester of clioquinol (5-chloro-7-iodo-quinolyl-2 ', 3 ', 4 ' -tri-O-acetyl-glucopyranoside) uronate was prepared according to the following reaction scheme:

5-chloro-8-hydroxy-7-iodo-quinoline (50mg, 0.164mmol), 1-bromo-1-deoxy-2, 3, 4-tri-O-acetyl-D-glucopyranoside uronic acid methyl ester (65mg, 0.164mmol), calcium sulfate monohydrate (35mg) and pyridine (1.5ml) were stirred at room temperature for 20 minutes. To the reaction mixture was added freshly prepared silver carbonate (35mg), and the suspension was stirred at room temperature in the dark for 20 hours. The reaction product was subsequently deacetylated with 1N aqueous sodium hydroxide solution.

The reaction mixture was diluted with dichloromethane (10ml), filtered and the solvent was evaporated under reduced pressure. The product was purified by flash chromatography (TLC: dichloromethane/methanol 99/1, eluent dichloromethane/methanol 99.5/0.5).

NMR(400MHz，CDCl₃)2.04(s，3H，Ac)，2.09(s，3H，Ac)，2.13(s，3H，Ac)，3.68(s，3H，Me)，3.99(d，1H，5’-H)，5.40-5.52(m，3H，2’，3’-，4’-H)，6.29(d，1H，1’-H)，7.56(m，1H，3-H)，7.99(s，1H，6-H)，8.52(d，1H，4-H)，8.93(s，1H，2-H)。

This compound is referred to below as clioquinol glucuronide.

Example 5

Nuclear Magnetic Resonance (NMR) spectroscopy for vitamin B study₁₂Interaction with clioquinol glucuronide prepared in example 4.

Since chloroiodohydroxyquine glucuronide is soluble in water, the study was performed in an aqueous buffered solution at a pH of 6.5. Three different solutions were prepared and recorded on a DRX 400MHz spectrometer at 20 deg.C¹H NMR spectrum. Solution a) contains free vitamin B in a concentration of 2.6mM₁₂(hydroxy cobalamin). Solution B) contains 2.6mM vitamin B₁₂And 10mM chloroiodohydroxyquine glucuronide mixture (molar ratio of about 1: 4). Solution c) contained 10mM clioquinol glucuronide.

In the figureIn 5, the aromatic region (5.5-9.8ppm) of the spectrum of the three solutions is indicated. The difference between them is very small but is evident in the expanded fig. 6 and 7, respectively. Vitamin B₁₂The partial resonances (corresponding to the benzimidazole moiety) of (a) produce a shift (see figure 6), and the same phenomenon is observed for both resonances (corresponding to the quinoline moiety) of clioquinol glucuronide (see figure 7).

The results show that chloroiodohydroxyquine glucuronide and vitamin B₁₂The interaction is generated probably due to vitamin B₁₂The benzimidazole moiety of (a) and the quinoline moiety of clioquinol glucuronide.

It is considered that vitamin B₁₂The hydrophobic combination with the chloroiodohydroxyquine glucuronide causes vitamin B₁₂Causes of excretion from the body together with clioquinol glucuronide, thereby preventing vitamin B₁₂Is finally absorbed, resulting in vitamin B₁₂And (4) lack. Therefore, it is considered that vitamin B₁₂The lack is at least to some extent responsible for SMON. Thus, when clioquinol is administered, it should be ensured that certain levels of vitamin B are present in the subject being treated₁₂To avoid its lack. This can be achieved by reacting clioquinol with vitamin B₁₂The co-administration is complete.

Example 6

Preparation of pharmaceutical compositions containing ubiquinone

Mixing 250g of ubiquinone and 200g of sapamine^(N- (4' -stearamido-phenyl) -trimethylammonium methylsulfate) and 1025g lactose monohydrate were mixed for 5 minutes. To a mixture of 100g corn starch and 100g cold water was added 300g of boiling water in one portion. The corn suspension cooled to 40 ℃ was added to the powder mixture containing the ubiquinone under continuous stirring. The mixture was granulated with a 2.5mm sieve and dried at 40 ℃ for 18 hours. The dried granules were mixed with 400g of corn starch and 20g of magnesium stearate. The final mixture was formulated into tablets having a diameter of 8.0mm and a weight of 200 mg.

Example 7

Preparation of a pharmaceutical composition containing ubiquinone, clioquinol and vitamin B₁₂Pharmaceutical composition of

Mixing 250g of pantoquine, 250g of clioquinol and 200g of sapamine^(N- (4' -stearamido-phenyl) -trimethylammonium methylsulfate) and 1025g lactose monohydrate were mixed for 5 minutes. To a mixture of 100g corn starch and 100g cold water was added 300g of boiling water in one portion. The corn suspension cooled to 40 ℃ was added to the powder mixture containing the ubiquinone and clioquinol with continuous stirring. Followed by the addition of 5g vitamin B₁₂An aqueous solution of (a). The mixture was granulated with a 2.5mm sieve and dried at 40 ℃ for 18 hours. The dried granules were mixed with 400g of corn starch and 20g of magnesium stearate. The final mixture was formulated into tablets having a diameter of 8.0mm and a weight of 200 mg.

The disclosures of the various publications cited herein are incorporated by reference.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims (22)

1. Use of ubiquinone in the manufacture of a pharmaceutical composition for the treatment or prevention of alzheimer's disease.

2. The use according to claim 1, wherein the ubiquinone is administered in an amount of 5mg to 250mg 1 to 3 times per day.

3. The use according to claim 2, wherein the ubiquinone is administered in an amount of 10mg to 50mg 1 to 3 times per day.

4. The use of any one of claims 1-3, wherein the pharmaceutical composition is formulated for oral, parenteral or transdermal administration.

5. Use of ubiquinone and clioquinol for the manufacture of a pharmaceutical composition for the treatment or prevention of alzheimer's disease.

6. The use of claim 5, wherein the pharmaceutical composition further comprises vitamin B₁₂。

7. The use of claim 5 or 6, wherein the pharmaceutical composition is formulated for oral, parenteral or transdermal administration.

8. A pharmaceutical composition comprising (a) an effective amount of a ubiquinone to treat or prevent Alzheimer's disease and (B) clioquinol and vitamin B₁₂A mixture of (a).

9. The pharmaceutical composition of claim 8, further comprising a pharmaceutically acceptable diluent, adjuvant, excipient or vehicle.

10. The pharmaceutical composition of claim 8 or 9, wherein the amount of the ubiquinone is 5 to 250 mg.

11. The pharmaceutical composition of claim 10, wherein the amount of the ubiquinone is from 10mg to 50 mg.

12. The pharmaceutical composition of claim 8 or 9, wherein the amount of clioquinol is from 5mg to 250 mg.

13. The pharmaceutical composition of claim 12, wherein the amount of clioquinol is from 10mg to 50 mg.

14. The pharmaceutical composition of claim 8 or 9 wherein vitamin B₁₂The amount of (B) is 5. mu.g-2 mg.

15. The pharmaceutical composition of claim 14 wherein vitamin B₁₂The amount of (B) is 0.5mg-1 mg.

16. The pharmaceutical composition of claim 8 or 9, wherein the composition is formulated for parenteral, transdermal or oral administration.

17. The pharmaceutical composition of claim 8 or 9, wherein the composition is formulated as a tablet.

18. A kit comprising in one or more containers an effective amount of a group consisting of ubiquinones and clioquinol for treating or preventing Alzheimer's disease and an effective amount of vitamin B for inhibiting the deleterious side effects of administration of clioquinol₁₂。

19. The kit of claim 18, wherein the ubiquinone is contained in one pharmaceutical composition, the clioquinol is contained in a second pharmaceutical composition, and the vitamin B is₁₂Is contained in the third pharmaceutical composition.

20. The kit of claim 18 wherein the ubiquinone is contained in a pharmaceutical composition and clioquinol and vitamin B₁₂Is contained in a second pharmaceutical composition.

21. The kit of claim 18 wherein the ubiquinone and clioquinol are contained in a pharmaceutical composition and the vitamin B is₁₂Is contained in a second pharmaceutical composition.

22. The kit of claim 18, wherein the ubiquinone and vitamin B₁₂Contained in one pharmaceutical composition and clioquinol contained in a second pharmaceutical composition.