CZ20003538A3 - The use of smilagenin and anzurogenin D for the treatment of Alzheimer's disease - Google Patents

Abstract

The use of smilagenin and anzurogenin D in the treatment of cognitive dysfunction and similar conditions. Pharmaceutical compositions are also described.

Description

Technical area

The present invention relates to smilagenin and anzurogenin D and their use for the treatment of cognitive dysfunction and related conditions and compositions for use in such treatment. More specifically, the present invention relates to the treatment of conditions characterized by a deficiency in the number or function of membrane-bound receptors. In the following description, the invention will be described in the context of the treatment of Alzheimer's disease (AD) and senile dementia of the Alzheimer type (SDAT), in which a deficiency in multiple receptor types has been demonstrated. However, it should be understood that the present invention relates generally to the treatment of conditions caused by intrinsic pathological conditions and/or adverse external influences that are characterized by a deficiency in the number or function of membrane-bound receptors or a deficiency in signal transduction at junctions between neurons or at junctions between neurons and effector cells.

State of the art

The conditions listed above include Parkinson's disease, dementia with Lewy bodies, postural hypotension, autism, chronic fatigue syndrome, myasthenia gravis, * Lambert-Eaton disease, Gulf War syndrome-related illnesses and conditions, organophosphate exposure, and problems associated with aging.

Alzheimer's disease (AD) and senile dementia of the Alzheimer type (SDAT) are serious and growing problems in all societies, where the demographic profile is shifting towards an older population due to increased life expectancy and control of acquired diseases. There is a need for agents that will treat or improve the treatment of AD/SDAT.

Age-associated memory impairment (AAMI) is a characteristic of elderly patients who experience memory loss despite normal psychological and physical status. It is an ill-defined syndrome, but agents effective in the treatment of AD/SDAT may also benefit these patients.

AD/SDAT research is conducted by traditional and conventional medical research methods. In conventional medicine, there are several approaches to the treatment of AD/SDAT. It is known that the biochemical processes of memory in the cerebral cortex are (at least in part) cholinergically mediated. It is known to those skilled in the art that cholinergically mediated mechanisms are directly caused by the action of acetylcholine on receptors and these are referred to as direct effects. Other clinically useful effects may also be caused by modulation of the release of acetylcholine from presynaptic nerve terminals or inhibition of acetylcholine degrading enzymes. These modulatory factors may act through neurons in which the mediator is noncholinergic; these effects are referred to as indirect effects. Several treatment trials have focused on the role of other mediators, such as 5-hydroxytryptamine, which is a mediator in other brain regions, such as the midbrain nuclei. However, because nerve fibers from these areas lead towards the cerebral cortex, in which the primary neurotransmitter is acetylcholine, attention has been focused on influencing this mediator and on searching for agents affecting this mediator.

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Cholinergic strategies for the treatment of AD/SDAT have targeted several sites in the pathway for the production, synaptic release, and clearance of released acetylcholine.

One approach involves treatment with high doses of lecithin and other acetylcholine precursors. This procedure has limited use in achieving long-term improvements in cognitive performance.

Another approach involves the use of herbal medicines such as Polygalae root extract, which has been shown to increase choline-acetylcholine transferase (CAT) activity and nerve growth factor (NGF) secretion in the brain. Oral administration of NGF has no effect on neurons in the central nervous system due to the high molecular weight of the protein, which does not cross the blood-brain barrier. However, blood-brain barrier-crossing agents that can stimulate NGF synthesis in the central nervous system have been proposed to improve memory-related behaviors.

The results of the third clinical strategy, which uses cholinesterase inhibitors such as tacrine hydrochloride, were somewhat better than those of the previous two strategies. Substances derived from plants used in Chinese and Western medicine, such as huperzine, galanthamine, and physostigmine, have all been shown to have some - albeit limited - beneficial effect in the treatment of AD/SDAT in clinical trials and laboratory models. All of these substances are acetylcholine esterase (AChE) inhibitors. Patients with AD/SDAT may have reduced acetylcholine (Ach) synthesis, reduced efficiency of Ach release from presynaptic vesicles, and reduced number or functionality of postsynaptic (Mi) receptors. A reduction in the number of presynaptic M ₂ receptors has also been observed. Favorable • · · ·

The effect of AChE inhibitors is attributed to an increase in the concentration of acetylcholine in synapses in the brain, which is caused by slowing the degradation of the released transmitter.

Agents that affect cholinergic function are known to affect memory and recall. For example, nicotine stimulates nicotinic acetylcholine receptors, and the short-term memory improvements of cigarette smoking are thought to be due to nicotine's effects. Scopolamine, an acetylcholine antagonist, produces amnesia and cognitive impairment, manifested in psychomotor tests as a prolongation of simple reaction time, probably due to impaired attention, and is used for this purpose as an adjunct to analgesic therapy. The amnestic effect of scopolamine can be antagonized by nicotine.

There are two subfamilies of nicotinic receptors (α and β), each containing four subtypes differing in ligand specificity. The role of nicotinic receptors in the CNS is not well understood at the molecular level. It is possible that agents that bind to nicotinic receptors may modify the turnover rate of muscarinic receptors in the brain. Nicotinic receptors are ion channels that open upon ligand binding, and their activation causes a rapid (on the order of milliseconds) increase in cellular permeability to Na ⁺ and Ca ⁺⁺ , depolarization, and excitation.

Another class of cholinergic receptors can be stimulated by muscarine. These muscarinic (M) receptors are G-protein coupled receptors. The response of muscarinic receptors is slower and can be excitatory or inhibitory. They are not necessarily linked to changes in ion permeability.

Cloning of cholinergic receptors revealed five types of muscarinic receptors, which were designated mi-ms.

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Pharmacological effects are associated with four cloned receptors, which are designated M1-M4 according to pharmacological specificity.

Using specific receptor proteins and monoclonal antibodies, it has been possible to further localize muscarinic receptors in the brain as mi (postsynaptic) and m ₂ (presynaptic). In the heart, M2 receptors are postsynaptic. Presynaptic muscarinic receptors are thought to be inhibitory because binding of Ach to these receptors inhibits the release of further Ach, causing a negative feedback loop for Ach release. Selective M ₂ receptor antagonists, which are preferentially distributed to the brain, may therefore be useful for the treatment of Alzheimer's disease.

It is known that in diseases such as AD/SDAT, there is a general reduction in the number of neurons and a deficit in the function of cholinergic nerves. It is hypothesized that the high-affinity nicotinic binding sites on the remaining neurons can be converted to low-affinity binding sites in the treatment of such diseases, which will slow down the release of the neurotransmitter. The reduction in the affinity of the nicotinic binding sites prevents rapid desensitization.

Activation of nicotinic receptors in the brain by agonists has a rapid onset and rapid off-effect. Reduced affinity of nicotinic receptors reduces desensitization. Schwarz, R.D. et al. (J. Neuro. Chem. 42 (1984), 1495-8) have shown that nicotinic binding sites are located presynaptically on cholinergic (as well as 5-hydroxytryptaminergic and catecholaminergic) axon terminals. Alterations in high-affinity binding sites in AD/SDAT may also induce changes in the modulatory effect of nicotinic binding sites on other transmitter systems.

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Presynaptic cholinergic mechanisms are also under inhibitory control mediated by GABAergic neurons, and this inhibition is thought to be enhanced in AD/SDAT. Removing or reducing this inhibition enhances presynaptic cortical cholinergic activity and improves cognitive function.

The interaction of interneuronal projections innervated by nicotine (reduction of binding affinity) and the removal of inhibition of GABA-ergic fibers are influenced by the presynaptic locus.

This is a simplified model of transmitter action in the central nervous system, but it provides a framework for understanding the attempts that have been made to increase the effective concentration of acetylcholine at central synapses. It also illustrates the concept of direct and indirect action. There are disadvantages associated with the three common therapeutic approaches for the treatment of AD/SDAT, which have been discussed above: delivery of Ach precursors, administration of agonists, and inhibition of acetylcholinesterase. These treatments can lead to a short-term increase in Ach availability, which can activate feedback mechanisms that can lead to desensitization of postsynaptic receptors. Theoretically, long-term improvement cannot be achieved, and if treatment is discontinued, any benefit of AD/SDAT and AAMI treatment will be lost and the condition may worsen.

Compounds with M1 agonist and M2/M3 antagonist activity have been shown to improve cognitive performance in patients with SDAT (Sramak et al., Life Sciences, Vol. 2, No.

3, 195-202, 1997). However, these compounds have unacceptable cholinergic side effects such as fatigue, diarrhea and nausea.

A more radical approach for the treatment of AD/SDAT and AAMI aims to increase the number of postsynaptic (Mi) receptors in the brain. Z • tftftf

Ί ·········«» ·· ·····«· ··· · ··· · ··· >· *« Chinese Patent No. CN1096031A, it is known that sarsapogenin (SaG) can increase the number of Mi cholinergic receptors in the brain and also reduce the number (i.e. towards normal levels) of β-adrenergic receptors, the number of which can be pathologically increased in AD/SDAT.

Patent applications have been published protecting the use of many steroidal sapogenins containing spirostane, furospirostane, spirosolane or solanidine structures for the treatment of various diseases including SDAT. Two patent applications are particularly relevant to the present invention:

Chinese Patent Application No. CN1096031A describes the use of the spirostane sapogenin, sarsapogenin, for the treatment of SDAT.

However, the description given in this application is brief. Another patent of importance for the present invention is patent application DE 4303241A1, which describes the use of a large number of saponins and sapogenins for the treatment of a wide range of diseases, which the inventors believe to be of viral origin. This invention is, however, of dubious value in that it is known that in many conditions characterized by deficits in synaptic transmission no infectious element is identified, thus undermining the basic premise of this invention.

Furthermore, this patent does not disclose any data of any type that would enable those skilled in the art to select a preferred compound from the large number of compounds that are protected in this invention.

The essence of the invention

The inventors have discovered that smilagenin (SMI) and anzurogenin-D (AZD) have the ability to regulate receptors. More specifically, it has been found that these compounds - and in particular SMI - increase the number of

M2 receptors in the brain. Therefore, in one aspect the invention comprises the use of smilagenin and/or anzurogenin D for the manufacture of a medicament for the treatment of conditions characterized by a deficit in the number or function of postsynaptic membrane-bound receptors.

Those skilled in the art will be aware of the relationship between saponins and their sapogenins and will appreciate that the desired effect of sapogenins can be achieved in patients by administering the respective saponins or mixtures thereof. Hydrolysis of at least a portion of the saponins occurs in the gastrointestinal tract. Those skilled in the art will also be aware of the epimerization of some sapogenins under acidic hydrolysis conditions.

The sapogenins of the present invention have the following general formula:

Referring to this general formula, the structure of AMI and AZD is shown in the following table:

Compound A/B circle cis/trans/nana carbonated state Stereochemical arrangement of C25 methyl (R or S) Hydroxy group on the spirostane ring Smilagenin cis R 3β-OH Anzurogenin D transgender R 3B-OH, 5α-OH, 6β-OH

Saponins and sapogenins according to the present invention are found in many plant species, especially from the Smilax family,

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Asparagus, Anemarrhena, Yucca and Agave. The most important species are Smilax regelii Kilip and Morton - commonly known as Honduran Asparagus; Smilax aristolochiaefolia Miller - commonly known as Mexican Asparagus; Smilax ornata Hooker - commonly known as Jamaican Asparagus; Smilax aspera - commonly known as Spanish Asparagus; Smilax glabra Roxburgh; Smilax febrifuga Kunth - commonly known as Ecuadorian or Peruvian Asparagus; Anemarrhena asphodeloides Binge; Yucca schidigera Roezl ex Ortigies; and Yucca brevifolia Engelm.

In another aspect, the present invention includes a pharmaceutical composition for improving cognitive function, which comprises an effective amount of smilagenin and/or anzurogenin D.

In another aspect, the present invention includes a pharmaceutical composition for improving cognitive function, which comprises an effective amount of smilagenin and/or anzurogenin D in the form of an extract obtained from plants of the genus Smilax, Asparagus, Anemarrhena, Yucca or Agave.

It is to be understood that the invention includes within its scope the use of the compositions set forth above. Accordingly, the invention includes in a fifth aspect a method for improving cognitive function comprising administering to a human or animal an effective dose of a composition of the present invention.

The invention also includes a method for improving cognitive function in a human or animal, comprising administering an effective dose of smilagenin and/or anzurogenin D.

The term cognitive function, as used herein, refers to functions such as thinking, reasoning, remembering, imagining, and learning.

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In identifying compounds that could be used for the treatment of SDAT and other diseases characterized by a reduction in the number of receptors or synaptic transmission, the inventors considered that it was necessary to identify those compounds that would have the desired effect without any estrogenic effects, since these effects are unacceptable, especially in men. Many of the compounds protected in patent application DE 4303214A1 have significant estrogenic activity and are therefore unacceptable.

However, smilagenin and anzurogenin D do not exhibit estrogenic activity. Furthermore, these compounds were tested for their effects on other steroid receptors and were found to have no activity at any of the following receptors:

Progesterone receptor;

Glucocorticoid receptor;

Testosterone receptor.

The selected compounds were also tested for their activity in a number of in vitro assays. The following assays/experiments were considered key to determining possible activity for increasing the number of membrane-bound receptors:

1. Chinese hamster ovary (CHO) cells were transfected with a DNA fragment encoding the muscarinic receptor. The cell line used for most experiments was a cell line expressing the m2 receptor.

2. Research into the influence of muscarinic receptor expression in a cultured cell line of neuronal origin.

3. Cardiac muscle cells obtained from neonatal Spraque-Dawley rats were cultured. Cardiac muscle cells express muscarinic receptors, usually m2. The concentration of these receptors decreases with long-term culture and the effect of selected compounds on preventing the decrease in receptor number was tested.

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The methods and results of these experiments are described in more detail below.

1. Experiments performed with CHO cell line

The effect of various compounds on the expression of m2 receptors in CHO cells transfected with m2 receptor DNA was investigated. The number of receptors was assayed by binding of tritiated QNB and subtraction of non-specific binding. The compounds were dissolved in DMSO and DMSO was used as a control. The compounds were tested at various final concentrations. The compounds were also tested in the presence and absence of tamoxifen to distinguish the estrogen receptor-mediated mechanism. The results are summarized in Table 2, in which the compounds used in the present invention are listed in bold and the data obtained for other sapogenins are given for comparison.

Table 2: Effect of compounds on M2 receptor expression on CHO cells

Compound Molar concentration of a compound Effect on receptor expression - expressed as % increase relative to control (negative values are listed in brackets) Sarsapogenin 10 ^points 34 10' ⁶ (14) Anzurogenin D 10 ⁵ 22 10 ^-6 (26) Sisalgenin 10' ⁵ NS 10 ⁶ NS Smilagenin 10* ^S 57 10' ⁶ 18 Diosgenin 10~ ^b NS 10 ⁶ NS

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Ruscogenin 10 ⁵ (22) 10' ⁶ NS Tigogenin 10 ⁵ NS 1O' ^Ď NS

NS = statistically insignificant effect

Experiments showed that smilagenin and anzurogenin D increased the number of muscarinic receptors expressed on the surface of CHO cells cultured in vitro. This effect was not antagonized by tamoxifen, suggesting that the estrogen receptor was not involved in the mechanism of action.

2. Effect of compounds on cell survival

Additional in vitro assays were used to determine the effects of smilagenin and anzurogenin D. Specifically, various neuroblastoma cell lines, including SKN-SN and SH-SY5Y cells, and a pheochromocytoma cell line, were cultured in vitro in the presence of β-amyloid fragments or serum depletion. A variety of techniques were used to demonstrate the efficacy of the compounds in protecting cultured cells. These techniques include trypan blue exclusion, chemiluminescence, and lactate dehydrogenase release. Most important was the observation that incubation of cells, specifically PC12 cells, with β-amyloid reduced muscarinic receptor number as measured by radiolabeled ligand binding. This reduction in receptor number was found to be attenuated by the use of smilagenin and anzurogenin D.

3. Effect of compounds on cultured cardiac muscle cells

Cardiac muscle cells were isolated from the ventricular muscle of neonatal Sprague-Dawley rats using standard techniques. The cells were cultured in vitro and the number of muscarinic receptors expressed on cell membrane fragments obtained after homogenization of cells collected at different time intervals was assessed using specific binding of tritiated QNB. Preliminary experiments showed that the number of expressed receptors began to decrease after 10 days of culture. Experiments were therefore designed to investigate the effect of various compounds on inhibiting this decrease in receptor number. The results of this experiment are summarized in Table 3, in which the compounds used in the present invention are listed in bold and the data obtained for other sapogenins are given for comparison.

Table 3: Effect of various compounds on muscarinic receptor expression on cultured cardiac muscle cells

Compound Concentration of compound causing a statistically significant increase in the number of receptors expressed on neonatal heart muscle after 10 days of in vitro culture Diosgenin NS Anzurogenin D _{iQ e m} Ruscogenin NS Sarsapogenin 1O ^'S M Tigogenin NS Astragaloside _{yes 5 meters} Smilagenin 10 ⁵ M

NS = statistically insignificant effect

It is believed that the active compounds of the present invention may act through the G-protein and that their effects on receptor numbers are secondary to their effects on the G-protein. Following stimulation of the membrane G-protein, two basic sets of events are initiated: the effector response; and receptor internalization. The subsequent processing of the receptor to a state in which it is again present on the cell surface or on another membrane and can interact with other ligands for the receptor is influenced by many factors. Many of these factors or mechanisms appear to be G-protein-mediated. There is evidence that activation of m ₃ receptors may affect the expression or concentration of G-proteins. It is believed that the mechanism of action of the compounds of the present invention may be through interaction with receptor regeneration processes, G-protein binding, or G-protein homeostasis.

An alternative hypothesis is that the compounds increase the synthesis or release or decrease the rate of degradation of neurotrophic factors such as brain growth factor and/or nerve growth factor. These effects on growth factors may be mediated by the action of the compounds on a cytosolic or nuclear receptor or by the binding of the compound to a promoter region, with subsequent direct effect on the rate of production of mRNA for the growth factor, or they may be mediated by increased production of another factor such as a G-protein, or these effects may be secondary to affecting the receptor or G-protein processing.

Claims (7)

Patent Nation Use of smilagenin for the manufacture of a medicament for the treatment of conditions associated with a deficiency in the number or function of membrane-bound receptors. Use of anzurogenin D for the manufacture of a medicament for the treatment of conditions associated with a deficiency in the number or function of membrane bound receptors. 3. A cognitive enhancing pharmaceutical composition comprising a pharmacologically active effective amount of smilagenin and / or anzurogenin D. A cognitive enhancing pharmaceutical composition comprising a pharmacologically active effective amount of smilagenin and / or anzurogenin D in the form of an extract obtained from a plant of the genus Smilax, Asparagus, Anemarrhena, Yucca or Agave. Use of an effective dose of the composition of claims 3 or 4 for the preparation of a medicament for improving cognitive function in a human or animal. 6. A non-therapeutic method for improving cognitive function in a human or animal, comprising administering an effective dose of smilagenin and / or anzurogenin D. Changed claims Use of a pharmacologically effective dose of smilagenin and / or anzurogenin D for the preparation of a medicament for improving cognitive function in a patient suffering from cognitive dysfunction associated with aging.