A method of treating alzheimer's disease and pharmaceutical composition based kliohinola for treatment

 

(57) Abstract:

The invention relates to pharmacology and medicine, in particular to a method of treatment of Alzheimer's disease and pharmaceutical compositions on the basis of climinal for her treatment. The technical result of the present invention is to provide new uses of known pharmaceutical compounds for the prevention, control and treatment of Alzheimer's disease at any stage of its development. 2 C. and 12 C.p. f-crystals, 1 tab., 1 Il.

The invention relates to a method of treating Alzheimer's disease by applying a chelating agent and pharmaceutical composition for treatment on the basis of this chelating agent.

Alzheimer's disease is a chronic neurodegenerative disorder, common especially among the elderly.

Clinical diagnosis of Alzheimer's disease confirmed by the presence and accumulation of amyloid deposits in the brain. Amyloid is found primarily in the terminal zones of neurons in the form of a morphologically heterogeneous deposits known as senile plaques. The formation of senile plaques associated with the appearance of symptoms and signs of this disease, including am the education of these plexuses of neurofibril related memory loss and other symptoms of dementia.

The main component of amyloid deposits is a polypeptide, referred to herein as A (amyloid-beta). A normal is a soluble component of the cerebrospinal fluid, where it is at a concentration of about 3-5 nm. A Mature form may be from 39 to 43 amino acids, usually 40 amino acids and is secreted in the form of a product of proteolytic cleavage of the protein cell surface protein called the amyloid precursor (BIA) (Kang et al., 1987). Currently, the function of A normal unknown, but may be, it forms cation-selective channels in cell membranes (Kawachara et al., 1997).

It was shown that the precipitation of synthetic A is influenced by several external factors, including low pH, high salt concentrations and the presence of metals such as zinc, copper and mercury (Bush et al., 1995). It was reported that A specific manner and to the full saturation binds to high-affinity zinc binding (KD=107 nm) in a molar ratio of 1:1 (zinc: (A) (Bush et al., A). This binding occurs at physiological concentrations of zinc (Bush et al., 1994b).

There is good reason to assume that the removal of amyloid deposits from the body of patients with bolezn the drug means to remove deposits of amyloid.

VO 93/10459 discloses a method of treating Alzheimer's disease by introducing agent that binds zinc. The preferred compounds mentioned phytic acid, desferrioxamine, sodium citrate, etc, 1,2-diethyl-3-hydroxypyridine-4-one and 1-hydroxyethyl-3-hydroxy-2-methylpyridin-4-one.

DE 3932338 discloses the use of a chelating agent such as 8-hydroxyquinoline, for the treatment of Alzheimer's disease.

US 5373021 describes disulfiram and its salts and analogues as compounds that can penetrate the blood-brain barrier. Described compounds can be used to reduce neurological disorders caused by, among others, Alzheimer's disease.

Known hitherto connection, proposed for the treatment of Alzheimer's disease, have several disadvantages that prevent their widespread use. For example, most of these compounds are able to penetrate through the blood-brain barrier and, therefore, can hardly reach those areas in which there is deposition of amyloid. Disulfiram, which is able to penetrate the blood-brain barrier, has the disadvantage that it is also the agent, restraint from alcohol.

yuushi effect etc in relation to zinc and copper is relatively weak. In addition, add does not penetrate the blood-brain barrier and is considered to be relatively toxic.

The purpose of this invention is the provision of new uses of known pharmaceutical compounds for the production of pharmaceutical compositions for the treatment of Alzheimer's disease; the specified connection has the ability to penetrate the blood-brain barrier, effectively helatirovat heavy metals to prevent aggregation of amyloid and dissolution of existing amyloid deposits. This goal is achieved through the use of climinal for this new application.

The term "treatment of Alzheimer's disease" as used herein refers to the prevention, control and treatment of Alzheimer's disease at any stage of its development.

Kliohinol has the chemical name 5-chloro-7-iodine-8-hydroxyquinolin and belongs to the group of hydroxyquinolines. It is known the use of climinal as a local antiseptic agent. Kliohinol particularly used in the treatment of amebiasis and infectious diarrhea. Kliohinol almost insoluble in water at pH 7-11. Outside these values, it is possible to achieve the desired concentrations of this compound. Espectrometria, the coordination number for climinal in the case of Co(II), Ni(II), cu(II) and Zn(II) is 2, while the coordination number of the Fe(III) is 3. Reportedly injected drugs climinal penetrate the blood-brain barrier, creating concentrations in the brain of the order of 20 µl/ml, with the doses of 10-20 mg/kg (Tateishi et al. , 1975, and Tamura 1975). It was found that the concentration of climinal also high in these areas of the brain, like the hippocampus, which is directly affected by Alzheimer's disease.

Using methods of microautoradiography on monkeys have shown that kliohinol forms chelates with zinc in the hippocampus. Chelates with Zn(II) were found mainly in axonometrically synaptic endings mossy fibers. Unconjugated kliohinol intravenous extremely quickly penetrates into the nervous system, almost without stopping the blood-brain barrier (Shivaki, 1979).

Although kliohinol has known chelating ability and the ability to penetrate the blood-brain barrier, it was impossible to assume that he also has the ability to dissolve precipitated zinc A. in Accordance with the present izobreteniya based on this unexpected discovery.

Now suppose that kliohinol and A competitive chelate zinc and other heavy metals. Kliohinol is the strongest chelating agent and will, therefore, be dominated by capturing heavy metal ions. Thus, from A precipitated complex zinc-A will be re-dissolve into the surrounding liquid, as kliohinol will capture zinc ions. Complex kliohinola and zinc will pass through the blood-brain barrier and is excreted from the body.

Because kliohinol is relatively strong chelating agent, it can also be helatirovat metal ions from enzymes or prosthetic groups. Therefore, it may be desirable to assign patients being treated with kliohinol, ions of metals belonging to the trace elements, or a prosthetic group, especially during a long course of treatment with this connection.

Vitamin B12contains cobalt. Previous studies subacute leeloominai neuropathy (PMON) in Japanese patients who took kliohinol in higher doses than recommended, and for a long period of time, showed that there is a correlation between long-term treatment with kliohinol, defic studies in vitro and in vivo. Thus, in order to prevent deficiency of vitamin B12may be appropriate introduction of this vitamin simultaneously or sequentially with kliohinol.

Kliohinol you can type in any number in any galenical form and in accordance with any scheme introduction.

Preferably, the daily number of climinal should be from 10 mg to 750 mg, depending on the condition of the patient. The usual daily dose is 100 mg Alternatively, you can enter from 10 mg to 250 mg kliohinola, preferably 100 mg, three times per day. Daily dose of up to 750 mg for two weeks is considered safe from the point of view of neurotoxicity or other side effects.

To prevent the appearance of symptoms and signs of Alzheimer's disease or for the inhibition of symptoms and signs during the development of this disease, you can enter daily dose kliohinola from 10 to 100 mg for an extended period of time, namely, up to ten years.

The specific amount of preparation must be determined by the attending physician and may depend on many factors, such as age, condition, history, etc., that a particular patient. The results presented in nastojachemu best results in dissolution. This window can be determined by a physician having ordinary experiments.

If kliohinol going to type for a long period of time, it is preferable to perform intermittent scheme introduction. In the first period kliohinol may be, for example, within one to three weeks, followed by a period free from the drug, during which may be the recovery of the body from any unwanted side effects kliohinola. The duration of the period without administration of the drug may be one to four weeks. During this period, preferably vitamin b12and other prosthetic groups, and/or metals. After this period you can repeat the first period. Long-term intermittent therapy will provide not only re-dissolution of the aggregates of zinc and A, but also prophylactic inhibition of formation of these aggregates.

Intermittent introduction of climinal will also reduce the toxic potential of this drug, which means that the treatment can be extended as the evolution of the disease.

Pharmaceutical composition comprising kliohinol may comprise any suitable herbal form for the different concentrations. If you need to quickly get high drug concentrations in the brain, preferably kliohinol in the form of intravenous injections. Because it is more convenient to introduce a pharmaceutical composition orally, this method of administration can be used, if there is no need rapid achievement of high concentrations of the drug in the brain. This pharmaceutical composition can also enter intradermally.

Pharmaceutical composition comprising kliohinol, may also contain other active ingredients. Specifically, this composition may contain metals, trace elements or prosthetic group, such as vitamin B12and/or any other therapeutic agent that can be used simultaneously for the treatment of Alzheimer's disease, to improve or relieve symptoms and signs of Alzheimer's disease, or delay the beginning of the manifestation of any symptoms and signs of Alzheimer's disease. Alternatively, the other active ingredient (ingredients) may be administered as separate pharmaceutical compositions (compositions) together with a pharmaceutical composition comprising kliohinol.

According to the present invention, a new use kliohinola inclusive,

2) prevention of this disease in patients with early or prodromal symptoms and signs, and/or

3) braking start or evolution or aggravation of symptoms or signs of Alzheimer's disease.

The drawing shows a solubilization-amyloid by kliohinol.

The present invention will be illustrated by the following examples.

Example 1. 5.3g climinal suspended under stirring in 200 ml of n-decane. Nerastvorim material was allowed to settle. Weighing dried nerastvorimogo kliohinola after blowing n-decane showed that only 2% of climinal dissolved in decane. 100 ml (light yellow) the supernatant was mixed with 100 ml of saline phosphate buffer, pH 7.4, and the phases were allowed to separate. Saline phosphate buffer (lower phase) was collected and filtered to remove the residue, which was formed at the interface between the phases after extraction with an organic solvent. Assuming that 2% of climinal dissolved in n-decane and that the distribution coefficient is 1/1750, with saline solution with phosphate buffer in mixtures of 1:1 of the Dean and clerinae, concentration kliohinola in physiologically is where the diagnosis of Alzheimer's disease was confirmed histologically. Two sample frontal lobes of the neocortex weighing 0.5 g of homogenized in 3 ml of climinal in physiological solution with phosphate buffer at 100%, 10% and 1% leaf extract saline phosphate buffer/kliohinol and only in physiological solution with phosphate buffer.

The resulting homogenates were centrifuged at 150000 x g for thirty minutes and the supernatant was collected and placed in ice (fraction S). Centrifugal precipitation were subjected to identical homogenization and centrifugation and re-collected supernatant (fraction "P").

1 ml of Each supernatant was treated with 200 μl of ice-cold 10% THU (trichloroacetic acid) to precipitate all of the protein, including A. the precipitate was washed with 100% ethanol and resuspendable in 100 μl of TBS (Tris 20 mm, NaCl 150 mm, pH 7,4).

of 7.5 μl of the Sample (S or R) was boiled for 5 minutes with an equal volume of Tris-technologo buffer containing 4% SDS (sodium dodecyl sulfate), and was loaded onto Novex pre-cast 10-20% Tris-trichinosis gel, and then transferred to nitrocellulose Western. The signal for A recorded using mAb W02 (increase compared with the remnants of A 5-16) and visualized using ECL (electrogenerating chemoluminescence THU, to 1 ml of physiological solution with phosphate buffer containing 10% bovine serum albumin, was added 1 μg of synthetic A 1-40 and the solution was processed as described above. The signal for A determined in loose sediment, but not in the supernatant.

The result is shown in the drawing.

Kliohinol in the studied concentrations was effective in accelerating the solubilization A. in Addition, there was an optimal concentration of 10%, which indicates greater solubility in physiological solution with phosphate buffer one aggregation forms A compared with others.

While the drawing shows only the data for one sample, all data for 19 other samples show the same trend, namely that kliohinol is effective in accelerating the solubilization A.

Example 2. In this experiment compares the chelating ability of climinal and etc.

Samples of synthetic A weight of 10 ng were placed in microtiter wells and caused aggregation by adding 25 μm ZnCl. The units are then transferred by filtration on a nylon filter hole size 0.2 μm. These aggregates were washed in 200 μl of TBS, TBS containing 2 μm transferred to the exposure on the film of the ECL. Measured transmittance of the film of the ECL and the relative strength of the signal was calculated based on 100% only for TBS. Relative signal strength was 66% for add and 49% for climinal.

The results indicate that kliohinol is more effective chelating agent for besieged by A zinc than add.

Example 3. This example demonstrates resolubilize effect kliohinola.

Prepare a 2.5 μm solution A with TBS pH 7.4. 95% maintained in A dissolved state. The addition of 30 microns of zinc resulted in precipitation of A dissolved and only 43% remained in solution. Then add 120 μm kliohinola to besieged by A zinc led to an increase in dissolved to A 70%.

These results show that kliohinol has the ability to re-dissolve precipitated zinc A.

Example 4. In this example examined the effect of climinal on vitamin B12in vitro NMR spectroscopy.

Because kliohinol practically insoluble in water at pH 7-11, the study was conducted at pH 13. It was prepared with 3 tubes. The first contained 1 mg kliohinola in 0.5 ml, the second contained 1.4 mg ciankobalamin (B12) in 0.5 ml and the third consisted of 0.5 is listed on the spectrophotometer DRX 400 MHz at 20oC. Comparison of the third spectrum from the first and the second showed that some of the resonances ciankobalamin had a shift, and the same was observed for two of the resonances kliohinola. These results suggest that there is interaction between kliohinol and ciankobalamin.

Example 5. In this example examined the effect of climinal on vitamin B12in vivo.

Six male mice pre-injected kliohinol for three days (50 mg/kg/day). The mice were divided into two groups, control group and the group treated with injections of [57With]-ciankobalamin. At 48 hours after injection, animals were killed, slaughtered the brain, liver and kidney and counted on a gamma counter as a thousand counts per minute per gram of tissue (wet weight) FROM the middle. The magnitude of radioactivity for each of the groups shown in the table.

Comparison of the results shows that significant changes in the amount of accumulation of radioactivity in brain and liver were observed. A distinct reduction of vitamin B12was observed in the kidneys. We can conclude that kliohinol affects the concentration of B12in some organs of mice.

The source of the hara M.; Arispe N.; Kuroda Y.; Rojas, E. (1997), Biophysical Journal 13/1, 67-75.

4. Bush, A. I. , Moir, R. D., Rosenkranz, K. M./ and Tanzi, R. E. (1995) Science 268: 1921-1923.

5. Bush, A. I. , Pettingell Jr., W. H., Paradis, M. D., and Tanzi, R. E. (1994a) J. Biol. Chem. 269: 12152-12158.

6. Bush, A. I., Pettingell Jr., W. H., Multhaup, G., Paradis, M. D., Vonsattel, J. P., Gusella, J. F., Beyre - uther, K., Masters, C. L., and Tanzi, R. E. (1994b) Science 265: 1464-1467.

7. Esler, W. P., Stimson, E. R., Jennings, J. M., Ghilardi, J. R., Mantyh, P. W., and Maggio, J. E. (1996) Neurochem. 66:723-732.

8. Kidani Y, et al., (1974) Jap. Analyst 23:1375-1378.

9. Tateishi J, et al., (1973) Psychiat. Neurol. Jap. 75:187-196.

10. Shirilogi H., et al., (1979) Handbook of Clinical Neurology, North Holland Publishing Company, 141-198.

11. Tamura Z, (1975), Jap. J. med. Sci.Biol. Suppl 28: 69-77.

1. A method of treating Alzheimer's disease, including the introduction of a patient in need of such treatment, a therapeutically effective amount of climinal.

2. The method according to p. 1, while kliohinol enter in the amount of 10 - 250 mg 1 - 3 times a day.

3. The method according to p. 1 or 2, wherein the metal trace elements and/or prosthetic group administered together or sequentially with the introduction of climinal.

4. The method according to p. 3, in which the prosthetic group is vitamin B12.

5. The method according to any of the previous items, wherein the pharmaceutical composition comprising kliohinol, enter PR is administered for one to three weeks, followed by a period of one to four weeks, during which the pharmaceutical composition is not administered.

7. The method according to p. 6, wherein the metal trace elements and/or prosthetic group administered during the period during which the pharmaceutical composition is not administered.

8. The method according to p. 7, wherein the prosthetic group is vitamin b12.

9. The method according to any of the preceding paragraphs, wherein the treatment is continued for a period of time up to ten years.

10. The method according to any of the previous items, wherein the pharmaceutical composition containing kliohinol made in the form for oral administration.

11. The method according to any of paragraphs. 1-9, wherein the pharmaceutical composition is made in a form for parenteral administration.

12. The method according to any of paragraphs. 1-9, wherein the pharmaceutical composition is manufactured in the form for intradermal injection.

13. Pharmaceutical composition for the treatment of Alzheimer's disease containing kliohinol and vitamin B12.

14. The pharmaceutical composition according to p. 13, in which the number of climinal is 10 to 250 mg.

 

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