EP1420772A2

EP1420772A2 - Use of beta-adrenoceptor agonists for the treatment of neurodegenerative diseases

Info

Publication number: EP1420772A2
Application number: EP02797607A
Authority: EP
Inventors: Josef Krieglstein; Carsten Culmsee; Vera Junker; Helmut Blum; Wolfgang Greb
Original assignee: EUCRO European Contract Research GmbH and Co KG
Current assignee: EUCRO European Contract Research GmbH and Co KG
Priority date: 2001-08-29
Filing date: 2002-08-22
Publication date: 2004-05-26
Also published as: WO2003020257A3; PL373490A1; WO2003020257A2; AU2002333510A1; JP2005505548A

Abstract

The invention relates to the use of beta -adrenoceptor agonists for restoring and/or maintaining the function of partially or fully damaged/degenerated cells of the central nervous system and/or other nerve cells. Using beta 2-adrenoceptor agonists enables astrocytes to be activated and endogenous neuroprotection processes to be stimulated, whereby the damage or destruction of nerve cells can be reduced and even prevented in some cases.

Description

Use of ß-adrenoceptor agonists for the treatment of neurodegenerative diseases

The present invention relates to the use of β-adrenoceptor agonists for the treatment of neurodegenerative diseases.

The human brain is a highly complicated organ with more than 100 billion nerve cells (= neurons) and around 10,000 connections (= synapses) per cell. The brain is the central organ of the conscious and unconscious processing of the stimuli acting on the human body, of thinking and feeling, of purposeful action, learning and memory. One of the most important services of the human brain is information processing in speech; also the control center for a large number of organ functions as well as breathing, heart rate and temperature control.

There are a variety of diseases that lead to the death of nerve cells and / or a reduction in the synapses and thus to a reduction in brain performance.

Examples of such clinical pictures are Alzheimer's disease, cerebrovascular dementias,

Parkinson's disease, Pick's disease, Huntington's chorus, amyotrophic lateral sclerosis, Lewy

Body dementia, stroke and brain trauma, such as contusio and commotio cerebri as well as brain and spinal cord injuries or cross-sectional injuries, spina bifida, as well as diseases of the inner ear, for example diseases associated with the occurrence of tinnitus, such as subacute or chronic tinitus, hearing loss, Crohn's

Meniere, and diseases with hearing impairment or

Decrease in eyesight, etc. A clinically established neuroprotective

So far there is no therapy for the clinical pictures mentioned. Only symptoms, but not their causes, are treated.

The goal of causal therapy is to prevent the destruction of nerve cells.

There is currently no established therapy with which it is possible to protect or regenerate the nerve cells from damage. An essential element of the currently applied therapy for the above diseases is indirect or secondary

To prevent damage to nerve cells, such as cerebral blood flow increase or restore them when a vessel is closed. However, this form of therapy, if at all, is only successful if it can be used quickly after the acute event.

The object of the present invention was to find drugs which protect nerve cells from damage and which can at least partially restore the function of partially or completely degenerated cells.

Surprisingly, it was found that the activation of astrocytes with drugs such as ß-adrenoceptor agonists sets in motion endogenous processes of neuroprotection, whereby the damage or destruction of nerve cells can be reduced and in some cases even prevented.

The present invention accordingly relates to the use of β-adrenergic agonists for restoring and / or maintaining the function of partially or completely damaged cells of the central nervous system and / or other nerve cells.

For the purposes of the present invention, “damaged cell” means that the cell has been damaged by external influences or is partially or completely destroyed in the sense of degeneration by processes taking place in the cell, which can be associated with an impairment of bodily functions. The expression “damage to the cell” includes both the damage to individual cells or cell types and the damage to the strands or pathways of nerve cells.

In addition to the cells of the central nervous system, the nerve cells also include the cells of the spinal cord and all other nerve cells in the body.

β-adrenoceptors respond in particular to adrenergic drugs. Examples of β-adrenergic agonists which are preferably used in the present invention because of their good activity are clenbuterol, formoterol, fenoterol, salbutamol, orciprenaline, isoetharine, cimaterol, ractopamine, reproterol, salmeterol, terbutaline, their isomers, acid addition salts, Analogues and any mixtures of the above.

The β-adrenergic agonists mentioned above are in part known medicinal substances. For example, clenbuterol is known from the prior art as a branch agent. Experiments have shown, for example, that the lipophilic ß-adrenoceptor agonists can permeate into the brain and stimulate the ß-adrenoceptors of the astrocytes there. The stimulation of these receptors in turn leads to activation of the astrocytes and, as a result, to an increased release of growth factors, such as NGF, which can protect nerve cells from damage.

The beta-adrenoceptor agonists are administered for therapy in the amounts customary for these medicaments, in particular in an amount of 0.01 to 100 mg / day, preferred ranges of amounts also being able to depend on the particular beta-adrenoceptor agonist. With substances such as clenbuterol, formoterol, fenoterol and salmeterol, a particularly good neuroprotective effect is obtained if they are administered in an amount of 0.01 to 5 mg / day. Terbutaline is preferably applied in an amount of 1.0 to 30 mg / day, salbutamol in an amount of 1.0 to 50 mg / day, and orciprenaline and reproterol in an amount of 1.0 to 100 mg / day.

Β1-adrenoceptor agonists, such as Dobuta in, can also activate astrocytes and thereby protect the neurons. In a possible embodiment, the β-adrenoceptor agonists used according to the invention are used in combination with the β1 and / or β2 adrenoceptor agonists.

In a further possible embodiment of the present invention, the β-adrenergic agonists are used in combination with NMDA antagonists, as a result of which a supplementary or further principle of action is used.

The NMDA antagonists, e.g. the adamantane derivatives are known compounds which are often used for the treatment of various diseases. For example, the dopaminergic influence of amantadine (1-adamantanamine) is known.

European patent application EP-392 059 describes the use of adamantane derivatives for the prevention and treatment of cerebral ischemia. According to this publication, the use of the adamantane derivatives prevents the destruction of brain cells after ischemia by using the adamantane derivatives as antagonists for the NMDA receptor channels of the nerve cells in the brain.

Adamantane derivatives with the formula I are preferably used

in which R ¹ and R ^{2 are the} same or different and represent hydrogen or a straight-chain or branched CC ₆ alkyl group or together with the N atom can represent a heterocyclic group with 5 or 6 ring atoms, R ³ and R ^{4 are the} same or different are and represent hydrogen, a straight-chain or branched CC ₆ alkyl group or a C ₅ -C ₆ cycloalkyl group or a vinyl group, and

R ^{5 represents} hydrogen or a straight-chain or branched CrCe alkyl group.

The adamantane derivatives with the formula I can be used in the form of the compounds described by the formula I or in the form of their pharmaceutically acceptable salts. Preferred pharmaceutically acceptable salts include the acid addition salts, such as the hydrochlorides, hydrobromides, sulfates, acetates, succinates, tartrates, the hydrochlorides being preferred.

Preferred compounds with the formula I are those in which R ¹ , R ² and R ^{4 are} hydrogen and R ³ and R ^{5 are} a methyl and / or ethyl group.

In a particularly preferred compound, R ¹ , R ² and R ^{4 are} hydrogen and R ³ and R ^{5 are} a methyl radical, or their hydrochloride. This connection is known as the INN Memantine.

The ß-adrenoceptor agonists used according to the invention and, if appropriate, other customary medicinal substances which do not negatively influence or support the therapy and usual ingredients, can be present in pharmaceutical dosage forms, in particular as a solution, suspension, emulsion, tablets, suppository, etc. it can be used in special formulations such as liposomes, nanosomes, slow-release pellets, etc. You can in the usual way, for example orally, parenterally, intravenously, inhalatively, nasally, rectally, intraventricularly, intraarterially, intraperitoneally and / or intramuscularly or as an implant. The mode of administration is preferably selected such that the impaired cells can be reached as quickly as possible by the drug according to the invention.

The β-adrenoceptor agonists used according to the invention are particularly suitable for the production of medicaments for the treatment of neurodegenerative diseases. Examples of such diseases are Alzheimer's disease, cerebrovascular dementias, Parkinson's disease, Pick's disease, Huntington's chorea, amyotrophic lateral sclerosis, Lewy-body dementia, stroke and brain trauma such as contusion and cerebral sprain, as well as brain and spinal cord injuries or cross-sectional injuries, spina bifida , as well as diseases of the inner ear, for example diseases that are associated with the occurrence of tinnitus, such as subacute or chronic tinitus, sudden hearing loss, Meniere's disease, and diseases that are associated with impaired hearing or impaired vision, etc.

In a further embodiment of the present invention, the β-adrenergic agonists are used to restore and / or maintain the function of cells of the central nervous system and / or other nerve cells which have been partially or completely damaged by encephalopathy. Encephalopathy is one of the pathological non-inflammatory brain changes with variable neurological and / or psychological symptoms. Examples of encephalopathies that can be treated according to the invention with β-adrenergic agonists are toxic encephalopathy, encephalopathia diabetica, encephalopathia hepatica, encephalopathia hypertensive, metabolic encephalopathy, such as encephalopathy caused by metabolic disorders, eg endogenous, renal encephalopathy, eg in case of enzyme disorders, enzymatic disorders, endogenous enzymes Liver diseases, disturbances in the water-electrolyte or acid-base balance, myclonic infantile encephalopathy (Kinsboorne syndrome), encephalopathia postictereca infantum (bilirubin encephalopathy), post-combinatory encephalopathy, encephalopathy caused by heavy metals, especially organic compounds, such as inorganic and organic compounds, especially from inorganic and organic compounds, especially from inorganic and organic compounds, especially from inorganic and organic compounds , Mercury and amalgam, thallium, bismuth, aluminum, nickel and any mixtures of these compounds and their metal alloys, toxic encephalopathy caused by alcohol, enzep spongiform bovine halopathy (BSE), supcortical progressive encephalopathy, traumatic encephalopathy. In a further embodiment of the present invention, the compounds used according to the invention are used for the prevention of the diseases mentioned above.

In a further embodiment of the present invention, the compounds used according to the invention are used as additive (s) for culture media to promote the growth and / or differentiation and / or protection of mammalian cells and human cells.

Examples

1) Activation of astrocytes. Primary cultures of astrocytes were obtained from the cerebral cortex tissue of newborn Fischer 344 rats within 24 hours after birth. The brains were dissected out of the skull cap under sterile conditions, the bark tissue (cortex) was isolated and the cells were dissociated by a close-meshed wire network. The cells were placed in cell culture bottles and cultured in serum-containing DMEM solution (containing fetal calf serum and a penicillin-streptomycin mixture) until the cells confluent. Oligodendrocytes and microglia were removed by washing with cold buffer solution. The confluent astrocytes were then detached from the bottom of the culture flasks with a trypsin solution and sown at a density of 20,000 cells / cm 2 in petri dishes on coverslips and cultivated in serum-containing medium until the cells re-confluent. Two days after confluence, the medium was changed with serum-free medium and after 24 hours another medium change, also with serum-free medium. Twenty-four hours after the second medium change, salmeterol was added. Six hours after the treatment, the astrocytes were photographed in 200x microscopic magnification to document the morphological changes (Fig. 1/7). The figure shows the astrocytes 6 hours after the start of treatment. The changes from the polygonal, flat and light-refractive cells in the controls compared to the activated, star-shaped and light-refracting astrocytes in the groups treated with salmeterol can be clearly seen.

2) Induction of NGF in cultured rat hippocampal neurons

Primary mixed cultures with a share of 50% neurons and astrocytes were obtained from the hippocampus of newborn Fischer 344 rats within 24 hours after birth. The hippocampus was isolated from the rat brain under sterile conditions and, after a brief incubation in a papain solution, carefully triturated using a glass pipette. The cells thus dissociated were seeded at a density of 3 × 10 ⁵ cells in 35 mm petri dishes and cultivated in a serum-containing medium (MEM with 10% NU serum and a penicillin-streptomycin mixture). Two days after cultivation, cytosine arabinofuranoside was added to the medium for 24 hours to inhibit the growth of the astrocytes. A medium change with fresh serum-containing medium was carried out every 3-4 days. After 14 days in culture there was a change of medium performed serum-free medium and added Clenbuterol after another 24 hours. The culture medium was collected four hours after the start of the Clenbuterol incubation. The NGF content in the medium was determined using a standardized enzyme-linked immune reaction (ELISA). For this purpose, the chambers of a multiwell plate were coated with an NGF antibody and then incubated in the individual chambers with the medium of the different groups. The NGF thus bound in the chambers from the medium was then incubated with a beta-galactosidase _: conjugated NGF antibody. This was followed by a beta-galactosidase _: catalyzed conversion of chlorophenol red beta-galactopyranoside to a red dye which was measured photometrically. In addition to the samples, standard dilutions of NGF were also measured to create a standard curve. The NGF content in the samples was determined using the standard curve (Fig. 2/7).

3) Neuroprotective effect of clenbuterol in vitro. Primary mixed cultures from the hippocampus of the rat were set up as described in Example 2 and, after 14 days in culture, were subjected to a medium change to serum-free medium. Twenty four hours after changing the medium, the adrenoceptor antagonist propranolol was added to the medium. A further 20 minutes later, the β ₂ -adrenoceptor agonist clenbuterol was added and the cells were incubated for 4 hours. Sister cultures of the hippocampal cells treated in this way received only vehicle, propranolol or clenbuterol alone. After 4 hours, the medium was changed and the cells were incubated for 1 hour with L-glutamate (1mM) in serum-free medium. The medium was then changed again with serum-free medium in order to remove the glutamate from the cultures. Propranolol and clenbuterol were added freshly with each change of medium and were thus present in the medium during the glutamate treatment and up to 18 hours afterwards. Eighteen hours after the damage to glutamate, the cells were incubated with a trypan blue solution, fixed, and the damaged, blue-stained neurons were quantified under 200 × microscopic magnification (Fig. 3/7).

4) Cerebroprotective effects of clenbuterol in a model of rat brain ischemia

Permanent occlusion of the middle cerebral artery (arteria cerebri media, MCA) induced ischemia in the cortex of male Long Evans rats. The surgical intervention was carried out under inhalation anesthesia (1.5% halothane in an oxygen / nitrous oxide mixture 30:70). The left was under deep anesthesia The temporal muscle is cut between the eye and ear, and the exposed skull is drilled open under stereomicroscopic control. The hard meninges were removed and the MCA was sutured in three places using bipolar electrocoagulation. The wound in the temporal muscle was then closed in order to maintain the function of the muscle for eating. The body temperature of the rats was regulated to 37 + 0.5 ° C during the operation and kept stable for 2 hours after the procedure with the help of a heat lamp at an ambient temperature of 30 ° C. Physiological parameters (blood pressure, plasma glucose level, arterial pH, CO ₂ and O ₂ partial pressures) were also checked and documented during the procedure. Seven days after the MCA was closed, the brains were removed and frozen. With the help of a cryomicrotome, coronal sections (20μM) of the brains were made at defined intervals of 0.5 mm. The brain sections were then stained with cresyl violet, whereby the area of the infarct showed only a weak color and could thus be distinguished from the healthy tissue. The infarct area of the individual sections was measured and the infarct volume was calculated from the area values and the defined distance between the subsequent sections. Clenbuterol was administered intraperitoneally in the various doses 3 hours before the MCA was closed (Fig. 4/7).

5) Neuroprotective Effect of Salmeterol in Vitro Primary mixed cultures from the rat hippocampus were set up as described in Example 2 and, after 14 days in culture, were subjected to a change in medium to serum-free medium. Twenty-four hours after changing the medium, the β-adrenoceptor agonist salmeterol was added and the cells were thus incubated for 4 hours. Sister cultures of the hippocampal cells treated in this way received only vehicle or salmeterol alone. After 4 hours, the medium was changed and the cells were incubated for 1 hour with L-glutamate (1 mM) in serum-free medium. The medium was then changed again with serum-free medium in order to remove the glutamate from the cultures. Salmeterol was added fresh each time the medium was changed and was thus present in the medium during the glutamate treatment and up to 18 hours afterwards. Eighteen hours after the damage to glutamate, the cells were incubated with a trypan blue solution, fixed, and the damaged, blue-stained neurons were quantified under a 200-fold microscopic magnification (Fig. 5/7). The values given are mean values and standard deviation from 5-6 cultures per group. * P <0.05; ** p <0.01; and * ^* * p <0.001 compared to the control treated with glutamate (analysis of variance, Scheffe test). 6) Neuroprotective effect of salmeterol in vivo

Focal cerebral ischemia of the mouse was produced by ligating the cerebral artery. Male NMRI mice (26-31 g, 10-12 animals per group) were used for the experiments. The animals were anesthetized by an intraperitoneal injection of tribromoethanol (600 mg / kg). The surgical field was then opened through a 2 cm incision between the left eye and ear, the temporalis muscle was removed with a thermocauter and the bone was removed with a fine bur to expose the cerebral artery. This artery and its two distal branches were permanently occluded. During the preparation, the body temperature of the mouse was measured and kept constant at 37 +/- 1 ° C by an infrared heat lamp. After the preparation, the animals were left for an additional two hours at an ambient temperature of 30 ° C. To determine the area of the infarct, the mice were anesthetized again with tribromoethanol 48 hours after occlusion of the cerebral artery and perfused with a 1.5% neutral red solution (0.5 ml intraperitoneally). As a result, the perfused brain tissue turned red and the infarcted area remained bright. The isolated brains were fixed with 4% formaldehyde buffer (pH 7.4) for at least 24 hours and then the unstained area on the brain surface (infarct area) became a computer -supported (NIH image software) measured. The salmeterol dissolved in 0.9% NaCI was injected interperitoneally 5 hours before the operation. The animals in the control group received only 0.9% NaCI solution (Fig. 6/7). The values are mean values and standard deviation of 15-16 animals per group. * p <0.05 compared to the control (analysis of variance, Duncan's test)

7) Neuroprotective effects of clenbuterol and memantine in vivo. A focal cerebral ischemia of the mouse was produced by ligating the arteria media. Male NMRI mice (26-31 g, 10-12 animals per group) were used for the experiments. The animals were anesthetized by an intraperitoneal injection of tribromoethanol (600 mg / kg). The surgical field was then opened through a 2 cm incision between the left eye and ear, the temporalis muscle was removed with a thermocauter and the bone was removed with a fine bur to expose the cerebral artery. This artery and its two distal branches were permanently occluded. During the preparation, the body temperature of the mouse was measured and kept constant at 37 +/- 1 ° C by an infrared heat lamp. After the preparation, the animals were left for an additional two hours at an ambient temperature of 30 ° C. To determine the infarct area, the mice were again treated with tribromoethanol 48 hours after occlusion of the cerebral artery anesthetized and perfused with a 1.5% neutral red solution (0.5 ml intraperitoneally). As a result, the perfused brain tissue turned red and the infarcted area remained bright. The isolated brains were fixed with 4% formaldehyde buffer (pH 7.4) for at least 24 hours and then the unstained area on the brain surface (infarct area) became a computer -supported (NIH image software) measured. The two pharmaceuticals to be investigated, Memantine and Clenbuterol, were dissolved in 0.9% NaCl for injection. Memantine (20 mg / kg) was injected interperitoneally 30 minutes before surgery and clenbuterol 2 hours after surgery. The animals in the control group received only 0.9% NaCI solution (Fig. 7/7).

Claims

claims

1. Use of ß-adrenoceptor agonists to restore and / or maintain the function of partially or completely damaged cells of the central nervous system and / or other nerve cells.

2. Use according to claim 1, characterized in that astrocytes and / or endogenous protective mechanisms are activated or stimulated.

3. Use according to claim 1 or 2, characterized in that the ß-adrenergic agonists are selected from clenbuterol, formoterol, fenoterol, salbutamol, orciprenaline, isoetharine, cimaterol, ractopamine, reproterol, salmeterol, terbutaline, their isomers, acid addition salts, Analogues and any mixtures of the foregoing.

4. Use according to one of claims 1 to 3, characterized in that the ß-adrenoceptor agonists in an amount of 0.01 to 100 mg / day.

5. Use according to claim 4, characterized in that substances such as clenbuterol, formoterol, fenoterol, and salmeterol in an amount of 0.01 to 5 mg / day,

Terbutaline in an amount of 1.0 to 30 mg / day, salbutamol in an amount of 1.0 to 50 mg / day and orciprenaline and reproterol in an amount of 1.0 to 100 mg / day.

6. Use according to one of claims 1 to 5, characterized in that β1-adrenoceptor agonists, such as dobutamine, are used.

7. Use according to one of claims 1 to 6, characterized in that NMDA antagonists are used.

8. Use according to one of claims 1 to 7, characterized in that the neurodegenerative diseases are selected from Alzheimer's disease, cerebrovascular dementias, Parkinson's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, Lewy body dementia, stroke and / or brain trauma , like Contusio and Commotio cerebri as well as brain and

Spinal cord injuries or cross-sectional injuries, spina bifida, as well Inner ear disorders, for example disorders associated with the appearance of tinnitus such as subacute or chronic tinitus, sudden hearing loss, Meniere's disease, and disorders associated with impaired hearing or impaired vision, etc.

Use according to one of claims 1 to 7, characterized in that the neurodegenerative diseases are selected from toxic encephalopathy, encephalopathy diabetica, encephalopathia hepatica, encephalopathia hypertensive, metabolic encephalopathy, such as encephalopathy caused by metabolic disorders, e.g. with enzymopathies, endogenous disorders, renal insufficiency

(Encephalopathia uraemica), liver diseases, disturbances in the water-electrolyte or acid-base balance, myclonic infantile encephalopathy (Kinsboorne syndrome), encephalopathia postictereca infantum (bilirubin encephalopathy), post-combinatory encephalopathy, in particular caused by schwalogenetic compounds, in particular by encephalopathy, encephalopathy how connections from

Lead, mercury as well as amalgam, thallium, bismuth, aluminum, nickel and any mixtures of these compounds and the metal alloys, toxic encephalopathy caused by alcohol, encephalopathy spongiformes bovine (BSE), supcortical progressive encephalopathy, encephalopathy traumatica.

10. Use according to one of claims 1 to 7, characterized in that the compounds are used for the prevention of neurodegenerative diseases.

11. Use according to one of claims 1 to 7 as an additive for culture media for promoting growth and / or differentiation and / or protection of

Mammalian and human cells.