UA59412C2 - Use of phanquinone for treatment of alzheimer's disease - Google Patents

Abstract

The use of phanquinone for the manufacture of a pharmaceutical composition for the prevention or the treatment of Alzheimer's disease is disclosed. Also methods of treatment or prevention of Alzheimer's disease are disclosed.

Description

Description of the invention

The present invention relates to the use of a known compound for the preparation of pharmaceutical composition 2 for the treatment or prevention of Alzheimer's disease. Further, this invention relates to a pharmaceutical composition for such treatment or prevention. The present invention also relates to methods of treating or preventing Alzheimer's disease.

Alzheimer's disease (AD), which is the single leading cause of dementia in adults in industrialized societies, is a degenerative brain disorder characterized clinically by progressive memory loss, confusion, dementia, and ultimately death. Histopathologically, Alzheimer's disease is characterized by the presence in the neocortex, in particular, in the hippocampus, of two brain lesions: neurofibrillary tangles (MET) of paired spiral filaments (PNE) in neurons and neuritic (senile) plaques in the extracellular space. The formation of senile plaques is associated with the appearance of symptoms and signs of this disease, including amnesia. After the formation of a senile plaque, neurofibrillary tangles are formed in the bodies of 12 neurons (perikaryons). The formation of neurofibrillary tangles is associated with the worsening of amnesia and other symptoms of dementia.

The main component of senile plaques is amyloid deposits. Cataracts are a disease that often occurs together with Alzheimer's disease. It is localized in the eyes and is also due to the deposition of amyloid. Thus, the treatment of patients with Alzheimer's disease, which is described in this description 20 of the invention and the claims of the invention, can equally be used for the treatment of cataracts. Therefore, as used in this description and claims, the term Alzheimer's disease also includes cataracts.

The main component of amyloid deposits is a polypeptide called A D (amyloid-beta). AB is usually a soluble component of cerebrospinal fluid, where it is found in a concentration of approximately 3-BNM. AB can have 39-43 amino acids, usually 40 amino acids, in the mature form and is formed as a 29 product of proteolytic cleavage from a cell surface protein called amyloid precursor protein (APP) (Kapa eyi a. 1987, Mayte 325: 733-736).

Many studies have shown that AB is toxic to mice when added directly to nerve cell cultures (Uapkpeg V.A., YuOshiyu G.K., Kigzsppeg O.A., Zsiepse 1990, 250 (4978): 279-282; Kop U .M., Mapd ..,

Soitap S.MU., Vgaiip Kez 1990, 533 (2): 315-320; and Rike S.)., Vyhaisk O., UaIepsemis: A.)., Siare S.0., Soitap from 30 SMU,, u). Meshhovsi 1993, 13(4): 1676-1687). -

It was determined that the neurotoxicity of AB is localized in the peptide sequence, between amino acid residues 25 and 35 (AB(25-35)). AB(25-35) induces nerve cell death with the same potency as full-length t

AVI(I-40) (UapKkpeg V.A., Oshpu G.K., Kigesppeg O.A., Zsiepse 1990, 250 (4978): 279-282). Normal function AV Ge)

Nowadays it is unknown, but maybe it consists in the formation of cation-selective channels through cellular

From the membrane (Kamzhapnaga M. ei a/!., 1997, Viorpuzisa! doigpa! 73/1, 67-75). i am

It was shown that the precipitation of synthetic AB is caused by several factors of the external environment, including low pH, high salt concentration, and the presence of metals, for example, zinc, copper, and mercury (Vyvp, A.I., 1995, Zsiepse 268: 1921 -1923). It was reported that AB itself specifically and "saturatingly" binds zinc with a high binding affinity (K p0-107 nM) at a molar ratio of 1:1 (zinc: Z7Z

AV) (Vyvzy, A.I. ei aiYi., 1994, 9. Vioi. Spet. 269: 12152-12158). This binding takes place at physiological concentrations of zinc (Viznp, A. I. ei aiP., 1994, Zsiepse 265:1464-1467). "from It is strongly suggested that removing amyloid deposits from patients suffering from the disease

Alzheimer's disease, will reduce the symptoms of Alzheimer's disease. Thus, several attempts have been made to prepare such a medicine, because persistent searches for ways to treat the disease are underway

Alzheimer's o International patent application, UMO publication 93/10459, describes a method of treating Alzheimer's disease by (Ce) administration of a zinc-binding agent. Phytic acid, desferrioximine, sodium citrate, EDTA, 1,2-diethyl-3-hydroxypyridin-4-one and 1-hydroxyethyl-3-hydroxy-2-methylpyridin-4-one are mentioned as preferred compounds. German patent application OE 39 32 338 describes the use of an agent capable of chelating aluminum, such as 8-hydroxyquinoline, for the treatment of Alzheimer's disease.

US Patent No. 5,373,021 describes the use of disulfiram and its salts and analogs. According to this patent, the compounds described above can be used to reduce the neurological damage caused by Alzheimer's disease.

International patent application, publication MO 98/06403, describes the use of clioquinol for the preparation of 99 pharmaceutical compositions for the treatment of Alzheimer's disease.

GF) The compounds known so far, which are proposed for the treatment of Alzheimer's disease, have several disadvantages that prevent their widespread use. Most of these compounds are unable to penetrate the blood-brain barrier and, therefore, cannot easily reach the areas where amyloid is deposited. Disulfiram, which can penetrate the blood-brain barrier, has the disadvantage that when it is combined by a patient with ethyl alcohol, it causes severe side effects, including headache, nausea, vomiting, sweating, thirst, weakness and low blood //- pressure. Cliquinol (5-chloro-7-iodo-8-hydroxyquinoline), which can also cross the blood-brain barrier, has a damning history of causing subacute myelo-optic neuropathy (5AM) as a side effect. for Fanquinone (4,7-phenanthroline-5,6-dione) has been used until now to treat a variety of disorders such as amoebiasis. Fanchinon was sold to SIVA-ZEIS under the trademark EMTOVEH. In contrast to clioquinol, with the use of fanquinone in the normal range of doses, no adverse side effects were detected.

In the past, an anti-amoebic pharmaceutical containing both clioquinol and phanquinone was marketed

SIVA-SEISU under the trademark Mehaiyog. However, the sale of this drug was stopped when it became clear that clioquinol causes IOM.

The object of this invention is to provide a new application of a known compound for the treatment or prevention of Alzheimer's disease. Another object of this invention is to provide an increased effect of treatment of Alzheimer's disease. Another object of this invention is to eliminate any harmful 7/0 side effect of treating or preventing Alzheimer's disease.

According to this invention, the use of fanquinone for the preparation of a pharmaceutical composition for the treatment or prevention of Alzheimer's disease is provided. Fanquinone can be administered in any amount effective to treat or prevent Alzheimer's disease. Preferably, phanquinone can be administered in the amount of mg - 25Omg and more preferably 1Omg - 5Omg, one to three times a day.

According to one embodiment of the invention, the compound or mixture of compounds selected from the group consisting of antioxidants, acetylcholine enhancers, trace metals, prosthetic groups, and clioquinol is administered prior to, concurrently with, or after the administration of fanquinone.

The present invention also relates to a pharmaceutical composition containing phanquinone and a compound or mixture of compounds selected from the group consisting of antioxidants, acetylcholine enhancers, trace metals, prosthetic groups, and clioquinol, provided that when clioquinol is selected, at least one additional compound is selected from this group.

Antioxidant is mainly vitamin C, vitamin E, 010 or their combination. Acetylcholine enhancers are mainly Mi agonists or anticholinesterase inhibitors (that is, substances that inhibit acetylcholinesterase). The preferred anticholinesterase inhibitors are tacrine (trademark Sodpeh) and schob donepezil (trademark Agiser)). The prosthetic group is mainly vitamin B."

In a preferred embodiment of this invention, phanquinone and clioquinol are used to prepare i) a pharmaceutical composition for the treatment or prevention of Alzheimer's disease. Preferably, a combination of phanquinone, clioquinol and vitamin B" is used to prepare this pharmaceutical composition.

The pharmaceutical composition can be prepared for oral, parenteral or intradermal (intradermal) administration. Further, the pharmaceutical composition can be prepared in the form of a single pharmaceutical composition or in the form of two or more separate pharmaceutical forms for -- sequential or essentially simultaneous administration. "IS

The present invention also relates to a method of treating a subject who has or is suspected of having Alzheimer's disease, which involves administering to the subject an amount of fanquinone effective for the treatment or prevention of Alzheimer's disease. yu

Figure 1 shows the effect of fanquinone on the response depending on the dose of AB(25-35) in PC12 cells.

Figure 2 shows the effect of fanquinone on AB(25-35)-induced toxicity in PC12 cells.

Figure C shows the effect of fanquinone and clioquinol on induced 7p2-- aggregation of AB. "

Figure 4 shows the effect of clioquinol and phanquinone on C-induced AB aggregation.

Figure 5 shows the NMR spectra of three solutions. Solution a contained vitamin B." (hydroxycobalamin) in - with a concentration of 2.6 mm. Solution B contained a mixture of 2.6 mM vitamin B-»o and 10 mM clioquinol glucuronide (molar a ratio of approximately 1:4). Solution c contained 10 mM clioquinol glucuronide. "» Figure 6 shows the right half of Figure 5 enlarged to facilitate comparison of resonance positions.

Figure 7 shows the left half of Figure 5 enlarged to facilitate comparison of resonance positions.

There is a need to market a drug that is effective in the treatment or prevention of Alzheimer's disease. According to the present invention, there is provided a known drug phanquinone. The examples show that in PC12 cells AB(25-35) has a pronounced effect on the redox activity of cells. In the method used in example 2, the redox activity of cells that have been exposed to imm AB(25-35) decreases to 6095 from the normal level (see Fig. 2, light circles). However, when shu 20 treated AB(25-35) cells are treated with μg/ml fanquinone or a higher concentration, normal redox activity is restored. This indicates that phanquinone, when administered to a mammal such as a human, will alleviate the harmful effects of AB.

Without pretending to limit the invention to any specific mechanism of action, the authors now believe that the effect of AB on the redox activity of cells is prevented by the antioxidant effect of fanquinone.

The antioxidant effect of fanquinone can be enhanced by co-administration of additional antioxidant(s), such as vitamin C, vitamin E, vitamin 010, or combinations thereof. o The presence of senile plaques in the extracellular space can prevent or inhibit the transmission of impulses (i.e.) in the cholinergic nervous system. The functioning of this nervous system depends on the mediator acetylcholine.

Thus, compounds that increase the level of acetylcholine, such as compounds that prevent or inhibit 60 the normal hydrolysis of acetylcholine by acetylcholinesterase, so-called anticholinesterase inhibitors, can be administered together with fanquinone to improve the effect. The preferred anticholinesterase inhibitors are tacrine and donepezil.

In the study reported in the examples, it was shown that phanquinone can reduce the aggregation of AB(1-40) induced by Cy?" and 7p2", 65 The following proposed mechanism of action of the present invention is not intended to limit the invention to this mechanism. The applicant now believes that phanquinone and AB competitively chelate zinc, copper and other heavy metals.

Fanquinone is able to penetrate through the blood-brain barrier. When fanquinone captures a heavy metal ion in the extracellular space, it moves into the blood and is excreted from the body. When the aggregate or salt of AB and heavy metal ion is in equilibrium with free AB and free heavy metal ion, fanquinone can continue to capture the free metal ion and move the heavy metal ion into the blood as long as aggregated AB, i.e. amyloid, is present. Free AV cannot penetrate through the blood-brain barrier by passive diffusion, rather it will be destroyed by proteolytic enzymes normally present in the extracellular space. Thus, free AB or its degenerate part can potentially damage nerve cells before it is broken down to a non-toxic state. Meanwhile, fanquinone will avert or inhibit 7/0 the potential cytotoxic effect of AB due to its ability to counteract the reduction of redox activity normally caused by AB.

The reduction of aggregation caused by fanquinone is 50-60956 for Si 25 1 1095 for 7p 27. Further, it is shown that clioquinol has the opposite tendency. Clioquinol reduces 7p2" induced aggregation of AB(1-40) by more than 60965, whereas Cy?" - the induced aggregation was reduced by approximately 3095. In other words, fanquinone is 75 better in reducing C" - induced aggregation of AB(1-40), and clioquinol is better in reducing 7n2" - induced aggregation.

Since Si 27 and 7p" are naturally present in the body of a patient who has Alzheimer's disease or a patient suspected of having Alzheimer's disease, it may be desirable to reduce or redissolve the aggregates induced by both ions. Thus therefore, it may be desirable to administer clioquinol prior to, concurrently with, or following the administration of phanquinone.

However, the introduction of clioquinol is problematic, because clioquinol causes a side effect of PMS. Previous studies (see UUO 98/06403) show a possible effect of clioquinol on vitamin B4.

It is known that clioquinol is excreted through the kidneys in the form of glucuronide or sulfate derivatives (Koyaki

N., ei ai.: Epiegoperaiiis sigstiaiop oi siodtsipoY ip (be hai, 9. RIaptasoIobiodup. 1984 Oype; 1(6): 420-5 p. 29 and diyta M. ei ai: Meiaboivt oi 14S-iodospiogpudgohudiipoipe ip She dod apa ( Shegai, U. Rpagtasoriodup.i (U 1984 Mages; 7(3): 164-790), for example, in the form of the compound methyl (5-chloro-7-iodhinolyl-2",3,4"-tri-O- acetylglucopyranoside)uronate For brevity, this compound is called glucuronide in the following.

Detoxification of hydrophobic substances such as clioquinol in the body mostly occurs in the liver. Thus, it is believed that the clearance of clioquinol occurs as follows: clioquinol is converted into glucuronide "ch- clioquinol in the liver. After its formation, the water-soluble clioquinol glucuronide is secreted into the bile. Bile enters the intestine, where the main amount of clioquinol glucuronide is removed by defecation. Some amount of clioquinol glucuronide is resorbed (absorbed) from the intestines into the blood. Clioquinol glucuronide is filtered from He»! blood to the kidneys and appears in urine, which is formed at the end.

By treating mice with clioquinol and then administering vitamin B 345, which contains the radioisotope o (2 Co|-cyanocobalamin), in international patent application, publication MO 98/06403, it is shown that the concentration of vitamin B4o in the brain and liver of mice treated with clioquinol remains at normal level, while the concentration of radioisotope vitamin B-" decreases in the kidneys of such mice compared to the normal level. 20 This finding suggests that the metabolism of vitamin VI is dependent on clioquinol. Further, this finding suggests that the kidney is the target organ in which clioquinol-dependent metabolism takes place. c To study the possible interaction between clioquinol glucuronide and vitamin B 32 was planned;" an experiment in which clioquinol glucuronide and vitamin B 412 were mixed in water. This mixture was analyzed by H-NMR. The H-NMR spectra, see Figures 5-7, show that some resonances of vitamin B 42 (corresponding to the benzimidazole part of the molecule) have been shifted, and the same is observed for the two resonances of the glucuronide axes clioquinol (corresponding to the quinoline part of the molecule). The applicant believes that similar results could be expected using free clioquinol, however, clioquinol cannot be dissolved in aqueous and NMR testing solutions. "g" These results indicate a hydrophobic interaction between clioquinol glucuronide and vitamin B42, possibly

Between the benzimidazole part of vitamin B." and the quinoline part of clioquinol glucuronide. - Vitamin B4o is normally actively resorbed from kidney plasma after its filtration. In this way, the body

Kz extracts most of the vitamin B 45 that would otherwise be lost in the urine. It was recently demonstrated that the resorption of vitamin B is mediated by the action of the membrane protein megalin (Moezighir 5. K. ei ai. Ros. Mai. Asai. si. 1996; 93(16): 8612-7). Megalin has been shown to have a strong affinity for

Binding of the complex formed by vitamin B 45 and the transport protein transcobalamin.

Based on the new discovery reported here that vitamin B 32 binds to clioquinol glucuronide,

F, the applicant believes that vitamin B0 does not bind to bilum megalin and/or transcobalamin when it is already bound to clioquinol glucuronide. Thus, the resorption of vitamin B42 will be absent and the body will suffer from a deficiency of vitamin B10 after a certain period of administration of clioquinol, if it is not supplied in the form of a sufficient amount of new vitamin B41" with a normal diet.

The only source of vitamin VI2 in the human body is food, because a person cannot synthesize it.

Thus, diets low in meat and/or micro-organisms will obviously cause vitamin B deficiency." If individuals are provided with diets too low in vitamin B 415 , then the introduction of clioquinol will worsen the condition, because resorption of vitamin B" is prevented by the competitive binding of clioquinol 65 With megalin. The fact that ZMOM was observed only in Japan and that the Japanese diet mostly consists of vegetables and grains, in particular, rice (Kgotpotsi O, eyi a): "Bod sopzitrip racegp ip (pe 19605 ip zemep sotspigev", At. y) . Siip. Myig. 49: 889-894, 1989) may explain why the disease of ZMOM was limited to

by Japan

To counteract the side effects of the introduction of fanquinone and/or clioquinol, it may be desirable to co-introduce trace amounts of metals and prosthetic groups. The predominant prosthetic group is vitamin B.12 (cyanocobalamin).

By way of example, but not limitation, the compositions, uses, and methods of the invention may be indicated for: 1) patients diagnosed with Alzheimer's disease at any clinical stage of the disease, 2) prevention of Alzheimer's disease in patients with early or prodromal symptoms, or signs and 3) /o Delays in the appearance or development or exacerbation of symptoms and signs of Alzheimer's disease. The methods and compositions of the present invention will, for example, be useful for treating Alzheimer's disease, ameliorating or alleviating any of the symptoms and signs of Alzheimer's disease, ameliorating any pathological or laboratory findings of Alzheimer's disease, delaying the onset of any symptoms and signs of Alzheimer's disease , preventing the onset of Alzheimer's disease and reversing the appearance of any symptoms and signs of Alzheimer's disease /5 or Parkinson's disease.

The subject or patient is an animal, such as a mammal and, in particular, a human, and may be a fetus, a child or an adult.

The dose of fanquinone, optimal for repeated solubilization of AB, can be determined by the doctor by conducting routine experiments. An example of such an experiment is the monitoring of soluble AV in cerebrospinal fluid (CSF) (MO 93/10459, May 26, 1993/ University of Melbourne).

Starting with relatively low doses (10 - 25 mg/day), the doctor can observe the amount of solubilized AB in

SZE of the patient. If there is no increase in solubilized AB in response to the introduction of fanquinone, which indicates re-solubilization of 7p-AB aggregates, then the dose can be increased until such an increase is observed. Another example is the monitoring of clinical signs and symptoms of this disease using clinical, behavioral and psychometric observations and measurements.

Before administration to humans, the effectiveness is mainly shown in animal models. Can be used i) any animal model for Alzheimer's disease known in the art.

Pharmaceutical compositions according to the present invention preferably contain one or more pharmaceutically acceptable carriers and an active component(s). The bearer(s) must be "accepted" within the meaning of their p

From compatibility with other ingredients of the composition and not harmful to their recipients. In a more preferred version, fanquinone and optional additional active components in this pharmaceutical composition are purified. --

It should be understood that the amount of phanquinone and optional additional active ingredients required for the above treatment or prevention will vary according to the route of administration, the disorder to be treated, the condition, age, history of the subject and the prescribed composition of a pharmaceutical (22) composition, etc. In the treatment of a patient with a diagnosis of Alzheimer's disease, the amount of fanquinone is mostly effective in increasing the solubility of AB aggregates in the patient's cerebrospinal fluid.

In general, a suitable therapeutically effective amount of fanquinone in a pharmaceutical composition is, for example, 5-250 mg, preferably 10-500 mg. A suitable amount of a compound or a mixture of compounds selected from the group consisting of antioxidants, M1 agonists, anticholinesterase inhibitors, trace metals, prosthetic groups and clioquinol in a pharmaceutical composition is, for example, 5mcg 250mg, preferably 0.5 ntsh) s 1 mg. If clioquinol and vitamin B. o are selected, then the amount of clioquinol is mostly effective in the treatment or . prevention of Alzheimer's disease, and the amount of vitamin B4.o is mostly effective for inhibiting any and? harmful side effects of clioquinol administration. The amount of clioquinol and vitamin B4" is preferably 5mg - 25OmgH, more preferably 10mg - 5Omg and 5mcg - 2mg, most preferably 0.5mg - 1mg, respectively.

The amounts of phanquinone and optional additional active ingredients such as clioquinol and vitamin B 45 actually administered may be determined by the treating physician. If this pharmaceutical composition, in addition to phanquinone, contains additional active components, they can be in the same composition for co-administration in combination or in various compositions for administration essentially at the same time, but separately, or sequentially.

Therapeutic compositions include compositions suitable for parenteral (including intramuscular and intravenous), oral, rectal or intradermal administration

From (intradermal) administration, although oral administration is the preferred route. Thus, pharmaceutical compositions can be prepared in the form of tablets, pills, syrups, capsules, suppositories, compositions for transdermal administration, powders, in particular, lyophilized powders for reconstitution in solutions with a carrier for intravenous administration, etc.

The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which the

F) therapeutic agent. Carriers in this pharmaceutical composition may contain a binder such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent such as alginic acid, cornstarch, etc.; a lubricant or surfactant such as magnesium stearate or sodium lauryl sulfate; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin and/or an agent that improves taste and smell such as peppermint, methyl salicylate or orange flavoring.

Therapeutic compositions suitable for oral administration, such as tablets and pills, may be prepared by compression or molding, optionally with one or more auxiliary ingredients.

Compressed tablets may be prepared by mixing the component(s) and pressing this mixture in a suitable device into tablets of a suitable size. Before mixing, the fanquinone may be mixed with a binder, a wetting agent, an inert diluent, and/or a disintegrating agent, and additional optional components may be mixed with a diluent, a wetting agent, and/or a surfactant.

In a preferred embodiment, the free-flowing powder of fanquinone is mixed with a binding agent, such as microcrystalline cellulose, and a surface-active agent, such as sodium lauryl sulfate, to obtain a homogeneous mixture. Then, another binder, such as polyvidone, is transferred into this mixture while stirring.

After obtaining a homogeneous distribution, add an aqueous solution of vitamin VI" with constant stirring. This 7/0 mixture is passed through granulating sieves and dried by dehydration before pressing into tablets in a standard pressing device.

In the second preferred option, the free-flowing powder of fanquinone is mixed with surface-active substances and/or emulsifying agents, such as Zaratipe (M-(4-stearoylaminophenyl)-trimethylammoniummethylsulfuric acid) and lactose monohydrate, to obtain a 7/5 homogeneous distribution of components. A second preparation containing a disintegrating agent, such as corn starch, is added to the fanquinone mixture with continuous stirring. Such a second preparation may be prepared by adding an excess of boiling water to cornstarch suspended in cold water.

The final mixture is granulated and dried as described above and mixed with corn starch and magnesium stearate, and finally compressed into tablets in a standard device.

The tablet may or may not be coated. An uncoated tablet may be rough. A coated tablet may be coated with sugar, shellac, film, or other enteric-dissolving agents.

Therapeutic compositions suitable for parenteral administration include sterile solutions or suspensions of active ingredients. An aqueous or oily carrier may be used. Such pharmaceutical carriers may be sterile liquids such as water or oils, including petroleum oils, oils of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Compositions for parenteral administration also include lyophilized powder, i) which contains phanquinone and optionally additional active components, which must be reconstituted by dissolving in a pharmaceutical acceptable carrier that dissolves these active components, for example, in an aqueous solution of carboxymethylcellulose and sodium lauryl sulfate. When the pharmaceutical composition is a capsule, it may contain a liquid carrier such as a fatty oil, such as cocoa butter. -

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, ethanol etc. Me

These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release preparations, etc. The composition can be prepared in the form of a suppository with conventional binders and carriers, such as triglycerides.

In yet another embodiment, the therapeutic compound may be delivered in a controlled release system. In one variant, a pump can be used (see I apdeg, above; Zeyop, SKS (gii. Kei. Vioted. Epao. 14: « 201 (1987); Vyspulaii ei a. Ziyigdegu 88: 507 (1980); Zatsdek ei ai. , M. Epoi. U. Mea. 321: 574 (19843). In another variant, polymeric materials can be used (see Medadis! Arriisaiope oi SopigoPei

Keyease, I apdeg apa MUiize (ed.), SKS Rhev., Vosa Kap, Riogiza (1974); SopigoPedy Ogoid Vioamayarishu, Ogyd;" Rgodisi YuOevzidp apa Repogtapse, ZtoYep apa VaiYi (Edv.), MMpeu, Mem Mogk (1984); Kapdeg apa Rerrazv, 3.

Masgotoi All together. Chem. Masgotoi Spent 23: 61 (1983); see also I emu ei ai.., Zsiepse 228: 190 (1985);

Oshgipd eyi am, App. Meigoi 25: 351 (1989); Nomaga ek ai., u). Meigwig 71: 105 (1989)). In yet another variant, the controlled release system can be placed close to the therapeutic target, i.e. the central nervous system, resulting in only a fraction of the systemic dose (see, e.g., Boozop, Meadis! Arriisakyopv oi SopigoYied Keyeaze, supra, my. 2 , pp. 115-138 (1984)). Other controlled release systems are discussed in a review of Apdeg I (Zsiepse 249: 1527-1533 (1984)).

In one variant of the pharmaceutical composition according to the present invention, fanquinone and additional active components are contained in the form of separate pharmaceutical forms. As an example, one form might contain

Ge phanquinone and clioquinol, and another form may contain vitamin B. These two forms can be administered simultaneously or sequentially. For example, a form containing phanquinone and clioquinol can be administered, followed by the introduction of vitamin B4" within a day, week or month after the introduction of clioquinol and phanquinone. If these two forms are administered sequentially, the form containing phanquinone and clioquinol is preferably administered for one to three weeks followed by a washout period of one to four weeks during which the form containing

F) contains vitamin B, but not the form containing clioquinol. After a washout period, this treatment can be repeated.

The present invention also provides a pharmaceutical package, or a set containing one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention.

Optionally associated with such containers may be a notice in a form written by a governmental agency that regulates the manufacture, use, or sale of pharmaceutical or biological products, which notice indicates approval by that agency of the preparation, use, or sale for human administration. 65 Other features and advantages of the present invention will be apparent from the following examples, which, in combination with the accompanying illustrative materials, explain, by way of example, the principles of the present invention.

EXAMPLES

Example 1.

The effect of fanquinone on the response depending on the dose of AB(25-35) in PC12 cells.

AB(25-35) was obtained from Waspet (Switzerland) or Zidt (USA) and dissolved in phosphate-buffered saline (PBS) at pH 7.4, 2 hours before use. The neurotoxicity of AB is localized to the sequence between amino acid residues 25 and 35 (AB(25-35)), and a decapeptide containing this region induces nerve cell death as strongly as full-length AB(1-40) (Mappeg , YuOiyu .K.,

Kivsppeg OA: Meigoihorpis apa peigoiohis ePesiv oi oatuid V rgoivip: gemegza! Bu (aspukipip /o peshgoreridev. Zsiepse 1990; 250 (4978): 279-282).

PC12 rat pheochromocytoma cells were grown in Dulbecco's modified Eagle's medium (OMEM) containing 1905 penicillin-streptomycin, 590 fetal calf serum, and 1095 horse serum in a humidified thermostat with 595 CO.

RS12 cells were seeded on 9b-well microtiter plates in 100 μl of the appropriate medium. After 24/5 hours, the indicated concentrations of peptide AB(25-35) were added separately or together with fanquinone in the indicated concentrations. Incubation continued for 24 hours. After incubation, the reduction of MTT was measured using a commercially available test kit in accordance with the manufacturer's instructions (Voepgipdeg MappNeit).

Test values obtained only with the carrier were taken as 10095.

MTT is a substrate for intracellular oxidoreductases and cytoplasmic membrane oxidoreductases and

It has been widely used to measure reductions in redox activity. It was found that a decrease in redox activity is an early indicator of AB-mediated cell death (Zpeaptap M.5., Kadap S..,

Imegvop 1.13: Ipyirbyop o oi RS1T2 sei gedoh asympyu iv a zresiiys, eapu ipaisag oi (She tespapizt oi

In-atuioiid-tediagea sei! Deaii, Rgos. May! Asad 5 BOTH 1994; 91(4): 1470-1474).

To analyze the effect of phanquinone on AB(25-35)-induced toxicity in PC12 cells, PC12 cells were incubated with concentrations of AB(25-35) peptide in the range from 0 to 100μM. In the absence of phanquinone (ie, with the addition of only the carrier), AB(25-35) caused a dose-dependent inhibition of the decrease in MTT (Fig. 1, bold circles). and)

Concentrations of AB(25-35) as low as O0.01 mM caused a significant decrease, and at concentrations of AB(25-35)

0.1 mM or greater MTT reduction decreased to a maximum level of approximately 50905.

In the presence of TOmkg of fanquinone per ml, the toxic effect of AB(25-35) was practically eliminated (Fig. 1, light circles). with

Even at a concentration of AB as high as 1 μM, the presence of fanquinone completely neutralized the toxic effect

AV. Only at the highest concentration of AB (1ΩμM) was there a slight inhibition of the reduction of MTT AB. However, this inhibition was moderate, only about 1095, compared to about 5095 in the absence of fanquinone. "

Example 2.

Effect of fanquinone on AB(25-35)-induced toxicity in PC12 cells. me)

In this study, the same materials and methods were used as in Example 1, except that PC12 cells were maintained with a fixed concentration of AB in μM, while the concentration of phanquinone was varied between 0 and 10 μg/ml. In the absence of fanquinone, AB(25-35) resulted in approximately 4095 inhibition of MTT reduction (Fig. 2, light circles). This inhibition was maintained in the presence of up to 0.1 μg of fanquinone per ml.

An increase in the concentration of phanquinone above 0.um kg/ml led to a sharply reduced toxic effect "

AB (25-35). At phanquinone concentrations of mcg/ml and higher, the toxic effect of AB(25-35) was completely eliminated. Example 3. Effect of phanquinone and clioquinol on 7p27- and C2"-induced AB aggregation. "» Initial solution of 5 mg/ml AB(1-40) (supplied from Waspet (Switzerland)) was prepared again before each experiment by dissolving of the lyophilized peptide in 0.01M Nei, followed by serial dilution 1:1 with 0.01M Mahon to obtain a neutral pH. Aliquots of AB(1-40) were diluted in SFR (pH 7.4) to 100 μM and were incubated in a total volume of 3 μL for 24 hours at room temperature. For coincubation experiments, the specified concentrations of metal ions and/or an aliquot of test compounds were added. The test compounds were added to a final molar concentration of 10 μg/ml. "d" Amyloid formation was quantified using a fluorometric assay with thioflavin T. Thioflavin binds specifically to amyloid and this shifts its emission spectrum, producing a fluorescent signal proportional to the amount of amyloid. After incubation, AB(1-40) - peptides were added to SFR

Kz (pH 6.0) 13 μM thioflavin T in the final volume tTml. Fluorescence was continuously measured at an excitation wavelength of 454 nm and an emission wavelength of 482 nm using a Fluoroscan II fluorometer (MoiIesshag Demises, UK). Temporal scanning of fluorescence was carried out and three values after attenuation reaching a plateau (about 5 minutes) were taken to calculate the average value after subtraction of background fluorescence

ZmMkM of thioflavin T. For experiments with coincubation, the fluorescence of one test compound was determined. samples

F, analyzed in three repetitions. The mean 50 (standard deviation) for a typical experiment is shown in the figures (Figs. 3 and 4).

Fanquinone and clioquinol were tested for their ability to prevent aggregation of AB(1-40) into amyloid structures. because Clioquinol is a known compound in the treatment of Alzheimer's disease (see International Publication No. UUO 98/06403) and was originally tested in these experiments for comparative purposes.

It was investigated whether these two compounds had any effect on metal ion-catalyzed AB aggregation, in particular, aggregation caused by 7p2" and Su". The results of these experiments are shown in Fig. With and 4.

Figures 3 and 4 show that C" and 7p2", to a lesser extent, increase the aggregation of AB into amyloid structures b5 relative to spontaneous aggregation. In the presence of phanquinone and clioquinol at a concentration of 1 TOmkg/ml, aggregation of AB induced by metal ions was significantly reduced.

At the tested concentrations of 1 µg/ml, phanquinone reduced Si?-induced aggregation by 50-6096, while 7p? -induced aggregation was only moderately inhibited at about 1095. Surprisingly, clioquinol showed the opposite trend. Clioquinol reduced the 7p2"-induced aggregation of AB(1-40) by more than 6096, while the aggregation catalyzed by C?" was reduced by about 30965. Thus, pharmaceutical compositions containing phanquinone in combination with clioquinol may have a greater utility than pharmaceutical compositions containing only one of these compounds.

Example 4

In this example, a clioquinol metabolite was synthesized.

It is known that clioquinol is excreted through the kidneys in the form of glucuronide derivatives of clioquinol (Koiaki N., ei a. "Epiegoperaiiis sigscatsyop oi siodtsipoI ip (ye ta, 9. РНаптасоиориодуп. 1984 ип; 7(6): 420-5 and

Uygita M. ei ai.: Meyaroiivt oi ""S-iodospiogpudgohuditsipoipe ip She dod apa (She hai, y). Rpagtasobiodup. 1984

Magesny; 7(3): 164-70). The conversion of clioquinol into the corresponding glucuronide presumably occurs in the liver. After the formation of clioquinol glucuronide in the liver, it is eventually transferred to the kidneys for excretion in the urine. 19 The glucuronide derivative of clioquinol in the form of methyl-(5-chloro-7-iodhinolyl-2",3'4"-tri-O-acetylglycopyranoside)uronate is obtained in accordance with the following reaction scheme:

Mmeso0 o se

OAe g | h

Ode AvgSOziSsa50, and NO pyridine! n si o cm mesoo (8)

Zh hu Z

Os v - " mo no Ole sch «- s «

Go to her

Zo Maon in NO 1M I 4 what) mmmeon a) nos a « 20 on z s no :z» Ge

A mixture of B-chloro-8-hydroxy-7-iodinoline (5mg, 0.164mmol), methyl 1-bromo-1-deoxy-2,3,4-tri-O-acetyl-O-glucopyranosiduronate (b5mg, 0, 1b4mmol), SabzoO).NiO (35ml) and cpyridine (1.5ml) were stirred at room temperature for 20 minutes. Freshly prepared Ad»CO3 (35 mg) was added to the reaction mixture and the suspension was stirred at room temperature for 20 minutes in the dark. Then the reaction product was deacetylated with 1 M aqueous Mmaon. The reaction mixture was diluted with СНоСи» (1 Oml), filtered, and the solvent was evaporated under reduced pressure.

The above-mentioned product was purified by flash chromatography (TLC: SNCI./MeonH 99/1, eluent: SNCI./Meon - 99.5/0.5).

Kz NMR (400 MHz, SOSIz) 2.04 (s, ZN, As), 2.09 (s, ZN, As), 2.13 (s, ZN, As), 3.68 (s, ZN, Me ), 3.99 (d, 1H, 5-H), 5.40-5.52 (m, ЗН, 2-3-4-H), 6.29 (d, 1H, 1-H), 7 .56 (m, 1H, ЗН), 7.99 (s, 1H, 6-H), 8.52 (d, 1H, 4-H), 8.93 (s, 1H, 2-H).

This compound is called clioquinol glucuronide in the following.

Example 5. (F) The interaction of vitamin B42" with clioquinol glucuronide, obtained in example 4, was studied using

GI nuclear magnetic resonance (NMR) spectroscopy.

Since clioquinol glucuronide is soluble in water, the study was carried out in buffered water at pH 6.5. bo Three different solutions were prepared and their TN-NMR spectra were recorded in a spectrophotometer OVEX 400MHz at 202С.

Solution a) contained free vitamin B. (hydroxycobalamin) at a concentration of 2.6 mM. Solution B) contained a mixture of 2.6 mM of vitamin B4o and 10 mM of clioquinol glucoronide (molar ratio of approximately 1:4). Solution c) contained 10 mM clioquinol glucuronide.

In fig. 5 presents the spectra of these three solutions for the aromatic region (5.5-9.8 million 7). The differences are 65 quite small, but are evident at the magnification shown in Fig.b and Fig. 1, respectively. Some of the resonances of vitamin B1 (corresponding to the benzimidazole part of the molecule) were mixed (see Fig. 6), and the same is observed for the two resonances of clioquinol glucuronide (corresponding to the quinoline part of the molecule) (see Fig. 7).

These results suggest an interaction between clioquinol glucuronide and vitamin B.42, possibly of a hydrophobic nature between the benzimidazole part of the vitamin B molecule." and the quinoline part of the clioquinol glucuronide molecule.

The applicant believes that the hydrophobic binding of vitamin B 42 to clioquinol glucuronide causes vitamin B.12 to be excreted from the body together with clioquinol glucuronide, thus preventing the resorption of vitamin B 415, which would eventually lead to vitamin B.4 deficiency." Thus, the applicant believes that vitamin B12 7 6 insufficiency is, at least to some extent, the underlying cause of PMS. As a result, any administration of clioquinol must ensure that the level of vitamin B in the subject undergoing treatment is sufficient to prevent its insufficiency. This can be achieved by the joint administration of clioquinol and vitamin B 49.

Example 6.

Obtaining a pharmaceutical composition containing fanquinone. 250g of fanquinone was mixed with 200g of Zaratiput (M-(4-stearoylaminophenyl)-trimethylammoniummethylsulphuric acid) and 1025g of lactose monohydrate over a period of 5 minutes. 300 g of boiling water was added in one portion to a mixture of 100 g of corn starch in 100 g of cold water. A suspension of corn starch, cooled to 40"C, was added to the powdery mixture containing fanquinone with continuous stirring. The mixture was granulated using a 2.5 mm sieve and dehydrated for 18 hours at 40"7C.

Dry granules were mixed with 400 g of corn starch and 20 g of magnesium stearate. The final mixture was formed into tablets with a diameter of 8.0 mm and a weight of 200 mg.

Example 7.

Obtaining a pharmaceutical composition containing fanquinone, clioquinol and vitamin B". 250g of fanquinone and 250g of clioquinol were mixed with 200g of Zaratip? tsp (M-(4-stearoylaminophenyl)-trimethylammoniummethylsulphuric acid) and 1025g of lactose monohydrate over a period of 5 minutes. 300 g of boiling water was added in one portion to a mixture of 100 g of corn starch in 100 g of i) cold water. A suspension of corn starch, cooled to 40°C, was added to the powdered mixture containing phanquinone and clioquinol with continuous stirring. Then an aqueous solution of the vitamin was added

Vl". The mixture was granulated using a 2.5 mm sieve and dehydrated for 18 hours at 40°C. The dry granules were mixed with 400 g of corn starch and 20 g of magnesium stearate. The final mixture was formed into tablets with a diameter of 8.0 mm and a weight of 200 mg.

With respect to the various publications cited herein, their complete descriptions are incorporated herein by reference.

Having thus described the present invention, it should be apparent that the same may vary Me

Q5 In numerous ways. Such variation should not be considered as a departure from the idea and scope of the present invention, and all such modifications, as should be apparent to a person of ordinary skill in the art, are included in the scope of the following claims.

Claims (43)

The formula of the invention h - c 1. A method of treating a subject, including a human, having or suspected of having Alzheimer's disease, wherein said subject is administered phanquinone in an amount effective to treat or prevent Alzheimer's disease. 2. The method according to claim 1, which differs in that fanquinone is administered orally, parenterally or intradermally. 3. A method of treating a subject, including a person who has or is suspected of having Alzheimer's disease, wherein said subject is administered phanquinone in an amount effective to increase the solubility of amyloid-beta (Ar) in the cerebrospinal fluid of this subject. 4. A method of treating a subject, including a person who has or is suspected of having Alzheimer's disease, wherein said subject is administered GI (a) phanquinone in an amount effective to treat or prevent Alzheimer's disease, and ( B) a compound or a mixture of compounds selected from the group containing antioxidants, acetylcholine enhancers, trace metals, prosthetic groups and clioquinol. 5B 5. The method according to claim 4, which is characterized by the fact that the total amount of the compound (compounds) in (B) is sufficient to increase the effect of preventing or treating Alzheimer's disease or to inhibit any harmful (F; side effect) of clioquinol administration. 6. The method according to claim 4, which differs in that the amount of compound (compounds) in (B) is equal to 5 μg - 250 mg. 7. The method according to claim 4, which is characterized by the fact that the antioxidant is selected from the group consisting of vitamin C, vitamin E, vitamin C or their combinations. 8. The method according to claim 4, which differs in that the acetylcholine booster is tacrine or donezepil. 9. The method according to claim 4, which differs in that the prosthetic group is vitamin B 12. 10. The method according to claim 4, which differs in that (a) fanquinone and (B) the compound (compounds) are in the same pharmaceutical composition. 65 11. The method according to claim 4, which is characterized by the fact that (a) fanquinone and (B) the compound (compounds) are introduced essentially simultaneously or sequentially. 12. The method according to claim 4, which differs in that fanquinone is administered orally, parenterally or intradermally. 13. A method of treating a subject, including a person who has or is suspected of having Alzheimer's disease, wherein said subject is administered (a) phanquinone in an amount effective to treat or prevent Alzheimer's disease, and (B) a mixture of clioquinol and vitamin B0", wherein the amount of clioquinol is effective for treating or preventing Alzheimer's disease, and the amount of vitamin B0 is effective for inhibiting the harmful side effect of administration of clioquinol. 70 14. The method according to claim 13, which differs in that the amount of clioquinol is 5 mg - 250 mg, preferably 10 mg - 50 mg. 15. The method according to claim 13, which differs in that the amount of vitamin B 4" is equal to 5 μg - 2 mg, preferably 0.5 mg - 1 mg. 16. The method according to claim 13, which is characterized by the fact that (a) fanquinone and (b) the compound (compounds) are in the same pharmaceutical composition. 17. The method according to claim 13, which is characterized by the fact that clioquinol and vitamin B are administered sequentially, and fanquinone is administered together with clioquinol, vitamin B or separately. 18. The method according to claim 13, which differs in that phanquinone and clioquinol are introduced at the first stage, and phanquinone and vitamin B 42 are introduced at the next, second stage. 19. The method according to claim 18, which differs in that the first stage is one to three weeks, and the second stage is one to four weeks. 20. The method according to claim 13, which is characterized by the fact that fanquinone is administered orally, parenterally or intradermally. 21. A pharmaceutical composition containing (a) phanquinone in an amount effective to treat or prevent Alzheimer's disease, and (5) a compound or mixture of compounds selected from the group consisting of antioxidants, acetylcholine enhancers, trace metals, prosthetic groups and clioquinol, and provided that when clioquinol is selected, the pharmaceutical composition contains at least one additional compound selected from this group. 22. Pharmaceutical composition according to claim 21, characterized in that the total amount of compounds (B) is sufficient to increase the effect of treatment of Alzheimer's disease or to inhibit any harmful side effect. - 23. Pharmaceutical composition according to claim 21, which differs in that the amount of the compound (compounds) (B) is equal to 5 g μg - 250 mg. 24. Pharmaceutical composition according to claim 21, which is characterized by the fact that the antioxidant is selected from the group consisting of vitamin C, vitamin E, О.0о or their combinations. yu 25. Pharmaceutical composition according to claim 21, which differs in that the acetylcholine booster is tacrine or donezepil. 26. Pharmaceutical composition according to claim 21, which is characterized by the fact that the prosthetic group is vitamin B 4". 27. Pharmaceutical composition according to claim 21, which differs in that the amount of fanquinone is 5 mg - 250 mg, preferably 10 mg - 50 mg. from the village 28. Pharmaceutical composition according to claim 21, characterized in that it contains clioquinol in an amount effective to improve the treatment or prevention of Alzheimer's disease. ;" 29. The pharmaceutical composition according to claim 21, which is characterized in that it contains vitamin B 325 in an amount effective to inhibit the harmful side effect of clioquinol administration. 30. Pharmaceutical composition according to claim 21, which differs in that the amount of clioquinol is equal to 5 mg - 250 mg, preferably 10 mg - 50 mg. 31. Pharmaceutical composition according to claim 21, which is characterized in that this composition is prepared for i, parenteral administration, intradermal administration or oral administration. their 32. Pharmaceutical composition according to claim 21, which differs in that this composition is prepared in the form of a tablet. - 33. Pharmaceutical composition according to claim 21, which differs in that clioquinol and vitamin B 32 are prepared in separate pharmaceutical forms, and fanquinone is prepared together with clioquinol, vitamin B.o or separately. 34. A pharmaceutical composition comprising (a) phanquinone in an amount effective to treat or prevent Alzheimer's disease and (B) a mixture of clioquinol and vitamin B42. 35. Pharmaceutical composition according to claim 34, which differs in that the amount of fanquinone is equal to 5 mg - 250 (F) mg, preferably 10 mg - 50 mg. ka 36. The pharmaceutical composition according to claim 34, which is characterized in that it contains clioquinol in an amount effective to improve the treatment or prevention of Alzheimer's disease. in 37. Pharmaceutical composition according to claim 34, which is characterized by the fact that it contains vitamin B 34" in an amount effective to inhibit the harmful side effect of clioquinol administration. 38. Pharmaceutical composition according to claim 34, which differs in that the amount of clioquinol is 5 mg - 250 mg, preferably 10 mg - 50 mg. 39. Pharmaceutical composition according to claim 34, which differs in that the amount of vitamin B 42 is equal to 5 μg - 2 65 μg, preferably 0.5 μg - 1 μg. 40. Pharmaceutical composition according to claim 34, which is characterized in that this composition is prepared for parenteral administration, intradermal administration or oral administration. 41. Pharmaceutical composition according to claim 34, which differs in that this composition is prepared in the form of a tablet. 42. Pharmaceutical composition according to claim 34, which differs in that clioquinol and vitamin B 42 are prepared in separate pharmaceutical forms, and fanquinone is prepared together with clioquinol, vitamin B.o or separately. 43. A kit containing in one or more containers phanquinone and clioquinol in an amount effective to treat or prevent Alzheimer's disease, and vitamin B 35 in an amount effective to inhibit 70 a harmful side effect of clioquinol administration. c schi 6) c "- " (o) I c) - and? 1 se) sh» - 70 Ko) ime) 60 b5