LV14230B - Pharmaceutical Composite & Imacration for Parkinson's Disease & Imacration Prevention & ā rst ē š - Google Patents

Abstract

Use of Meldonium dihydrate (3-(2,2,2-trimethylhydrazinium)propionate dihydrate) or its pharmaceutically acceptable salts for prevention and/or treatment of Parkinson's disease.

Description

Parkinson's disease is a progressive neurodegenerative disease of the brain, which is a disease of motor disorders. Symptoms include muscle stiffness, tremor, slow movements (bradykinesia), and even loss of movement (akinesia). Symptoms of Parkinson's disease are caused by the death of dopamine-producing (dopaminergic) cells in the basal ganglia or nigrostriatal nucleus, the striatum and substantia nigra.

Today's traditional treatment for Parkinson's disease is based on the dopaminergic hypothesis. Therefore, pharmacotherapeutic strategies are focused on the use of substances that can either stimulate dopamine synthesis (levodopa) or act as dopamine receptor agonists (ropinirole, pramipexole). Although levodopa is one of the first-line preparations for Parkinson's disease, its use is limited by motor complications - levodopa-induced dyskinesia, which significantly worsens the patient's quality of life. Dopamine receptor agonists also cause hallucinations of side effects, mainly somnolence or insomnia. Moreover, dopamine receptors progressively lose their sensitivity and thus the intensity of symptoms increases. Other substances, such as COMT and MAO inhibitors (tolcapone and selegelin) are used as second line formulations.

Recently, there is evidence in the literature that reveals the effects of levodopa. In the early stages of Parkinson's disease, levodopa appears to act as a neurotoxic molecule, causing undesirable effects (Caudle W.M., Colebrooke R.E., Emson P.C., Miler G.W. Altered vesicular dopamine storage in Parkinson's disease: Trends in Neurosciences330, 200830). Consequently, there is a growing interest not only in novel non-dopaminergic pathogenesis aspects of Parkinson's disease (Ahlskog J.E. Beating a dead horse: dopamine and

Parkinson's disease. Neurology, 2007, 69, 1701-1711), but also on new non-dopaminergic pharmacostatic strategies for Parkinson's disease. It is therefore an object of the present invention to provide a novel pharmaceutical composition with non-dopaminergic mechanisms of action and low toxicity for the treatment of central nervous system, particularly Parkinson's disease.

We unexpectedly discovered that the known low toxicity cardiovascular preparation, Meldonium, exhibits neuronal protective activity in a model of Parkinson's disease in rats.

The neurotoxin 6-hydroxydopamine (6-OHDA), whose intrastriatal administration causes the death of dopamine-producing cells, has been used to develop a model for Parkinson's disease. The neuronal protection revealed by Meldonium was completely unexpected from the point of view of dopaminergic mechanisms, as in previous studies meldonium (50 mg / kg intraperitoneally in rats) did not alter the dopamine content of brain structures, including striatum (Germane K., Berzins DA Influence of catechol-induced alterations). content and organ in rat.

Experiment Klin. Pharmacother., Vol.19, Riga Science, 991, 51-56).

Description of the Invention

This invention demonstrates for the first time a new activity of the cardiovascular preparation of meldonia in a model of Parkinson's disease in rats, which manifested itself in the protection of brain cells. First, meldonium exhibits pronounced neuroprotective activity in a model of Parkinson's disease in rats produced by the neurotoxin 6-hydroxydopamine (6-OHDA), whose intrastriatal administration causes degeneration of dopamine-producing cells. In this model, Meldonium prevents dopamine-producing cells from being killed.

We determined the effect of Meldonium on the expression of two marker molecules:

1) tyrosine hydroxylase (TH), a key enzyme that converts the amino acid tyrosine (precursor) to 3,4-dihydroxy-L-phenylalanine (L-DOPA), which in turn is converted to dopamine in neurons;

2) markers of astrocytes of glial fibrillary acidic protein (GFAP).

Astrocytes are believed to interact between neurons, glia and blood vessels (Allen

NJ, Barres BA, Glia - more than just brain glue. Nature, 2009, 457, 675-677).

TH expression indicates that the cell lives and synthesizes dopamine, whereas normal GFAP expression indicates cellular homeostasis and contacts between neuronal and glial cells.

Prolonged and overexpression of GFAP is indicative of reactive astrogliosis, which usually goes hand in hand with the progression of Parkinson's disease.

The neuroprotective activity of Meldonia is characterized by the results obtained.

Materials and methods

The experiments were performed on male Wistar rats (from Riga Stradins University

Laboratory of Experimental Animals, Riga, Latvia). Rat weight at start of experiment 230-330 g. All experimental procedures were carried out in accordance with the guidelines of the European Directives 86/609 / EEC (1986). These procedures were approved by the Animal Ethics Commission of the Food and Veterinary Department of the Republic of Latvia

Riga, Latvia).

Meldonium [3- (2,2,2-trimethylhydrazinium) propionate dihydrate] was obtained from JSC “Grindeks” (Riga, Latvia). It was dissolved in saline to make a 2% solution. 6-Hydroxidopamine or 6-OHDA (from Sigma-Aldrich, St. Louis, MO, USA) was prepared ex tempore in 0.2% ascorbic acid solution and kept in the dark. 6-OHDA solution containing 20 pg / ml was used. Meldonia was given to rats twice weekly by intraperitoneal injection at a dose of 50 mg / kg / day.

Experimenta design

The rats were adapted to the experiment and then divided into four groups of 8 animals per group.

Meldonia or saline (1 ml / kg) was administered intraperitoneally for two weeks. At week 3 (day 15), neurotoxin 6-OHDA (control cerebrospinal fluid, aCSF) was administered to the corpus striatum

Groups:

Group 1: Saline (FS) intraperitoneally (i.p.) for two weeks, then aCSF intrastrially (i.s.)

Group 2: Meldonium at 50 mg / kg i.p. for two weeks, then aCSF intrastriatively (i.s.)

Group 3: FS i.p. for two weeks, then 6-OHDA i.s.

Group 4: Meldonium at 50 mg / kg i.p. for two weeks, then 6-OHDA i.s.

Surgical procedures

After two weeks of intraperitoneal administration of meldonia or FS, rats were anesthetized with ketamine 75 mg / kg + xylazine 10 mg / kg and placed in a stereotaxic apparatus (StoeltingInc., USA). Imipramine (20 mg / kg intraperitoneally) was administered to rats 30 minutes prior to this general anesthesia to protect adrenergic neurons from damage caused by 6-OHDA. Anesthetized rats with Stoelting microinjection were given aCSF or 3 μΐ of 6-OHDA solution (6-OHDA concentration 20 pg / ml) at a rate of 1 μ (/ min (total dose of 6OHDA was 20 pg) on the right side of the striatum. After injection, the cannula was held at the injection site for another 2 minutes, then slowly withdrawn. Intrastriatal Administration Coordinates (Patinos & Watson): AP, -0.24; L, -3.0;

DV, -5.0 mm from bregma.

Rotation

On days 14, 21 and 28 after surgical manipulation and administration of 6-OHDA, 0.2 mg / kg of apomorphine (a dopamine receptor agonist) was administered subcutaneously and the rat recorded asymmetric (contralateral) rotations within 30 min. Rotation intensity indicates the degree of denervation of the striatal lesion.

Preparation of brain tissue

After completion of the rotation test (day 28), rats were anesthetized with ketamine (150 mg / kg) and perfused through the ascending aorta with 50 ml of saline, followed by

250 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain was removed and fixed at -80 ° C. Brain structure corpus striatum and substantia nigra tissue were incised in 24 cryostat sections at 10 µm thickness at -20 ° C (Leica CM1850, Leica Microsystems, Germany). The sections were placed on polylysine-coated slides (3 slides on each slide). Slides with slices were dried at room temperature for 15 minutes, then fixed in acetone (10-15 minutes), then left again at room temperature for 2 hours. Slides were then wrapped in aluminum foil and stored at -20 ° C (until immunohistochemistry).

Im monohistochemistry

EnVision kits (DAKO, Glostrup, Denmark) were used for immunohistochemistry studies.

The formulations were fixed in ice-cold acetone solution for 5 minutes, then washed with Tris-buffered saline (TBS). The sections were incubated with peroxidase blocking reagent (0.5% H2O2 / PBS) for 5 min to inhibit endogenous peroxidase activity. The preparations were then incubated with primary polyclonal rabbit antibodies to TH (Millipore, clone LNC1) for 18 hours at 4 ° C, and incubated with GFAP antibodies for one hour at room temperature.

Primary antibody binding was determined by incubating the preparations (sections) in a humid chamber for 30 min with EnVision reagent, then rinsing with TBS (pH 7.6) three times for 5 min. The preparations were then incubated with chromogenic 3'3-diaminobenzidine-tetrahydrochloride (DAB) for 7 min, then with hematoxylin for 2 min. Spinal ganglion tissue was used as a positive control; preparations not incubated with the primary antibody were used as a negative control.

The brain nucleus - corpus striatum and substantia nigra tissue was studied with a light microscope connected to a video imaging system (Motic Image Advanced 3.2,

China, Xyameri). Tissue evaluation was performed by two researchers using the "blind" method. Results were expressed as cells / mm ² .

Statistical analysis

GraphPadPrism 4 version software was used for data analysis. Results are expressed as means ± SEM. Statistical reliability was determined at p values <0.05.

Meldonia. effect on apomorphine-induced rotations

In rats not given 6-OHDA, neither saline nor meldonium nor apomorphine induced rotation (data not shown). Rotations occurred in rats on days 14, 21, and 28 after 6-OHDA injection; this effect was particularly pronounced on day 21 (Fig. 1). Meldonium (50 mg / kg) significantly reduced apomorphine rotations in rats given 6-OHDA, and this effect was statistically significant on day 21 after OHDA administration (78.8 ± 13.5 vs.147.0 ± 20.9, p = 0.003, Fig. 1). ); The results for days 14 and 28 for the Meldonium + 6-OHDA group did not differ from the data for the 6OHDA group (Fig. 1).

Effect of meldonium on tyrosine hydroxylase (TU) expression

The obtained results show that 6-OHDA significantly reduces (approximately four-fold) the number of TH-positive neurons in the striatum (corpus striatuni) compared to the saline control group (5 ± 2 vs. 21 ± 10 cells / mm ² , p = 0.03, 2). .att.). Meldonium per 50 mg / kg does not affect TH expression in the striatum. However, the two-week premedication of meldonia at this dose completely reversed the 65 OHDA-induced TH-positive cell depletion in the striatum (27 ± 4 vs. 5 ± 2 cells / mm ² , p = 0.0006).

Also in the nigral nucleus (substantia nīgra), 6-OHDA decreased the number of TH positive neurons, but this was not statistically significant compared to control group (44 ± 14 vs. 95 ± 30 cells / mm, p = 0.1.3). . Meldonium per se dose 50 mg / kg had no effect on TH expression in the substantia nigra, whereas meldonium showed a tendency to increase 6-OHDA-induced decrease in TH cells (64 ± 13 vs. 44 ± 14, p = 0.3; Fig. 3).

Effect of Meldonia on GFAP (glial fibrillary acidic protein) expression

In the striatum (corpus striatum), 6-OHDA significantly increased (approximately four-fold) the number of GFAP-positive astrocytes compared to the control group (42 ± 6 vs. 11 ± 2 cells / mm, p = 0.0011, Fig. 4), indicating reactive astrogliosis. Meldonium at 50 mg / kg per se did not affect GFAP positive cell counts, but meldonium reduced to 6OHDA-induced increase in GFAP cell counts (20 ± 4 vs. 42 ± 6 cells / mm ² , p = 0.01,

Figure 4).

In the nucleus, injection of 6-OHDA of the substantia nigra also increased (approximately three-fold)

GFAP positive astrocyte cell count (35 ± 5 vs. 10 ± 2 cells / mm ² , p = 0.002). Meldonium alone did not affect these cells at the dose of 50 mg / kg, but administration of meldonium for two weeks showed a tendency to decrease the 6-OHDA effect (25 ± 4 vs. 35 ± 5, p = 0.1, Fig. 5).

As can be seen from the data presented in this invention, meldonium at 50 mg / kg administered intraperitoneally every day for two weeks prior to the intrastriatal injection of the neurotoxin 6-OHDA completely protected the degeneration of the 6-OHDA-induced dopamine-producing neurons in the damaged striatum. cell death; to a lesser extent, these effects were expressed in another (intact) structure, the substantia nigra.

Meldonium in the defective structure (striatum) also protected OHDA-induced astrocyte activation, demonstrating the ability of meldonium to regulate synaptic activity between neurons and between neurons and astrocytes, whose function (although not yet fully elucidated) is associated with the maintenance of signaling cascade homeostasis in neurons. -glandular-vascular level.

Thus, the neuroprotective activity of meldonia, which is evident in the model of Parkinson's disease in rats, points to the potential of meldonia as a novel type of anti-parkinson drug in the prevention and treatment of Parkinson's disease. For the treatment of Parkinson's disease and other neurodegenerative diseases, meldonia may be administered orally or by injection. Meldonium structure-based pharmaceutical compositions may be prepared using meldonium dihydrate or a pharmaceutically acceptable salt form. Oral meldonium can be administered in the form of tablets, lozenges, granules, solution, powder or capsules containing 100 mg - 1500 mg / day, preferably 500-1000 mg / day. Pharmaceutical formulations and excipients which are authorized for pharmaceutical use may be used in the preparation of Meldonium dosage forms following standard manufacturing procedures. Intravenous or intramuscular injection of Meldonium is prepared as a 1040% solution in water, isotonic sodium chloride solution, glucose / water solution or pH 6.5-8.0 buffer solution. For the treatment of Parkinson's disease patients, the recommended daily dose of meldonia is 100 mg -1000 mg, preferably 250-500 mg.

The above Meldonium pharmaceutical compositions may be used for the prevention and treatment of various neurological disorders requiring protection of dopamine-producing neuronal degeneration, primarily to stimulate tyrosine hydroxylase expression in the nigrostriatal system of the brain.

Claims (5)

1/5 O Day14 □ Day21 HDay28 [ Fig. Number of contralateral rotations induced by apomorphine in rats (recorded 30 min). Rotation test was performed after apomorphine (0.2 mg / kg subcutaneously) on days 14, 21 and 28 after 6OHDA injection (20 pg) in the right striatum. Saline (Saline, 1 ml / kg) or Meldonium at 50 mg / kg (M50) was administered intraperitoneally two weeks prior to 6-OHDA injection. ** p <0.01, M50 + 6-OHDA vs. Saline + 6-OHDA, Day 21, unpaired t-test. A pharmaceutical composition comprising as active ingredient a compound selected from the group consisting of 3- (2,2,2-trimethylhydrazinium) propionate dihydrate and 3- (2,2,25-trimethylhydrazinium) propionate for pharmaceutical use, as well as pharmaceutically acceptable salts thereof. a pharmaceutically acceptable excipient or diluent for use as an agent for neuroprotection and for the prophylaxis and prevention of dopamine-producing cell degeneration in diseases of the central nervous system. Fig. 2 Number of tyrosine hydroxylase (TH) positive neurons in the striatum (cells / mm ² ). Saline (Saline, 1 ml / kg) and Meldonium 50 mg / kg (M50) were administered intraperitoneally every day for two weeks prior to intrastriatic (right) injection of 6-OHDA or artificial cerebrospinal fluid (aCSF). * p = 0.03, Saline + 6-OHDA vs. Saline + aCSF; *** p <0.0001, M50 + 6-OHDA vs. Saline + 6-OHDA. Mann-Whitney U-tests. 2/5 Pharmaceutical compositions according to claim 1. Use according to claim 1, wherein the composition is 10 used to stimulate tyrosine hydroxylase expression in the nigrostriatal system. Fig. 3 Number of tyrosine hydroxylase (TH) positive neurons in the nigral nucleus - substantia niger (cells / mm ² ). Saline (Saline, 1 ml / kg) and Meldonium 50 mg / kg (M50) were administered intraperitoneally every day for two weeks prior to intrastriatic (right) injection of 6-OHDA or artificial cerebrospinal solution (aCSF). 3/5 3. Pharmaceutical compositions according to claim 1. Use according to claim 1, wherein the composition is used to regulate expression of glial fibrillar acidic protein in nigrostriatal nuclei. Fig. 4 GFAP (glial fibrillary acidicprotein) positive astrocytes in the striatum (cells / mm ² ). Saline (Saline, 1 ml / kg) and Meldonium 50 mg / kg (M50) were administered intraperitoneally every day for two weeks prior to intrastriatic (right) injection of 6-OHDA or artificial cerebrospinal fluid (aCSF). * p = 0.0011, Saline + 6-OHDA vs. Saline + aCSF; ** p = 0.01, M50 + 6-OHDA vs. Saline + 6-OHDA. Mann-Whitney U-tests. 5/5 4/5 4. Pharmaceutical compositions according to claim 1. Use according to claim 1, wherein the central nervous system disease is Parkinson's disease. Fig. 5 Number of GFAP (glialfibrillary acidicprotein) positive astrocytes in substantia nigra (cells / mm ² ). Saline (Saline, 1 ml / kg) and Meldonium 50 mg / kg (M50) were administered intraperitoneally every day for two weeks prior to intrastriatic (right) injection of 6-OHDA or artificial cerebrospinal fluid (aCSF). p = 0.002, Saline + 6-OHDA vs. Saline + aCSF. Mann-Whitney U-test.