N-(FURAN-2-YLMETHYL)-7H-PURIN-6-AMINE FOR TREATMENT OF CIRCADIAN RHYTHM DISEASES, DISORDERS AND DYSFUNCTIONS

Abstract

N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof, for prevention and/or treatment of circadian rhythm disorders, circadian rhythm diseases and/or circadian rhythm dysfunctions is disclosed. The disorders, diseases and dysfunctions include, inter alia, jet-lag, social jet-lag, shift-work disorder, and circadian rhythm disturbance induced by neurodegeneration is also disclosed.

Claims

1-17. (canceled)

18. A method of prevention and/or treatment of circadian rhythm disorders, circadian rhythm diseases and/or circadian rhythm dysfunctions, comprising the step of administering N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof, to a subject in need of such prevention and/or treatment.

19. The method according to claim 18, wherein the circadian rhythm disorder, circadian rhythm disease and/or circadian rhythm dysfunction to be treated is selected from jet lag, social jet lag and circadian dysfunction due to shift-work.

20. The method according to claim 19, wherein N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof, is administered in a single-dose or in multiple doses over a time period no longer than 5 days.

21. The method according to claim 18, wherein the circadian rhythm disorder, circadian rhythm disease and/or circadian rhythm dysfunction to be treated is selected from sleep disorders and diseases of sleep.

22. The method according to claim 18, wherein the circadian rhythm disorder, circadian rhythm disease and/or circadian rhythm dysfunction to be treated is selected from the group comprising depression, unipolar depression, bipolar disorder, seasonal affective disorder, dysthymia, anxiety disorder, schizophrenia, ADHD, sleep disturbance due to neurodegeneration such as Alzheimer’s, Parkinson’s and Huntington’s disease, Smith-Magenis syndrom, REM sleep disorders, advanced sleep phase syndrome/disorder - ASPD, age related and hereditary forms of ASPD - FASPD/FASPS, delayed sleep phase syndrome, irregular sleep phase syndrome, free-running sleep phase syndrome, hypersomnia, parasomnia, narcolepsy, nocturnal enuresis, metabolic syndrome, obesity, hyperinsulinemia, non-alcoholic steatohepatosis, diabetes, in particular type 2 diabetes, restless legs syndrome.

23. The method according to claim 18, wherein the circadian rhythm disorder, circadian rhythm disease and/or circadian rhythm dysfunction to be treated is selected from REM sleep disorders, advanced sleep phase syndrome/disorder - ASPD, age related and hereditary forms of ASPD - FASPD/FASPS, delayed sleep phase syndrome, irregular sleep phase syndrome, free-running sleep phase syndrome.

24. The method according to claim 18, wherein N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof, is administered together with light therapy.

25. The method according to claim 18, wherein the circadian rhythm disorder, circadian rhythm disease and/or circadian rhythm dysfunction to be treated is selected from advanced sleep phase syndrome/disorder - ASPD, age related and hereditary forms of ASPD -FASPD/FASPS, REM sleep disorder, irregular sleep phase syndrome, free-running sleep syndrome, sleep dysfunction due to neurodegeneration such as Alzheimer’s, Huntington’s and Parkinson’s disease, Smith-Magenis syndrom, bipolar disorder and ADHD, and wherein N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof, is administered in multiple doses over a period of more than 5 days.

26. A method of improving sleep quality, comprising the step of administering N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof to a subject.

27. A method of stabilization of circadian rhythms after their disturbance due to abnormal light conditions or due to a neurodegenerative disease such as Alzheimer’s, Parkinson’s and Huntington’s disease, comprising the step of administering N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof to a subject.

28. The method according to claim 18, wherein the N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof, is administered together with a chronoterapeutic selected from the group consisting of CSNK1D inhibitors, CSNK1A inhibitors, CSNK1E inhibitors, GSK3beta inhibitors, ALK5 inhibitors, AMPK inhibitors, SIRT1 activators, CRY1/CRY2 ligands, PPARG agonists, BMAL1 expression regulators, vasopresin receptor ligands, REV-ERBα/β agonists, RORα/γ agonists, harmin, resveratrol and agonists of melatonin receptors including melatonin, ramelteon, tasimelteon, agomelatin.

29. The method according to claim 18, wherein the N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof, is administered together with a chronoterapeutic selected from melatonin, ramelteon, tasimelteon, agomelatin, harmine and resveratrol.

30. A method of treatment of proliferative diseases, comprising adjusting circadian clock of a patient by administering N-(furan-2-ylmethyl)-7H-purin-6-amine to the patient for optimal timing of administration of the antineoplastic.

31. The method according to claim 30, wherein the said proliferative disease is cancer, in particular selected from carcinomas, sarcomas and leukemias.

32. The method according to claim 30, wherein the antineoplastic is selected from the group of compounds damaging DNA and/or blocking cell cycle.

33. The method according to claim 30, wherein the antineoplastic is selected from the group consisting of melphalan, busulphan, mitoxanthrone, cyclophosphamide, ifosfamide, karmustine, lomustine, bendamustine, uramustine, cisplatin, carboplatin, oxaliplatin, dacarbazine, mitozolomide, temizolomide, ribociclib, palbociclib, abemaciclib, vincristine, vinblastine, vinorelbine, docetaxel, paclitaxel, ixabepilone, irinotecan and topotecan.

34. A combined preparation containing N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof, and at least one further chronotherapeutic in concentrations effective for prophylactic or therapeutic modulation of circadian rhythms and/or sleep of mammals including humans.

35. A combined preparation containing N-(furan-2-ylmethyl)-7H-purin-6-amine, or a pharmaceutically acceptable salt or solvate thereof, and at least one antineoplastic.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0042] FIG. 1: N-(furan-2-ylmethyl)-7H-purin-6-amine (compound 1) regulates the expression of Bmal1 gene and increases the period of the circadian rhythm of human cells U2OS transduced by transcriptional circadian reporter mBmal1-Luc (Example 1).

[0043] FIG. 2: N-(furan-2-ylmethyl)-7H-purin-6-amine (compound 1) increases the period and modulates the amplitude of circadian expression of transcriptional clock reporter mPer2-Luc in mouse cells NIH3T3 (Example 2).

[0044] FIGS. 3A-3C: N-(furan-2-ylmethyl)-7H-purin-6-amine (compound 1) increases the period and modulates the amplitude in organotypic explants of hypotalamic suprachiasmatic nucleus (SCN). Representative luminescence recordings directly show PER2 protein levels, black arrows indicate the time when the compound was added to the medium (FIG. 3A). The circadian period (FIG. 3B) and amplitude (FIG. 3C) after application of the compound in the given* concentration. (Example 3).

[0045] FIG. 4: N-(furan-2-ylmethyl)-7H-purin-6-amine (compound 1) modulates the expression of mBmal1-Luc reporter during the first 5-15 hours after the application into the medium. (Example 4).

[0046] FIG. 5: N-(furan-2-ylmethyl)-7H-purin-6-amine (compound 1) modulates the expression of mPer2-Luc reporter during the first 1-12 hours after the application into the medium. (Example 5).

[0047] FIGS. 6A-6F: N-(furan-2-ylmethyl)-7H-purin-6-amine (compound 1) modulates the effect of other chronotherapeutics on the expression of mBmal1-Luc reporter in U2OS cells. In the medium, a compound modulating circadian rhythm was present together with N-(furan-2-ylmethyl)-7H-purin-6-amine (50 .Math.M, black line) or equivalent amount of DMSO vehicle (gray). Depending on the type of the added compound, an additive (FIG. 6A) or synergistic (FIGS. 6B, 6C, 6D, 6E) effect on the period increase was observed. In the case of harmine (FIG. 6F), the addition of N-(furan-2-ylmethyl)-7H-purin-6-amine caused a decrease of the period. Mechanisms of the compounds’ actions were as follows: PF670 (PF-670462) - non-selective CK1d/e inhibitor; T0901317 - ROR agonist; KL001 - CRY1 degradation inhibitor; rosiglitazon - PPARG agonist, SR9009 - REV-ERBa/b agonist; harmin -DYRK1 inhibitor and modulator of BMAL1 transcription (Example 6).

[0048] FIG. 7: Effect of the combination of N-(furan-2-ylmethyl)-7H-purin-6-amine (+) or DMSO vehicle (-) with antineoplastics on the viability of the cancer cell line A549 (Example 7).

[0049] FIG. 8: Effect of N-(furan-2-ylmethyl)-7H-purin-6-amine on the BJ fibroblasts growth. The cell count in the wells of a 24-well plates after 12 days (Example 9).

EXAMPLES OF CARRYING OUT THE INVENTION

Example 1: N-(furan-2-ylmethyl)-7H-purin-6-amine Prolongs the Circadian Rhythm Period of Human Cells

[0050] Human osteosarcoma U2OS cells were grown in standard DMEM with 10% fetal calf serum. U2OS cells were transduced with lentiviral particles containing the reporter pLV6-Bmal1-Luc (S. Brown, Addgene plasmid # 68833) and selected under Blasticidin. The cell line was clonally expanded and a single monoclonal cell line was used for further experiments. Cells were cultured in a 384-well plate in growth medium to 90+% confluence. Test compound or vehicle (DMSO) was applied to recording medium with 100 U / ml penicillin, 100 ug / ml streptomycin, 1x GlutaMAX (ThermoFisher, Waltham, MA, USA), 10 % fetal calf serum (Sigma) and 0.1 mM Luciferin-EF (Promega Madison, WI, USA). Luminescence was recorded every hour for an additional 144 hours in Luminoskan Ascent (ThermoFisher). Circadian rhythm analysis was performed using cosinor analysis. The results are shown in FIG. 1.

[0051] This experiment demonstrates that N-(furan-2-ylmethyl)-7H-purin-6-amine extends the circadian rhythm period of human cells and can be used to manipulate the circadian rhythm phase, for example, to synchronize the endogenous circadian clock with solar time. The overall effect on circadian oscillations indirectly but unequivocally demonstrates that N-(furan-2-ylmethyl)-7H-purin-6-amine can also be used to modulate the concentration and activity of other oscillator components, including the critical components BMAL1, PER2, CRY and CLOCK over time, as these are an essential part of the mechanism for generating rhythmic expression of the used luminescent reporter. The concentration and level of other proteins, whose rhythmic expression is controlled by an oscillator in the brain and peripheral tissues (so-called clock-controlled genes), is also necessarily affected. The effect of N- (furan-2-ylmethyl) -7H-purin-6-amine on the activity of oscillator components and subordinate genes also means influencing the physiological processes of the cell controlled by these genes and proteins.

Example 2: N-(furan-2-ylmethyl)-7H-purin-6-amine Prolongs the Circadian Rhythm Period of Mouse Cells

[0052] Mouse embryonic fibroblasts NIH3T3 cells were grown in standard DMEM with 10% fetal calf serum. NIH3T3 cells were transfected with 1 .Math.g of Per2-Luc reporter (Meng, 2009) using GeneJuice (Novagen) and selected under hygromycin. The cell line was clonally expanded and a single monoclonal cell line was used for further experiments. Cells were cultured in a 96-well plate in growth medium to 90+% confluence. Test compound or vehicle (DMSO) was applied to recording medium with 100 U / ml penicillin, 100 ug / ml streptomycin, 1x GlutaMAX (ThermoFisher, Waltham, MA, USA), 10% fetal calf serum (Sigma) and 0.1 mM Luciferin-EF (Promega Madison, WI, USA). Luminescence was recorded every hour for an additional 144 hours in a Luminoskan Ascent (ThermoFisher). Circadian rhythm analysis was performed using cosinor analysis. The results are shown in FIG. 2.

[0053] The experiment demonstrates that N-(furan-2-ylmethyl)-7H-purin-6-amine prolongs the circadian rhythm period of mouse cells and can be used to manipulate the circadian rhythm phase, for example, to synchronize the internal circadian clock with solar time. The overall effect on circadian oscillations indirectly, but unequivocally, demonstrates that N- (furan-2-ylmethyl) -7H-purin-6-amine can also be used to modulate the concentration and activity of other oscillator components, including the critical components Email, Per2, Cry and Clock, as these are an essential part of the mechanism for generating the rhythmic expression of the luminescent reporter used. The concentration and level of other proteins, whose rhythmic expression is controlled by an oscillator in the brain and peripheral tissues (so-called clock-controlled genes), is also necessarily affected. The effect of N-(furan-2-ylmethyl)-7H-purin-6-amine on the activity of oscillator components and subordinate genes also means influencing the physiological processes of the cell by these genes and proteins controlled.

Example 3: N-(furan-2-ylmethyl)-7H-purin-6-amine Prolongs the Period and Modulates the Amplitude of Circadian Expression of the PER2::Luc Clock Reporter in the Explanted Suprachiasmatic Nuclei (SCN) of the Hypothalamus, the Central Clock of Mammals, of the Transgenic Mouse Line

[0054] Male and female mPer2Luc mice (strain B6.129S6-Per2tm1Jt / J, JAX, USA) (Yoo et al., 2004) were maintained in a light / dark cycle with 12 hours of light and 12 hours of darkness (LD12: 12), killed between 12:00 and 15:00, i.e. 6-9 hours after turning on the lights, by rapid cervical dislocation under isoflurane anesthesia, their brains were removed and 250 .Math.m thick SCN slices in ice-cold HBSS medium were prepared using a vibratome (Leica, Wetzlar, Germany). Two explants containing SCN from each brain were prepared. Individual SCN explants were then placed on Millicell Culture Inserts (Merck, Darmstadt, Germany) inside 35 mm Petri dishes with test compound or DMSO vehicle in 1 ml air-buffered recording medium supplemented with 100 U / ml penicillin, 100 ug / ml streptomycin (Sigma-Aldrich, St. Luis, MO, USA), 1x GlutaMAX (ThermoFisher, Waltham, MA, USA), 5% fetal calf serum (Sigma) and 0.1 mM Luciferin-EF (Promega Madison, WI, USA). The dishes were sealed with vacuum Vaseline and glass coverslips and placed inside a LumiCycle (Actimetrics) for bioluminescence recording. Rhythm analysis was performed in Lumicycle Analysis software (Actimetrics).

[0055] The results are shown in FIGS. 3A-3C.

[0056] The example demonstrates that N-(furan-2-ylmethyl)-7H-purin-6-amine prolongs the circadian rhythm period and modulates the period in the complex tissue of the central pacemaker and can be used to manipulate the circadian rhythm phase, for example to synchronize the internal circadian clock with solar time. The overall effect on the circadian oscillator indirectly, but unequivocally, demonstrates that the compound can also be used to modulate the concentration and activity of other components of the oscillator, including the critical components Bmal, Per2, Cry and Clock over time. The concentration and level of other proteins, whose rhythmic expression is controlled by an oscillator in the brain and peripheral tissues (so-called clock-controlled genes), is also necessarily affected. The effect of N-(furan-2-ylmethyl)-7H-purin-6-amine on the activity of the oscillator components and subordinate genes also means influencing the physiological processes of the cell controlled by these genes and proteins.

Example 4: N-(furan-2-ylmethyl)-7H-purin-6-amine Modulates the Expression of the Bmal1 Gene and Protein

[0057] Human osteosarcoma U2OS cells were grown in standard DMEM with 10% fetal calf serum. U2OS cells were transduced with lentiviral particles containing the reporter pLV6-Bmal1-Luc (S. Brown, Addgene plasmid # 68833) and selected under Blasticidin. The cell line was clonally expanded and a single monoclonal cell line was used for further experiments. Cells were cultured in a 384-well plate in growth medium to 90+% confluence. Test compound or vehicle (DMSO) was applied to recording medium with 100 U / ml penicillin, 100 ug / ml streptomycin, 1x GlutaMAX (ThermoFisher, Waltham, MA, USA), 10% fetal calf serum (Sigma) and 0.1 mM Luciferin-EF (Promega Madison, WI, USA). Luminescence was recorded every hour for additional 144 hours in Luminoskan Ascent (ThermoFisher). The results are shown in FIG. 4.

[0058] The example demonstrates that N-(furan-2-ylmethyl)-7H-purin-6-amine affects Bmal1 gene expression in human cells. A change in the level of Bmal1 mRNA affects its protein level. Because this protein controls the expression of other circadian oscillator genes and directly activates the expression of a number of other genes containing the E-box in their regulatory DNA region and exhibiting circadian oscillations, N-(furan-2-ylmethyl)-7H-purin-6-amine also affects their expression.

Example 5: N-(furan-2-ylmethyl)-7H-purin-6-amine Regulates Per2 Gene and Protein Expression

[0059] Mouse embryonic fibroblasts NIH3T3 cells were grown in standard DMEM with 10% fetal calf serum. NIH3T3 cells were transfected with 1 .Math.g of Per2-Luc reporter (Meng, 2009) using GeneJuice (Novagen) and selected under hygromycin. The cell line was clonally expanded and a single monoclonal cell line was used for further experiments. Cells were cultured in a 96-well plate in growth medium to 90+% confluence. Test compound or vehicle (DMSO) was applied to recording medium with 100 U / ml penicillin, 100 ug / ml streptomycin, 1x GlutaMAX (ThermoFisher, Waltham, MA, USA), 10% fetal calf serum (Sigma) and 0.1 mM Luciferin-EF (Promega Madison, WI, USA). Luminescence was recorded every hour for additional 144 hours in Luminoskan Ascent (ThermoFisher). The results are shown in FIG. 4.

[0060] The example demonstrates that N-(furan-2-ylmethyl)-7H-purin-6-amine regulates Per2 gene expression in mouse cells. The change in the mRNA level also affects the level of the PER2 protein. Because this protein controls the expression of other circadian oscillator genes and inhibits the expression of a number of other genes exhibiting circadian oscillations, N-(furan-2-ylmethyl)-7H-purin-6-amine also affects their expression.

Example 6: N-(furan-2-ylmethyl)-7H-purin-6-amine Modulates the Response of Cells to Other Stimuli Affecting Circadian Rhythm

[0061] Human osteosarcoma U2OS cells were grown in standard DMEM with 10% fetal calf serum. U2OS cells were transduced with lentiviral particles containing the reporter pLV6-Bmal1-Luc (S. Brown, Addgene plasmid # 68833) and selected under Blasticidin. The cell line was clonally expanded and a single monoclonal cell line was used for further experiments. Cells were cultured in a 384-well plate in growth medium to 90+% confluence. Test compounds or vehicle (DMSO) were applied to recording medium with 100 U / ml penicillin, 100 ug / ml streptomycin, 1x GlutaMAX (ThermoFisher, Waltham, MA, USA), 10% fetal calf serum (Sigma) and 0.1 mM Luciferin-EF (Promega Madison, WI, USA). Luminescence was recorded every hour for additional 144 hours in Luminoskan Ascent (ThermoFisher). Circadian rhythm analysis was performed by cosinor analysis.

[0062] The example demonstrates that N-(furan-2-ylmethyl)-7H-purin-6-amine in combination with compounds that extend the circadian period by different mechanisms of action has an additive or even synergistic effect on the length of the circadian period in human cells and can be used to enhance the effect of these compounds. Conversely, depending on the mechanism of action, some compounds (in this case, harmine) shorten the period after the addition of N-furan-2-ylmethyl) -7H-purin-6-amine, so it can be used to negatively modulate the effect of this type of compound.

Example 7: N-(furan-2-ylmethyl)-7H-purin-6-amine Sensitize Cancer Cells to Antineoplastics

[0063] Resazurin is a blue weakly fluorescent compound that is irreversibly reduced into red highly fluorescent resofurin by metabolically active cells and can be therefore used for viability tests. In this assay, the effect of antineoplastics on viability of cancer cell line A549 pretreated with N-(furan-2-ylmethyl)-7H-purin-6-amine (final concentration 100 micromol) was evaluated.

[0064] Human cancer cell line A549 was maintained in standard cultivation medium DMEM with 10% fetal bovine serum. 1000 cells were seeded into the wells of 384-well plate in 30 microliters of RPMI medium. After 24 hours, dexamethasone (final concentration 100 nM) was added in order to synchronize circadian oscillator together with N-(furan-2-ylmethyl)-7H-purin-6-amin (final concentration 100 .Math.M) or DMSO vehicle. After 12 hours, DMSO vehicle or the evaluated antineoplastics (the final concentration 3.3 .Math.M) were added. The compounds were dosed by ECHO system, the DMSO solutions were 1000-times concentrated. After 72 incubation, 1000-times concentrated solution of resazurin in DMSO to a final concentration of 0.0125 mg/ml was added. Fluorescence was measured after 3-hour incubation. Results are shown in FIG. 7. It was demonstrated that pretreatment with N-(furan-2-ylmethyl)-7H-purin-6-amine increases activity of antineoplastics. An equivalent cytotoxic effect can be therefore achieved by an application of lower dose of antineoplastics.

Example 8: N-(furan-2-ylmethyl)-7H-purin-6-amine is not Toxic for Human Non-Cancer Cell Lines in Resazurin Test

[0065] Resazurin is a blue weakly fluorescent compound that is irreversibly reduced into red highly fluorescent resofurin by mitochondria. It is used for viability testing of eukaryotic cells. The effect of the compounds in several concentrations (maximum concentration of 100 microM and 3 three-fold dilutions) on viability of skin fibroblasts BJ and retinal epitelium cells ARPE-19 was evaluated after 72 hour treatment. The cells were maintained in standard cultivation medium DMEM with 10% fetal bovine serum. 5000 cells were seeded into 96-well plates 24 hours prior to the addition of test compound. DMSO vehicle was used as a negative control. After 72 hours, 1000-times concentrated solution of resazurin in DMSO was added to the cells zo the final concentration of 0.0125 mg/ml.

[0066] Fluorescence (ex = 570 nm, em= 610 nm) was measured after 1 hour (ARPE-19) or 3 hours (BJ) of incubation. IC50 values were calculated from dose response curves using drc library for R programming environment.

[0067] Following results were obtained: Resazurin test after 3 day exposure - ARPE-19 IC.sub.10 >100 uM and BJ IC.sub.10> 100 microM. N-(furan-2-ylmethyl)-7H-purin-6-amine has a favorable toxicity profile for human non-cancer cells. This is an important advantage in comparison with many other N6-substituted purines and their biosters many of which are toxic sometimes because of the inhibition of cyclin dependent kinases (Voller et al. 2010 [doi: 10.1016/j.phytochem.2010.04.018], Jorda et al. 2012 [doi: 10.2174/138161212800672804]).

Example 9: N-(furan-2-ylmethyl)-7H-purin-6-amine is not Toxic for Normal Human Cell During a Long-Term Cultivation

[0068] Human fibroblasts BJ were maintained in standard cultivation medium DMEM with 10% fetal bovine serum. The experiment was performed in 24-well plates. 10,000 cells in DMEM medium with 10% fetal bovine serum were seeded into the wells. The test compound was added to a final concentration in the range of 12.5-100 .Math.M after 24 hours. DMSO vehicle was used as a negative control. In order to obtained better idea of test variability, 8 control wells were used (columns A and D). The cultivation medium containing either test compound or DMSO was replaced twice a week. The cells were trypsinized on the 12th day and counted on Coulter Z2 apparatus. The test compound in the evaluated concentration range and exposure time did not have a negative effect on cell growth. The results are shown in FIG. 8.

[0069] N-(furan-2-ylmethyl)-7H-purin-6-amine has a favorable toxicity profile in human noncancer cells. This is an important advantage in comparison with many other N6-substituted purines and their biosters many of which are toxic sometimes because of the inhibition of cyclin dependent kinases (Voller et al. 2010 [doi: 10.1016/j.phytochem.2010.04.018], Jorda et al. 2012 [doi: 10.2174/138161212800672804]).

Example 10: N-(furan-2-ylmethyl)-7H-purin-6-amine has Favorable Pharmacokinetic Parameters

Analytical Chemistry

[0070] Quantification of N-(furan-2-ylmethyl)-7H-purin-6-amine in the samples was performed using RapidFire RF300 system (Agilent Technologies) interfaced with QTRAP 5500 mass spectrometer fitted with an electrospray ionization source (AB Sciex, Concord, Canada) and running in multiple - reaction monitoring mode.

[0071] This system is further referred to as RF-MS. Preparation of the lyophilized samples for the analysis is described in the sections dedicated to the individual methods. Lyophilized samples were dissolved in the mobile phase (95% water, 5% acetonitrile, 0.1% formic acid) with respective internal standards. The dissolved samples were aspired directly from 96-well plates into a 10 .Math.L sample loop and passed through a C4 cartridge (Agilent Technologies) with solvent A (95% water, 0.01% formic acid, 5% acetonitrile) at a flow rate of 1.5 mL/minute for 3 seconds. After the desalting step, the analyte retained on the cartridge was eluted with solvent B (95% acetonitrile, 5% 0.01% formic acid) to the mass spectrometer at a flow rate of 0.4 mL/minute for 7 seconds. Mass spectrometry was carried out using electrospray ionization in the positive ion mode. Daughter ion peaks were identified using a multiple - reaction monitoring protocol.

Evaluation of Stability in Human Plasma

[0072] Test compound (a final concentration of 2 .Math.M) was incubated with human plasma (Transfusion Department, University Hospital Olomouc, Olomouc, Czech Republic) for 0, 15, 30, 60 and 120 min at 37° C. The reactions were stopped by addition of acetonitrile-methanol mixture (2:1). Samples were stored at -80° C. overnight and centrifuged (2811x g, 6 min, 4° C.). Supernatants were lyophilized.

Evaluation of Microsomal Stability

[0073] The reaction mixtures of test compounds (2 .Math.M), human liver microsomes (ThermoFisher Scientific, 0.5 mg/mL), NADPH generating system (NADP.sup.+ - 0.5 mM, isocitrate dehydrogenase - 6 U/mL, isocitric acid - 4 mM, and MgSO.sub.4- 5 mM) in 0.1 mol/L K.sub.3PO.sub.4 buffer. The reactions were stopped by the addition of acetonitrile-methanol mixture (2:1) after 0, 15, 30, and 60 min at 37° C. The samples were centrifuged (2811x g, 6 min, 4° C.) and the supernatants were lyophilized.

[0074] Calculations: The intrinsic clearance was calculated as CL.sub.int = V*(0.693/t.sub.½), where V is the volume of the reaction in .Math.L related to the weight of the microsomal protein in mg per reaction. Elimination half-life was calculated using the equation t.sub.½ = 0.693/k, where k is the slope of linear regression of natural logarithm of percent substrate remaining plotted versus incubation time.

Evaluation of Permeability Through Passive Diffusion - PAMPA

[0075] The parallel artificial membrane permeability assay (PAMPA) was performed using the Millipore MultiScreen filter MultiScreen-IP Durapore 0.45 .Math.m plates and receiver plates (Merck Millipore) according to the manufacturer’s protocol PC040EN00. The test compounds were dissolved in PBS (pH 7.4) to the final concentration of 20 .Math.M and added to the donor wells. The filter membrane was coated with 10% lecithin (Sigma Aldrich) dissolved in dodecane and the acceptor wells were filled with PBS (pH 7.4). The acceptor filter plate was carefully placed on the donor plate. Following 18-hour incubation at the room temperature aliquots of acceptor and donor solutions were removed and lyophilized.

[0076] Calculations: The relative permeability logPe was calculated as logPe = log{Cx-1n(1-drug.sub.A/drug.sub.E)}, where C = (V.sub.A × V.sub.D)/{(V.sub.D+V.sub.A) x A × T}. V.sub.D and V.sub.A are the volumes of the donor and acceptor solutions, respectively, A is the active surface area in cm.sup.2 and T is time of the incubation in seconds. DrugA and drugE is the mass of the compound in the acceptor solution and in the solution in theoretical equilibrium (as if the donor and acceptor were combined), respectively.

Evaluation of Plasma Protein Binding

[0077] The assay is based on the rapid equilibrium dialysis (RED). The RED plate inserts (Thermo Scientific™, Rockford, USA) consist of two chambers separated by a semipermeable membrane. For each compound, 10 .Math.M in 10% human plasma was transferred into the one chamber and the other was filled with PBS buffer (pH 7.4). The equal volumes of the solutions from either compartment were transferred into microtubes after the 4-hour incubation with shaking (250 rpm). Either 10% plasma or PBS buffer (pH 7.4) was added so that all the samples had the same matrix. The reactions were stopped by the addition of acetonitrile-methanol mixture (2:1). The samples were centrifuged (2811x g, 6 min, 4° C.) and the supernatants were lyophilized.

Evaluation of Transport Across Caco-2 and MDR-MDCK Monolayers

[0078] Caco-2 (American Tissue Type Collection) a MDR1-MCDK (Netherlands Cancer Institute) were cultured in DMEM medium with 10% fetal bovine serum. In order to generate cell monolayers for transport studies, the cells were trypsinized and seeded on tissue culture polyester membrane filters (pore size 0.4 .Math.m for Caco-2 and 1 .Math.m for MDR1-MCDK) in 96-well Transwell® plates (Corning, NY, USA). The culture medium was added to both the donor and the acceptor compartments and the cells were allowed to differentiate and form the monolayers. The culture medium was changed every other day.

[0079] Caco-2 and MDR1-MCDK differentiated monolayers were used only if they were intact, which was confirmed by Lucifer Yellow Rejection Assay. Prior to the experiment, the cells were washed twice with Hank’s balanced buffer solution (HBBS) (Gibco, Waltham, USA) and pre-equilibrated with HBSS buffer at pH 7.4 for 1 h. After removing the medium, the cells were treated with 10 .Math.M test compounds in HBSS (pH 7.4) for 1 and 2 h, for MDCK and Caco-2, respectively. Thereafter, the samples were removed from both donor and acceptor compartments and lyophilized. All experiments were done in duplicate.

[0080] Calculations: The apparent permeability coefficient was calculated as P.sub.app = (dQ/dt)/(C.sub.0 x A), where dQ/dt is the rate of permeation of the drug across the cell monolayer, Co is the donor compartment concentration at time t = 0 and A is the area of the cell monolayer. The efflux ratio R was defined as ratio P.sub.BA/P.sub.AB where P.sub.BA and /P.sub.AB represent the apparent permeability of test compound from the basal to apical and apical to basal side of cell monolayer, respectively. The compounds with the efflux ratio of 2 or higher were considered as potential P-gp substrates.

TABLE-US-00001 The results of the panel of in vitro pharmacokinetics tests. Values P.sub.app >20 x10.sup.-6 for Caco-2 and P.sub.app >10x10.sup.-6 for MDR-MDCK indicate good oral and CNS bioavailability, respectively MDR1-MDCK permeability (P.sub.app x10.sup.-6)/ Caco-2 permeability (P.sub.app x10.sup.-6) PAMPA permeability (log Pe) Mikrosomal stability (60 min) [% intact] Plasma stability (120 min) [% intact] 315.31 76 -5.3 90 91

[0081] N-(furan-2-ylmethyl)-7H-purin-6-amine is highly stable in human plasm and after an exposure to human microsomal fraction. The data from the Caco-2 model of gut wall predict that it will be orally available. Exceptionally high permeability in the MDR1-MDCK model of blood-brain barrier predict excellent CNS permeability probably through a transporter. CNS penetration is key for attaining the effects on the central pacemaker.

Example 11: Dry Capsules Containing N-(furan-2-ylmethyl)-7H-purin-6-amine and Melatonin

[0082] 50 capsules, each containing 500 mg of N-(furan-2-ylmethyl)-7H-purin-6-amine and 5 mg melatonin as active ingrediences, are prepared as follows: [0083] Composition: [0084] N-(furan-2-ylmethyl)-7H-purin-6-amine 25 g [0085] Melatonin 250 mg [0086] Talc 100 mg [0087] Magnesium stearate 100 mg [0088] Protocol: Homogenized compounds are passed through a sieve with 0.6 mm openings. An amount of 0.51 g of the mixture is put into a gelatin capsule.