A formulation for use in the treatment of alzheimer's disease

Abstract

The invention relates to a drug formulation for use in the treatment of Alzheimer's disease through inhibition of the acetylcholinesterase enzyme and a preparation method thereof The formulation of the invention comprises Etoperidone and Queuine as active ingredients, and the cholinergic effect of said substances is based on the inhibition of acetylcholinesterase. Moreover, the presence of Etoperidone and Queuine active ingredients in salt form in the formulation that is the subject of the invention creates a synergistic effect in inhibition of acetylcholinesterase enzyme

Description

[0001] SPECIFICATION

[0002] A FORMULATION FOR USE IN THE TREATMENT OF ALZHEIMER'S DISEASE

[0003] Technical Field

[0004] The invention relates to a drug formulation for use in the treatment of Alzheimer's disease through inhibition of the acetylcholinesterase enzyme and a preparation method thereof. The formulation of the invention comprises Etoperidone and Queuine in salt form as active ingredients, and the cholinergic effect of said substances is based on the inhibition of acetylcholinesterase. Moreover, the presence of Etoperidone and Queuine active ingredients in salt form in the formulation that is the subject of the invention creates a synergistic effect.

[0005] State of the Art

[0006] Alzheimer's disease (AD) is a degenerative disease that occurs with cognitive impairment, emotional and behavioral changes, physical and functional declines, and eventually decline in mental functions, behavioral problems, and decreased functionality due to disorders in brain functions.

[0001] . Alzheimer's disease is one of the most common causes of mental deterioration in the elderly, accounting for approximately 50-60% of overall dementia cases in people over 65 years of age [2], and AD is considered a multifactorial disease: The cause of AD is based on two main hypotheses: cholinergic and amyloid-beta plaque.

[0007] Cholinergic synapses are ubiquitous in the human central nervous system. Their high density in the thalamus, striatum, limbic system, and neocortex suggests that cholinergic transmission is critical for memory, learning, attention, and other higher brain functions. Several studies suggest additional roles for cholinergic systems in overall brain homeostasis and plasticity, and thus the brain's cholinergic system plays a central role in ongoing research regarding normal cognition and age-related cognitive decline, including dementias such as Alzheimer's disease. The cholinergic hypothesis of Alzheimer's disease focuses on the progressive loss of limbic and neocortical cholinergic innervation. Neurofibrillary degeneration in the basal forebrain is believed to be the primary cause of dysfunction and death of cholinergic neurons in the forebrain, leading to widespread presynaptic cholinergic denervation. Cholinesterase inhibitors increase the availability of acetylcholine at synapses in the brain and are one of the few drug therapies proven to be clinically useful in the treatment of Alzheimer's disease dementia, thus confirming the cholinergic system as an important therapeutic target in the disease [3].

[0008] The current therapeutic strategy in the treatment of Alzheimer's disease is based on the restoration of cholinergic function using compounds that block enzymes that break down acetylcholine [4], Cholinesterase inhibitors in the state of the art are designed to prevent the breakdown of acetylcholine and maintain its activity at cholinergic synapses. FDA-approved cholinesterase inhibitors currently available for the treatment of Alzheimer's disease are donepezil, rivastigmine, and galantamine. While Donepezil, one of these drugs, is of synthetic origin, Galantamine and Rivastigmine are of natural origin, but synthetic-based production is preferred in their industrial production. While these FDA-approved drugs provide a therapeutic effect of no more than 50% in reducing symptoms and delaying progression in Alzheimer's patients in the early stages, they have achieved almost no success in patients in advanced stages. Moreover, these approved drugs available on the market cause serious side effects due to their limited therapeutic effects, non-target specificity, low bioavailability, and high hepatotoxicity values [5]. However, these drugs have been shown to significantly improve cognition, daily and global function, and some behavioral symptoms of Alzheimer's disease compared with placebo treatment [6]. For this reason, cholinesterase inhibitors are often considered symptomatic treatments of Alzheimer's disease. In particular, long-term data show that cholinesterase inhibitor use in Alzheimer's disease reduces the risk of nursing home placement by approximately 30% per year of treatment [7] . Additionally, Alzheimer's patients treated with a higher average dose of cholinesterase inhibitors have been shown to be placed in nursing home care later than patients receiving a lower average dose [8]. Additional data from both laboratory-based studies and clinical studies suggest that cholinesterase inhibitors may have a broader mechanism of action than simply enhancing cholinergic activity, and these drugs are associated with a greater stabilizing effect on the course of Alzheimer's disease dementia than would be expected with cholinesterase inhibition alone [9]. However, the mentioned drugs may cause side effects such as vomiting, nausea, seizures, and diarrhoea, which are associated with toxicity to the cardiovascular and gastrointestinal systems [10, 1 1 ]. The active substance called Etoperidone in the state of the art is an atypical antidepressant that was introduced in Europe in 1977. It is a phenylpiperazine substituted triazole derivative with a composition that classifies said active substance as an analog of trazodone and offers a similar pharmacological profile. Etoperidone has been researched in the treatment of depression, Parkinson's tremors, extrapyramidal symptoms, and male impotence. The patent application numbered US4132791 A in the state of the art relates to the new application of two known drugs, whose generic names are "trazodone" and "Etoperidone", respectively. Said study describes that the adrenolytic drugs trazodone and Etoperidone can inhibit the extrapyramidal tremors that occur in Parkinson's disease.

[0009] Another active substance called Queuine in the state of the art is a derivative of 7- deazaguanine and is abundant in tomatoes, wheat, coconut water, and human, cow and goat milk. It is also stated that Queuine contributes to the production of various important biochemicals such as tyrosine, serotonin, dopamine, epinephrine, norepinephrine, nitric oxide, lipids, and others

[0012] , In the patent application numbered US2021052589A1 in the state of the art, compounds, compositions, and methods for the treatment of Alzheimer's disease are described. In said document, Queuine is used as a neuroprotective agent in the prevention or treatment of Alzheimer's disease by preventing the misfolding of proteins involved in the pathological mechanism that leads to the formation of amyloid plaques.

[0010] It has become necessary to develop a new formulation to be used in the treatment of Alzheimer's disease due to reasons such as the limitations and inadequacies of the solutions in the state of the art, the low effectiveness of the active ingredients used in the treatment of Alzheimer's disease, causing toxicity problems, high dose requirement and low bioavailability.

[0011] Brief Description and Aims of the Invention

[0012] In the invention, a drug formulation for use in the treatment of Alzheimer's disease through inhibition of the acetylcholinesterase enzyme and a preparation method thereof are described. The formulation of the invention comprises Etoperidone and Queuine in salt form as active ingredients, and the cholinergic effect of said substances is based on the inhibition of acetylcholinesterase. Moreover, the presence of Etoperidone and Queuine active ingredients in salt form in the formulation that is the subject of the invention creates a synergistic effect.

[0013] The aim of the invention is to inhibit high levels of acetylcholinesterase enzyme in the treatment of Alzheimer's disease. The combined use of Etoperidone and Queuine active ingredients contained in the formulation of the invention shows a synergistic effect and inhibits the acetylcholinesterase enzyme, thereby providing treatment for Alzheimer's disease.

[0014] Another aim of the invention is to reduce drug toxicity in the treatment of Alzheimer's disease. Etoperidone and Queuine, which are metabolite and nutraceutical-derived, respectively, contained in the formulation that is the subject of the invention, create lower levels of toxic response in the body than the active ingredients in the state of the art, which are completely synthetic in origin.

[0015] Description of Drawings

[0016] Figure 1 Percentage graph of cell index of SH-SY5Y cells (RTCA analysis results) compared to the control group after exposure to different concentrations of Queuine (A), Etoperidone (B)

[0017] Figure 2 Percentage graph of cell viability of SH-SY5Y cells (MTT results) compared to the control group after exposure to different concentrations of Queuine (A) and Etoperidone (B).

[0018] Figure 3 The effect of the safe doses determined in the invention on cell index in the use of Queuine and Etoperidone alone and in combination in the SH-SY5Y cell line. (RTCA synergistic effect results)

[0019] Figure 4 Acetylcholinesterase enzyme inhibition results by Ellman method.

[0020] Detailed Description of the Invention

[0021] The invention relates to a drug formulation for use in the treatment of Alzheimer's disease through inhibition of the acetylcholinesterase enzyme and a preparation method thereof. The formulation of the invention comprises Etoperidone hydrochloride and Queuine hydrochloride as active ingredients, and the cholinergic effect of said substances is based on the inhibition of acetylcholinesterase. In addition, the presence of Etoperidone and Queuine active ingredients in the formulation that is the subject of the invention creates a synergistic effect in the inhibition of the acetylcholinesterase enzyme, and by means of this effect, the treatment of Alzheimer's disease is provided more effectively. The reason for using salt forms of said active ingredients in the invention is that they are in a form suitable for cell culture.

[0022] The safe dose ranges determined for each drug molecule separately for the drug formulation of the invention are 50-70 pM for Etoperidone and 0.25-1.25 pM for Queuine. The formulation of the invention comprises 0.05-0.07% Etoperidone hydrochloride, 0.0025-0.0125% Queuine hydrochloride, and 99.91 -99.95% medium by volume, where the medium content is explained in Table 1. The drug formulation subject to the invention contains 0.7 pl of Etoperidone hydrochloride with a concentration of 100 mM and 0.125 pl of Queuine hydrochloride with a concentration of 10 mM in 999.175 pl of the medium specified in Table 1 , with a total volume of 1 ml.

[0023] The content of the culture medium prepared for the SH-SY5Y (a cell line isolated from a bone marrow biopsy taken from a four-year-old female with neuroblastoma) cell line used in the invention and the components included in the content are listed in Table 1. Said culture content is also used to dilute the stock solutions used in the invention.

[0024] Table 1. SH-SY5Y cell line culture medium components and quantities.

[0025] In the invention, first of all, in order to prepare Etoperidone hydrochloride stock solution, the stock was prepared at a concentration of 100 mM and 120 pl DMSO was used as the solvent. The reason for choosing DMSO is to prevent any possible contamination risk since this is a study conducted on the cell line (SH-SY5Y). That is, at this stage, 5 mg Etoperidone hydrochloride was dissolved in 120pl DMSO to obtain a stock solution with a concentration of 100 mM. Afterward, to prepare the Queuine hydrochloride stock solution, 500 pg of Queuine hydrochloride was dissolved in 180 pl DMSO to obtain a stock solution with a concentration of 10 mM.

[0026] Etoperidone hydrochloride and Queuine hydrochloride obtained in the invention were subjected to Real-Time Cell Analysis (RTCA) and 3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide Analysis (MTT) for determining the safe dosage used for synergistic effects. The results for RTCA are presented in Figure 1 , and the results for MTT are presented in Figure 2. The same techniques were applied to other drugs (Donepezil hydrochloride, Galantamine hydrobromide, Thiamine hydrochloride), and their safe dosages were determined. Both RTCA and MTT results are consistent with each other. In Figure 1 , based on the cell index values measured using the RTCA technique, no cytotoxicity was observed for Queuine at 1 ,25pM and Etoperidone at 70 pM. 'In Figure 2, it is demonstrated that no cytotoxicity was observed for Queuine at 1.25pM and Etoperidone at 70 pM based on the cell viability values measured using the MTT technique. Similarly, no cytotoxicity was observed for Donepezil at 15 pM, Galantamine at 10 pM, and Thiamine at 4800 pM using the same technique. Cytotoxicity was not observed at concentrations of 5 pM, 10 pM, and 15 pM for Donepezil, 5 pM and 10 pM for Galantamine, 0.25 pM and 1.25 pM for Queuine, 50 pM, 60 pM, and 70 pM for Etoperidone, and 2400 pM for Thiamine for 48 hours. However, after 48 hours, cells treated with 100 pM Galantamine showed a significantly lower survival rate compared to the control group (p < 0.001 ). It was observed that 25pM Donepezil, 25-50pM Galantamine, 5 pM Queuine, and 100pM Etoperidone significantly reduced cell viability compared to the control group (p<0.001 , where higher concentrations for each reagent showed significant cytotoxic effects on cell viability (p < 0.001 ). As a result of RTCA and MTT analyses, concentrations of 70 pM for Etoperidone hydrochloride and 1 .25 pM for Queuine hydrochloride were determined as safe doses. A combination of Etoperidone hydrochloride at a concentration of 70 pM and Queuine hydrochloride at a concentration of 1 .25 pM was created and RTCA analysis was performed to first examine the effect of their synergistic effects on the cell index. The amount of both active substances used during the analysis is shown in Table 2 below.

[0027] Table 2. Amounts of Etoperidone hydrochloride (ETO), Queuine hydrochloride (QUE) and the medium used in RTCA analysis.

[0028] The medium mentioned in Table 2 is the medium the components of which are explained in Table 1 , and according to Table 2, Queuine hydrochloride with a concentration of 1.25 pM was prepared as Queuine (1000 pl), Queuine + Etoperidone (1000 pl), Queuine + Thiamine (1000 pl), Queuine + Donepezil (1000 pl) with a total volume of 4000 pl. In this table, the calculation for the Queuine + Etoperidone combination effect is given. By taking 999.3 pl of 4000 pl 1.25 pM Queuine solution and adding 0.7 pl of 100 mM Etoperidone stock, a Queuine+Etoperidone combination with a total volume of 1000 pl was prepared. In this preparation stage, vortex and spindown processes are applied, and the aim here is to prevent loss of active ingredient by ensuring equal distribution of the components.

[0029] In the RTCA analysis conducted according to Table 2 explained above, the results were positive, and no toxic effects were observed compared to the control group of the double combination drug and Donepezil, which was preferred as the positive control group (Figure 3). Then, the Ellman experiment was carried out to observe the inhibition effect of the drug that is the subject of the invention on the AChE (Acetylcholinesterase) enzyme, and the experimental results are shown in Figure 4. In the Ellman method mentioned in the invention, while the AChE enzyme hydrolyses acetylthiocholine iodide, the BChE enzyme hydrolyses butyrylthiocholine chloride, resulting in the formation of thiocholine. Thiocholine reacts with Ellman's reagent or 5,5'-Dithiobis (2- nitrobenzoic acid) (DTNB) to produce yellow 2-nitro-5-thiobenzoate (TNB), which can be detected at 405 nm. The yellow colour production rate was measured at 405 nm to indicate enzyme activity. AChE activity was determined in the mixture comprising 30 pL of AChE solution at a concentration of 2pg / pl in 0.1 M phosphate buffer (pH 8.0), 30 pL of a 3.3mM solution of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in 0.1 M phosphate buffered solution (pH 7.0) (containing 6 mM NaHCO3) and 110 pL of phosphate buffer (pH 8.0). As substrate, 30 pL of 0.1 mM acetylthiocholine iodide solution chloride was added and AChE activity was evaluated using a spectrophotometer as absorbance change at 405 nm for 3 min on the microplate at 25°C, and according to the results of acetylcholinesterase enzyme inhibition by the Ellman method (Figure 4). Figure 4 illustrated that Etoperidone exhibited acetylcholinesterase inhibition comparable to that of Donepezil, while Queuine demonstrated acetylcholinesterase inhibition similar to Galantamine. Notably, the individual usage of Etoperidone and Queuine resulted in significantly lower acetylcholinesterase inhibition compared to the inhibition rate observed when they were used in combination. Based on this, the combined use of both active ingredients in the drug of the invention creates a synergistic effect.

[0030] In the invention, 1 ,200,000 SH-SY5Y cells were planted in each of 9 petri dishes of 6 cm2. The total volume of the petri dishes is 3 ml. These were incubated for 24 hours in 5% CO2 medium at 37°C, and after 24 hours, the old medium in the petri dish was discarded. For each drug (Donepezil, Galantamine, Thiamine, Etoperidone, Queuine, Donepezil + Queuine, Etoperidone + Queuine, Thiamine + Queuine) the application was made at the safe concentration determined after the cytotoxicity study. These were incubated for 48 hours in a 5% CO2 medium at 37°C, and the experiment was stopped at the end of 48 hours to isolate the protein. After placing the 1 .5 ml tubes in ice, all steps in the isolation were carried out in ice to prevent protein degradation. Cell lysis buffer solution was prepared according to Table 3, with a total volume of 900 pl, 100 pl for each petri dish.

[0031] Table 3. Cell lysis buffer content for SH-SY5Y cell line

[0032] RIPA (Radioimmunoprecipitation assay) buffer is a common buffer used for protein isolation. RIPA buffer increases the solubility of the protein. The SDS contained in the buffer helps dissolve proteins by dissolving the protein's cell membranes and allowing the proteins to be released. The protease inhibitor added later to the cell lysis buffer preserves the stability of the protein and prevents its destruction. First, SDS was added into RIPA, then the protease inhibitor was added by pipetting slowly and mixed by pipetting slowly. Petri dishes were washed twice. The washing process was carried out by dispersing the old medium over 1 ml of PBS and making a rotational movement. At this stage, it should be ensured that PBS is taken completely. The cells to be added afterwards dilute the lysis buffer and reduce the activity of the lysis buffer. 100 pl of the prepared cell lysis buffer was added into the petri dish and the protein isolation process was carried out by moving it in one direction with a scraper without pressing too much. Then, the collected samples were quickly transferred into a 1 .5 ml tube and placed on ice. This procedure order was followed for all samples (8 different drugs and / or drug combinations). Then, the openings of the tubes containing the samples were covered with parafilm and spun in the homogenizer twice for 30 seconds at 6000 rpm. At the end of the first spin, the petri dish was immediately placed in ice, and the process was repeated a few minutes later. After a short spin-down, the parafilms were removed and kept in the heater at 95°C for 5 minutes. In the invention, BCA (Bicinchoninic Acid) kit was used for protein (acetylcholinesterase) concentration process. Spectrophotometry results after this application are given in Table 4 below. There is no active ingredient in the control group mentioned in Table 4. However, since there is a certain concentration of acetylcholinesterase in the cell, there is a certain value (2 pg / pl). The values in the table show the acetylcholinesterase concentration and the change in the values here reflects the effect of the active ingredients.

[0033] Table 4. UV spectrophotometry results of 8 different drugs and / or drug combinations

[0034] The preparation method of the formulation that is the subject of the invention comprises the process steps: i. Adding 0.125 pl of Queuine hydrochloride at a concentration of 10 mM into 999.175 pl of medium and mixing, ii. Adding 0.7 pl of 100 mM concentration Etoperidone hydrochloride to the solution obtained in the process step (i), and iii. Vortexing the resulting 1000 pl solution and processing it in a spin-down device.

[0035] ICso values in Table 5 were obtained as a result of MTT experiments. RTCA experiments along with MTT were performed simultaneously on each active SHSY5Y cell line and compared. Safe doses were determined according to MTT and RTCA experiments and IC50 values were calculated according to MTT results. Safe doses determined from synergistic effect studies are taken as reference. Synergistic effect studies were conducted with RTCA experiments using the values found here. The RTCA results (Figure 3) resulting from the synergistic effect studies were compared with the previous RTCA experiments (Figure 1 ), and the two experimental results were compatible with each other. %acetylcholinesterase inhibition values were calculated using the Ellman method alone, and the results are given in Table 6.

[0036] Table 5. IC50 values of active substances calculated as a result of MTT tests

[0037] Table 6. %acetylcholinesterase inhibition values calculated by Ellman method

[0038] MTT Analysis After 12,500 cells were planted for each well in a 96-well plate, the cells were incubated for 24 hours at 37°C and 5% CO2 medium. After 24 hours, drugs prepared by serial dilution method within the determined concentration range by taking the old medium were applied to three wells in 100 pl DMEM, and one well was selected as the control group (the well without drug application). The plate was then incubated for 48 hours at 37°C and 5% CO2 medium. After incubation, 110 pl of 10% MTT (Roche) solution in DMEM was added to each well, and two empty wells containing only MTT solution were added for background measurement. After 4 h, 100 pl of solvent buffer was added to each well. After the plate was incubated for 16 hours at 37°C and 5% CO2 medium, the number of cells in each sample was determined by measuring it with a UV spectrophotometric device at a wavelength of 480 nm and a background of 570 nm using a microplate reader (OMEGA). Data were calculated as percentages relative to control and a dose-dependent cell viability graph was created. (Figure 2). MTT test findings are similar to RTCA results. Cell viability was close to 15 pM for Donepezil, 10 pM for Galantamine, 1.25 pM for Queuine, and 70 pM for Etoperidone, indicating no cytotoxicity at these concentrations. Thiamine, on the other hand, did not show a cytotoxic effect at any concentration, as in the RTCA results. However, consistent with RTCA findings, it was observed that cell viability was reduced at concentrations of 15 pM for Donepezil, 25 pM for Galantamine, 5 pM for Queuine, and 100 pM for Etoperidone, and the effects of Donepezil, Galantamine, Queuine, Etoperidone and Thiamine on cell density were also examined in the SH-SY5Y neuroblastoma cell line using the MTT assay. Cell index percentage graphs obtained from the RTCA device are compatible with the graphs obtained from MTT results (Figure 2) for Etoperidone and Queuine (Figure 1 ). RTCA results showed that Etoperidone and Queuine molecules had no adverse effects on cell growth or viability. That is, MTT and RTCA results confirmed that Etoperidone and Queuine molecules were effective in crossing the cell barrier and acting on the cell.

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Claims

CLAIMS1. A drug formulation for use in the treatment of Alzheimer's disease, comprising 0.05-0.07% Etoperidone hydrochloride, 0.0025-0.0125% Queuine hydrochloride and 99.91 -99.95% medium by volume,2. A drug formulation according to Claim 1 , comprising 999.175 pl of medium, 0.7 pl of Etoperidone hydrochloride with a concentration of 100 mM, and 0.125 pl of Queuine hydrochloride with a concentration of 10 mM, with the total volume amounting 1 ml.

3. A drug formulation according to Claim 1 or Claim 2, wherein said medium comprises 15% FBS (Fetal Bovine Serum), 1 % L-glutamine, 1 % sodium pyruvate, 100 pg / ml primocin and DMEM (Dulbecco's modified cell medium).

4. A method of preparing a drug formulation for use in the treatment of Alzheimer’s disease, comprising the process steps of:(i) Adding 0.125 pl of Queuine hydrochloride at a concentration of 10 mM into 999.175 pl of medium and mixing,(ii) Adding 0.7 pl of 100 mM concentration Etoperidone hydrochloride to the solution obtained in the process step (i), and(iii) Vortexing the resulting 1000 pl solution and processing it in a spin down device.

5. Drug formulation prepared by a method according to claim 4.

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