Ionic-liquid-based formulations for the prevention or treatment of neurological diseases
12514845 · 2026-01-06
Assignee
Inventors
- Mara Guadalupe FREIRE MARTINS (Aveiro, PT)
- Ana Catarina ALMEIDA SOUSA (Aveiro, PT)
- Mukesh SHARMA (Aveiro, PT)
- João Manuel COSTA ARAÚJO PEREIRA COUTINHO (Ílhavo, PT)
- Ana Clara BRAZ CRISTÓVÃO (Covilhã, PT)
- Dina Batista Pereira (Covilhã, PT)
Cpc classification
C07D233/96
CHEMISTRY; METALLURGY
A61K31/4166
HUMAN NECESSITIES
C07D239/20
CHEMISTRY; METALLURGY
International classification
C07D239/20
CHEMISTRY; METALLURGY
A61K31/4166
HUMAN NECESSITIES
C07D233/54
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to an ionic liquid (IL)-based formulation comprising inhibitors of NADPH oxidases enzymes (Nox's), preferably isoforms 1 and 4, in particular the specific inhibitor 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin, for the treatment, therapy or prevention of neurological diseases, in particular Parkinson's diseases.
Claims
1. An ionic liquid comprising an anion with formula ##STR00003## wherein R is an alkyl group, or a cycloalkyl group; and a cation selected from the list consisting of cholinium, tetralkylammonium, tetraalkylphosphonium, or 1-alkyl-3-methylimidazolium cation families.
2. The ionic liquid according to claim 1 wherein the molar ratio of the anion and the cation ranges from 1:2 to 2:1 mol:mol.
3. The ionic liquid according to claim 1 wherein R is a cycloalkyl group from C3-C7.
4. The ionic liquid according to claim 1 wherein R is an unsubstituted cycloalkyl group from C3-C7.
5. The ionic liquid according to claim 1 wherein R is a cyclohexyl group.
6. The ionic liquid according to claim 1 wherein the cation is cholinium.
7. The ionic liquid according to claim 1, wherein the anion is 3-substituted 5-benzylidene-1-methyl-2-thiohydantoin or 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin.
8. The ionic liquid according to claim 1 for use in medicine or as medicament.
9. The ionic liquid according to claim 1 for use in slowing down the progression of Parkinson's Disease.
10. The ionic liquid according to claim 1 for use to prevent motor dysfunction in Parkinson's disease.
11. The ionic liquid according to claim 1 for use in the prevention or treatment of amyotrophic lateral sclerosis.
12. A pharmaceutical composition comprising a therapeutically effective amount of the ionic liquid according to claim 1 and a pharmaceutically acceptable carrier.
13. The pharmaceutical composition according to claim 12, wherein the therapeutically effective amount ranges from 0.005 mM to10 mM.
14. The pharmaceutical composition according to claim 12 wherein the composition is an injectable form, an intranasal form, an intrathecal form or a brain intraventricular form.
15. The pharmaceutical composition according to claim 12 for administration of a daily dose to a person with a disease or disorder of the central nervous system.
16. The pharmaceutical composition according to claim 15 wherein the dosage amount is less than 1000 mg/day.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
(1) The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.
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DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
(25) The present disclosure relates to the development of an IL-based formulation of a specific inhibitor of the NADPH oxidases enzymes (Nox's), particularly Nox1 and Nox4 for therapeutic application in Parkinson's disease (PD) or other neurological diseases. The strategy used consists on the conversion of Nox1(4)-specific inhibitor 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) into a formulation comprising cholinium 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate ([Chol].sub.2[N1(4)inh]+N1(4)inh, 1:1).
(26) The present disclosure refers to the conversion of the Nox1(4) specific inhibitor into an IL-based formulation, [Chol].sub.2[N1(4)inh+N1(4)inh, to be used in the context of PD and other neurological diseases and comprises the following steps: (1) Conversion of the specific inhibitor N1(4)inh into an IL-based formulation, namely cholinium 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate ([Chol].sub.2[N1(4)inh]+N1(4)inh, 1:1, mol:mol), comprehending the following procedure: a) Selection of the IL cation source and selection of the anion source; b) Synthesis of [Chol].sub.2[N1(4)inh] by metathesis reaction using a cholinium salt as the cation source and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) as the anion source; c) Characterization of [Chol].sub.2[N1(4)inh]+N1(4)inh and N1(4)inh purity by Nuclear Magnetic Resonance (.sup.1H NMR and .sup.13C NMR); d) Determination of [Chol].sub.2[N1(4)inh]+N1(4)inh solubility in water and PBS; e) Characterization of [Chol].sub.2[N1(4)inh]+N1(4)inh thermal stability by thermogravimetric analysis (TGA). (2) Evaluation of the biological effect of [Chol].sub.2[N1(4)inh]+N1(4)inh in dopaminergic neurons in vitro, by means of: a) Evaluation of [Chol].sub.2[N1(4)inh]+N1(4)inh cytotoxicity in immortalized rat dopaminergic neural cell line (N27); b) Evaluation of the reformulation's biological effect in the context of a therapy for PD, by assessing its dopaminergic neuroprotective capacity in PD in vitro models.
(27) Unless otherwise stated, the [Chol].sub.2[N1(4)inh]+N1(4)inh mixture used in the examples of the present disclosure were in the proportion 1:1 (mol:mol) (Chol]2[N1(4)inh]:N1(4)inh).
(28) In the present disclosure, [Chol].sub.2[N1(4)inh]+N1(4)inh can also be called [Chol].sub.2[N1(4)inh]+Nox1(4)inh or [Chol].sub.2[N1(4)inh]+Nox1(4).
(29) Conversion of the specific inhibitor N1(4)inh into an IL-based formulation:
(30) In an embodiment, the synthesis of the IL-based formulation starts with the selection of the components, i.e., the cation source and the anion source.
(31) In an embodiment, the cation source should belong to the class of cholinium salts, preferably cholinium bicarbonate.
(32) In an embodiment, the anion source should belong to the group of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoin based compounds, preferably it should be 3-cyclohexil-5-(2,4-dihydroxybenzylidene)-1-methil-2-thiohidantoin (N1(4)inh).
(33) In an embodiment, the IL-based formulation ([Chol].sub.2[N1(4)inh]+N1(4)inh) was synthesized by metathesis reaction with 1:1 mole ratio of cholinium bicarbonate (80% (m/v) in water) and N1(4)inh solution prepared in a minimum amount of absolute ethanol.
(34) In an embodiment, the N1(4)inh was mixed by drop-wise addition into cholinium bicarbonate solution under dark and cold conditions (using an ice-bath at 5 C.) and with continuous stirring.
(35) In an embodiment, the reaction mixture was further kept at 5 C. under continuous stirring for 2 h.
(36) In an embodiment, the excess solvent and water were removed under continuous flow of nitrogen gas until complete drying (2-3 h of nitrogen gas flow with the IL containing vial maintained in an ice-cold water bath (10 C.)).
(37) In an embodiment, the mixture comprising the synthesized [Chol].sub.2[N1(4)inh] and N1(4)inh was finally collected from the ice-cold water bath and stored under dried, cool and dark conditions.
(38) In an embodiment, the purity of the [Chol].sub.2[N1(4)inh] and N1(4)inh was evaluated by means of .sup.1H NMR and .sup.13C NMR as disclosed in
(39) In an embodiment, the obtained NMR spectra allowed to confirm that the [Chol].sub.2[N1(4)inh]+N1(4)inh was successfully synthetized and that it is pure.
(40) In an embodiment, the [Chol].sub.2[N1(4)inh]+N1(4)inh solubility in water and phosphate buffered saline (PBS) solution were determined.
(41) In an embodiment, the solutes N1(4)inh and mixture comprising [Chol]2[N1(4)inh]+N1(4)inh were added in excess to a fixed volume (500 L) of water and PBS solution. These mixtures were incubated at 37 C., under constant agitation 1150 rpm and for a minimum of 72 h, using an Eppendorf Thermomixer Comfort equipment. Throughout this process, solute was added to the mixture, whenever necessary, i.e., until the solution saturation. All samples were filtered using syringe filters (0.45 mm) to remove possible suspended solid particles.
(42) In an embodiment, the quantification of N1(4)inh and [Chol].sub.2[N1(4)inh] was carried by UV-spectroscopy, using a UV-spectrophotometry (SYNERGY|HT microplate reader, BioTek) at a wavelength of 416 nm and 480 nm, respectively. The interference of the PBS with the quantification method was also ascertained and blank control samples were always used.
(43) In an embodiment, the solubility of the mixture [Chol].sub.2[N1(4)inh]+N1(4)inh in water was 0.4050.053 mg/mL, which is 36 higher than N1(4)inh alone (0.0110.002 mg/mL). An increase in the IL-based formulation solubility was also noticeable in PBS, with a 21 fold increase (from 0.0080.001 mg/ml to 0.1680.002 mg/mL) (
(44) These results clearly demonstrate that the reformulation of N1(4)inh into [Chol].sub.2[N1(4)inh]+N1(4)inh allows to overcome one of the most important problems associated with Nox's specific inhibitors, i.e. low solubility, reinforcing the potential of this new reformulation to be used in the context of PD and other neurological diseases.
(45) In an embodiment, the decomposition temperature of [Chol].sub.2[N1(4)inh]+N1(4)inh was evaluated by thermogravimetric analysis (TGA) and the results disclose that the mixture is thermally stable up to 200 C., as disclosed in
(46) (2) Evaluation of the Biological Effect of [Chol].sub.2[N1(4)Inh]+N1(4)Inh in Dopaminergic Neurons In Vitro.
(47) In an embodiment, the cytotoxicity of [Chol].sub.2[N1(4)inh]+N1(4)inh in Immortalized rat dopaminergic neural cell line (N27) was evaluated.
(48) The cells were exposed to 20 and 30 M of [Chol].sub.2[N1(4)inh]+N1(4)inh, or to 20 M of cholinium chloride. Cholinium chloride was solubilized in saline and [Chol].sub.2[N1(4)inh]+N1(4)inh was solubilized in PBS 1 (Phosphate-saline buffer).
(49) The cells were kept for 24 h under the stimulus and cell viability was evaluated using the CCK-8 kit (Cell Counting Kit-CCK-8; Dojindo Molecular Technologies). [Chol].sub.2[N1(4)inh]+N1(4)inh did not exert a toxic effect on N27 dopaminergic cells when exposed to 20 or 30 M of the mixture, since no statistically significant differences were observed between cell viability values in cultures exposed to [Chol].sub.2[N1(4)inh]+N1(4)inh (20 M {circle around (2)} or 30 M {circle around (3)}
(50) In an embodiment, the dopaminergic neuroprotective capacity of [Chol].sub.2[N1(4)inh]+N1(4)inh was evaluated in PD in vitro models.
(51) The neuroprotective evaluations were performed using 20 M [Chol].sub.2[N1(4)inh]+N1(4)inh or 20 M cholinium chloride in order to exclude the possibility that the neuroprotection was due to the cholinium salt and not the IL-based formulation.
(52) The neurotoxins 6-hydroxydopamine (6OHDA) and 1-methyl-4-phenylpyridinium (MPP+) were individually added 2 h 30 after [Chol].sub.2[N1(4)inh]+N1(4)inh or cholinium chloride, and the stimulus maintained for 24 h, followed by the evaluation of cell viability using the CCK-8 kit.
(53) The working concentration of [Chol].sub.2[N1(4)inh]+N1(4)inh chosen was 20 M, based on results previously obtained with the non-reformulated inhibitor (N1(4)inh).
(54) In an embodiment, when comparing the condition of cells treated with 6OHDA, it is possible to observe a reduction of 45% in the viability of dopaminergic neurons, which is statistically different from the control condition (CTR {circle around (1)}) (
(55) In an embodiment, the results presented in
(56) In an embodiment, [Chol].sub.2[N1(4)inh]+N1(4)inh also plays a neuroprotective role in the prevention of MPP+ dopaminergic neurotoxicity, since it significantly prevented the 45% loss of N27 cells viability exposed to this neurotoxin (
(57) Then, the biological effect of [Chol].sub.2[N1(4)inh]+N1(4)inh was evaluated in vivo. All animal experiments were performed in accordance with institutional animal house, national and European Community regulations (86/609/ECC; 2010/63/EU).
(58) First the toxicity of [Chol].sub.2[N1(4)inh]+N1(4)inh (1:1) was evaluated in healthy mice, using two different routes of administration 1) 7 days brain intraventricular infusion of 0.2 mg/kg/day of [Chol]2[N1(4)inh]+N1(4)inh; 2) twice a day intranasal administration of 0.16; 0.08, 0.04 or 0.02 mg/kg/day of [Chol]2[N1(4)inh]+N1(4)inh during 14 days.
(59) In an embodiment, male C57BL/6 mice with 8-12 week-old were housed in a temperature/humidity-controlled environment under 12 h light/dark cycle with free access to water and food. 1) Mice were anesthetized with an intraperitoneal (i.p.) injection of ketamine (0.67 mL/kg of mouse weight) and xylazine (0.33 ml/kg of mouse weight) in saline, before they were placed in the digital stereotaxic frame (51900 Stoelting). Once the mouse skull was exposed and, using a digital coordinates system, the infusion sites were determined setting the zero at the bregma point. The delivery of [Chol].sub.2[N1(4)inh]+N1(4)inh (1:1) at a dose of 0.2 mg/kg/day, was performed by its direct intraventricular infusion during 7 days, performed using Alzet osmotic pump (ref. 1007D) connected to an Alzet catheter (Ref. Brain infusion Kit 3), in the coordinates medial-lateral (ML): 1.1 mm; anterior-posterior (AP): 0.5 mm and dorsoventral (DV): 2.5. On day 7, animals were anesthetized with an intraperitoneal (i.p.) injection of ketamine (0.67 mL/Kg of mouse weight) and xylazine (0.33 mL/Kg of mouse weight) in saline, and transcardiacly perfused first with saline, and after with buffered formaline (experiments end-point). The brains were frozen by liquid nitrogen and kept at 80 C. Subsequently, brains were incorporated in a gel of ideal cutting temperature (OCT) to be sectioned in a cryostat (Leica CM 3050S, Leica Microsystems). Coronal sections with a thickness of 30 m from the front pole to the end of the midbrain were collected at 20 C. Sections corresponding to the ST and SN of each animal were collected and stored sequentially in free-floating 24-well plate compartments (Orange Scientific), containing a cryopreservation solution of 30% glycerol (v/v) and 30% (v/v) ethylene glycol in phosphate buffer (PB). The plates were kept at 20 C. properly identified for later use in immunohistochemistry to evaluate the number of dopaminergic neurons in the susbtantia nigra (SN) by stereological count of the neurons immune-positive for the specific cellular marker tyrosine hydroxylase (TH). The results presented in
(60) Second the toxicity of [Chol]2[N1(4)inh]+N1(4)inh was evaluated in healthy rat using two different routes of administration 1) 30 days of brain intraventricular infusion of 0.007 mg/kg/day of [Chol]2[N1(4)inh]+N1(4)inh and 2) daily intranasal administration of 0.062 mg/kg/day of [Chol]2[N1(4)inh]+N1(4)inh during 30 days.
(61) In an embodiment, male wistar rats with 8-12 week-old were housed in a temperature/humidity-controlled environment under 12 h light/dark cycle with free access to water and food. 1) Rats were anesthetized with an intraperitoneal (i.p.) injection of ketamine (90 mg/kg) and xylazine (10 mg/kg) in saline, before they were placed in the digital stereotaxic frame (51900 Stoelting). Once the rat skull was exposed and, using a digital coordinates system, the infusion sites were determined setting the zero at the bregma point. The delivery of [Chol]2[N1(4)inh]+N1(4)inh (1:1) at a dose of 0.007 mg/kg/day, was performed by its direct intraventricular infusion during 30 days, performed using Alzet osmotic pump (ref. 2004) connected to an Alzet catheter (Ref. Brain infusion Kit 2), in the coordinates medial-lateral (ML): 1.5 mm; anterior-posterior (AP): 1.0 mm and dorsoventral (DV): 4.0. Toxicity was evaluated by analysing animal weight at day 0 and day 30 and their behavior motor performance at day 21. Motor coordination, balance and grip strength were analyzed using a rat rotarod apparatus model 47700 (Ugo Basile, Comerio, Italy). All rats were pretrained on the rotarod to learn and reach a consistent performance. The training was performed in 2 consecutive days, on each day 4 test trials were assessed, each lasting 5 min. Rats were trained at 12 RPM (fixed mode) first at 24 RPM (fixed mode) for 2 test trials and then at 4-40 RPM (accelerated mode) for other 2 test trials. The animals were allowed to rest approximately 30 min between each trial. The rotarod test was performed under an accelerating protocol (4-40 RPM) for 5 min, and the latency to fall was recorded using a specific software. The trial stopped when the mouse fell (activating a switch that automatically stopped the timer) or when 5 min had been completed. Each animal was given four independent trials with approximately 30 min inter-trial period to reduce stress and fatigue. The results presented in
(62) In an embodiment the overall results obtained related with the in vivo toxicity of [Chol]2[N1(4)inh]+N1(4)inh, prove that the IL-based formulation is not toxic either when administrated via brain intraventricular route nor via intranasal route.
(63) Afterward, the neuroprotective capacity of [Chol].sub.2[N1(4)inh]+N1(4)inh was evaluated in two in vivo model of PD, one induced by the intracerebral injection of 6OHDA and the other induced by the exposure to a chronic low dose of paraquat (PQ).
(64) In an embodiment, the biological neuroprotective effect of [Chol].sub.2[N1(4)inh]+N1(4)inh+N1(4)inh when administrated via brain intraventricular infusion route was evaluated in an mouse model for PD induced by the intracerebral injection of 6OHDA, and the number of dopaminergic neurons in the substantia nigra counted.
(65) In an embodiment, Male C57BL/6 mice were anesthetized and placed in the digital stereotaxic frame. Once the mouse skull was exposed, and using a digital coordinates system, injection and infusion sites were determined setting the zero at the bregma point. Hereupon, an injection of 6-OHDA (10 g/2 L of ascorbic acid 0.1% v/v) was performed in the right striatum (ST) of each animal using the coordinates: medial-lateral (ML): 2 mm; anterior-posterior (AP): 0.6 mm; dorsoventral (DV): 3.0 mm, using a Hamilton syringe at speed of 0.2 L/min. The delivery of 0.2 mg/kg/day of [Chol].sub.2[N1(4)inh]+N1(4)inh+N1(4)inh was performed as describe above. On day 7, animals were anesthetized with an intraperitoneal (i.p.) injection of ketamine (0.67 mL/kg of mouse weight) and xylazine (0.33 mL/kg of mouse weight) in saline, and transcardiacly perfused first with saline, and after with buffered formaline (experiments end-point). The brains were frozen by liquid nitrogen and kept at 80 C. Subsequently, brains were incorporated in a gel of ideal cutting temperature (OCT) to be sectioned in a cryostat (Leica CM 3050S, Leica Microsystems). Coronal sections with a thickness of 30 m from the front pole to the end of the midbrain were collected at 20 C. Sections corresponding to the ST and SN of each animal were collected and stored sequentially in free-floating 24-well plate compartments (Orange Scientific), containing a cryopreservation solution of 30% (v/v) glycerol and 30% (v/v) ethylene glycol in phosphate buffer (PB). The plates were kept at 20 C. properly identified for later use in immunohistochemistry to evaluate the number of dopaminergic neurons in the SN by stereological count of TH+ neurons. Two experimental groups were used: 1) mice injected with 6-OHDA in the right striatum and saline (0.9% m/v NaCl) in the left striatum and 2) mice injected with 6-OHDA in the right striatum, saline in the left and [Chol].sub.2[N1(4)inh]+N1(4)inh in the lateral ventricle. The hemispheres injected with saline were considered controls for 6OHDA in group 1, and [Chol].sub.2[N1(4)inh]+N1(4)inh only in group 2. In group 2, the intraventricular and stereotaxic procedures were performed in the same day. As depicted in
(66) In an embodiment, the biological neuroprotective effect of [Chol].sub.2[N1(4)inh]+N1(4)inh+N1(4)inh when administrated via brain intraventricular infusion route was evaluated in a rat model for PD induced by the exposure to a low chronic dose of PQ, through the analysis of the progression of behavior dysfunction. In this model, the animals are exposed to the toxin during 30 days (4 weeks) and then keeped alive during more 30 days to allow the pathology to progress (Total 8 weeks). The administration of [Chol].sub.2[N1(4)inh]+N1(4)inh started after the first 30 exposure to PQ, via brain intraventricle or intranasal route and lasted the second 30 days. Animals were euthanized at week 8.
(67) In an embodiment, male wistar rats 8-12 week-old were housed in a temperature/humidity-controlled environment under 12 h light/dark cycle with free access to water and food and subjected to subcutaneous chronic administration of PQ, that was carried out using osmotic minipumps (Alzet Durect, Cupertino, CA) at a dose of 2.5 mg/kg/day with a fluid delivery rate of 0.25 L/hr for a period of four weeks (Alzet model 2004, large pumps). Control groups were implanted with minipump filled with sterile saline, the vehicle used to dissolve PQ. The pumps were implanted subcutaneously on the back, slightly posterior to the scapulae (shoulder blades), for that rats were anesthetized with intraperitoneal (i.p.) injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Four weeks after been exposed to PQ, the intraventricular infusion of [Chol].sub.2[N1(4)inh]+N1(4)inh+N1(4)inh started and lasted during 4 more weeks. The experimental paradigm involved a total of 8 weeks, the first 4 weeks are for PQ exposure and the last 4 weeks are for IL-based formulation administration. For that rats were anesthetized and placed in the digital stereotaxic frame (51900 Stoelting). Once the rat skull was exposed and, using a digital coordinates system, the infusion sites were determined setting the zero at the bregma point. The delivery of [Chol]2[N1(4)inh]+N1(4)inh (1:1) at a dose of 0.007 mg/kg/day, was performed by its direct intraventricular infusion during four more weeks (30 days), performed using Alzet osmotic pump (ref. 2004) connected to an Alzet catheter (Ref. Brain infusion Kit 2), in the coordinates medial-lateral (ML): 1.5 mm; anterior-posterior (AP): 1.0 mm and dorsoventral (DV): 4.0. To evaluate the neuroprotective function of the IL-based formulation when infused in brain ventricle, behavior function was evaluated. Motor coordination, balance and grip strength were analyzed using a rat rotarod apparatus model 47700 (Ugo Basile, Comerio, Italy). All rats were pretrained on the rotarod to learn and reach a consistent performance. The training was performed in 2 consecutive days, on each day 4 test trials were assessed, each lasting 5 min. Rats were trained at 12 RPM (fixed mode) first at 24 RPM (fixed mode) for 2 test trials and then at 4-40 RPM (accelerated mode) for other 2 test trials. The animals were allowed to rest approximately 30 min between each trial. The rotarod test was performed under an accelerating protocol (4-40 RPM) for 5 min, and the latency to fall was recorded using a specific software. The trial stopped when the mouse fell (activating a switch that automatically stopped the timer) or when 5 min had been completed. Each animal was given four independent trials with approximately 30 min inter-trial period to reduce stress and fatigue. The open-field test was used to measure exploratory behaviors and general activity. Animals were transported to the testing room and left undisturbed for 30 minutes to 1 hour before the test. All around the arena is a beam break system, which is interrupted with the animal movement inside the arena. This information is processed in a specific software allowing to analyze several parameters such as distance, speed, rears and enters in the center. Each animal was placed in the center of the rectangular arena and allowed to explore freely for 10 min. The timer was started at the exact same time as the animal was placed in the arena. The operator has leaved the room. Between each animal, the arena was wiped with 70% (v/v) ethanol, and the next animal was placed. All behavior test were performed at week 8. The results presented show that brain intravetricle administration of [Chol].sub.2[N1(4)inh]+N1(4)inh significantly prevented the motor dysfunction (
(68) As depicted in
(69) As for the rats distance travelled and speed results depicted in
(70) In an embodiment, the biological neuroprotective effect of [Chol].sub.2[N1(4)inh]+N1(4)inh+N1(4)inh when administrated via intranasal route was evaluated in rat model for PD induced by the exposure to a low chronic dose of PQ and through the analysis of the progression of behavior dysfunction. In this model, the animals are exposed to the toxin during 30 days (4 weeks) and then keeped alive during more 30 days to allow the pathology to progress (Total 8 weeks). The administration of [Chol].sub.2[N1(4)inh]+N1(4)inh started after the first 30 exposure to PQ, via brain intraventricle or intranasal route and lasted the second 30 days. Animals were euthanized at week 8.
(71) In an embodiment, male wistar rats 8-12 week-old subjected to subcutaneous chronic administration of PQ, that was carried out using osmotic minipumps (Alzet Durect, Cupertino, CA) at a dose of 2.5 mg/kg/day with a fluid delivery rate of 0.25 L/hr for a period of four weeks (Alzet model 2004, large pumps). Control groups were implanted with minipump filled with sterile saline, the vehicle used to dissolve PQ. The pumps were implanted subcutaneously on the back, slightly posterior to the scapulae (shoulder blades), under anesthesia induced by ketamine (90 mg/kg) and xylazine (10 mg/kg). Four weeks after been exposed to PQ, the intranasal delivery of [Chol].sub.2[N1(4)inh]+N1(4)inh has started and the administration was performed daily during 4 more weeks. The experimental paradigm involve a total of 8 weeks, the first 4 weeks are for PQ exposure and the last 4 weeks are for IL-based formulation administration. General anesthesia was induced by inhalation of 3-4% (v/v) isoflurane for 4 minutes, to enable the intranasal administration of 0.062 mg/kg/day of [Chol]2[N1(4)inh]+N1(4)inh in rats once a day during 4 additional weeks (30 days). Inline, rats were placed lying face up with elevated snout, and were instilled using a polyurethane catheter (Introcan Safety; 24G; 0.719 mm) attached to a 50 l microliter syringe (Hamilton, USA) with 40 L of the [Chol]2[N1(4)inh]+N1(4)inh per 260 g of rat's body weight. The catheter was inserted about 1.7 mm deep into the right and left nostrils, enabling the delivery of the formulation towards the roof of the nasal cavity. Animals were kept in the above-mentioned position until they woke up, to prevent the IL-based formulation to enter the respiratory tract and stay longer in the nasal cavity. The following experimental groups were used: 1) rats exposed to salaine (control group); 2) rats exposed to PQ and 3) rats exposed to PQ and to [Chol].sub.2[N1(4)inh]+N1(4)inh. To evaluate the neuroprotective function of the IL-based formulation when administrated via intranasal route, behavior function was evaluated. Motor coordination, balance and grip strength were analyzed using a rat rotarod apparatus model 47700 (Ugo Basile, Comerio, Italy). All rats were pretrained on the rotarod to learn and reach a consistent performance. The training was performed in 2 consecutive days, on each day 4 test trials were assessed, each lasting 5 min. Rats were trained at 12 RPM (fixed mode) first at 24 RPM (fixed mode) for 2 test trials and then at 4-40 RPM (accelerated mode) for other 2 test trials. The animals were allowed to rest approximately 30 min between each trial. The rotarod test was performed under an accelerating protocol (4-40 RPM) for 5 min, and the latency to fall was recorded using a specific software. The trial stopped when the mouse fell (activating a switch that automatically stopped the timer) or when 5 min had been completed. Each animal was given four independent trials with approximately 30 min inter-trial period to reduce stress and fatigue. Behavior tests were performed at week 8. The results show that the intranasal administration of [Chol].sub.2[N1(4)inh]+N1(4)inh significantly prevented the motor dysfunction (
(72) As depicted in
(73) In an embodiment, these results prove that [Chol].sub.2[N1(4)inh]+N1(4)inh is not toxic, unlike its insoluble precursor, exerting, however, the same neuroprotective efficacy exerted by N1(4)inh alone in the context of PD.
(74) In an embodiment, these results prove that [Chol].sub.2[N1(4)inh]+N1(4)inh reduces the progression of motor dysfunction in PD context.
(75) These results clearly demonstrate that the present disclosure allows us to transform a toxic molecule into a non-toxic mixture. The reformulation did not interfere with the effectiveness of the precursors, radically transforming their potential applicability as a differentiating therapeutic approach, which allows reduce/halt the progression of the pathological condition associated with PD and as such reducing the speed of increase in the degree of disability over time.
(76) The present disclosure allows the current available therapies that modulate the symptoms to have an effect for longer time and more constantly, greatly increasing the quality of life of patients and their families, as well as reducing the negative socio-economic impact that the disease has.
(77) Examples: In the central nervous system (CNS), oxidative stress is one of the main contributors to the development of diseases and aging. Recent studies have revealed that several isoforms of NADPH oxidase (Nox) enzymes, whose sole function is to produce reactive oxygen species (ROS), are also in the CNS. There, they play a key role in regulating ROS-dependent cellular mechanisms, but also, when at high levels, lead to cell dysfunction and death, accelerating the aging process characteristic of the pathological processes of neurodegenerative diseases.
(78) Previous studies have demonstrated the importance of Nox's mediated oxidative stress in inducing neuronal loss in various neurological disorders, as is the case of PD, amyotrophic lateral sclerosis (ALS) and stroke. Those studies strengthen the importance of developing specific inhibitors for these enzymes, and the goal of several research laboratories, as well as pharmaceutical companies, such as GenKyoTex, a Swiss company, which is dedicated exclusively to the development of Nox inhibitors for future therapeutic application. For example, the synthesis of an inhibitor that specifically inhibits Nox's isoforms 1 and 4 (Bae et al., Synthesis and biological evaluation of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins as potent NADPH oxidase (NOX) inhibitors. Bioorg Med Chem 2016; 24:4144-4151) has been recently reported, but which is only soluble in a solvent containing Triton-X/Ethanol/PBS, which we found to be a vehicle that induces toxicity in dopaminergic neurons. The present disclosure put together the technology of ionic liquids and the inhibition of Nox's, to develop a new therapeutic strategy to target specific deleterious ROS productions involved in the pathological process of neurodegeneration. The following are three examples of the applicability of the invention:
Example 1: Parkinson's Disease (PD)
(79) In an embodiment, PD is a chronic neurodegenerative disorder that affects more than 6.1 million people and its efficacy tends to decrease as the disease progresses to more debilitating stages, reducing the therapeutic options. According to the 2018 and 2020 reports on PD (The Parkinson's Disease Market: Pipeline Review, Developer Landscape and Competitive Insights, 2018, pp. Report ID: 4586296. Global Parkinson's Disease Market and Competitive Landscape, 2020, pp. Report ID: 5023386), the absence of preventive therapies to halt the progression of PD remains one of the main unmet needs in this area. In view of this, a paradigm shift towards the search/development of progression preventive therapies for the disease is occurring. In fact, 53% of drugs under development by pharmaceutical companies are disease-modifying agents that aim to prevent the progression of PD, and only 32% are symptomatic. The pathogenesis of PD is highly influenced by oxidative stress. Nox1-ROS generation plays a crucial role in the process of dopaminergic cell death and alpha-synucleinopathy that occurs in this disease and its inhibition in animal models prevents disease progression as described in the documents (Cristovao et al., The role of NADPH oxidase 1-derived reactive oxygen species in paraquat-mediated dopaminergic cell death. Antioxidants & redox signaling 2009; 11:9: 2105-18. Choi et al., NADPH Oxidase 1-Mediated Oxidative Stress Leads to Dopamine Neuron Death in Parkinson's Disease. Antioxidants & redox signaling 2012; 16 (10): 1033-45. Cristovao et al., NADPH oxidase 1 mediates -synucleinopathy in Parkinson's disease. Journal of Neurosciences 2012; 32 (42): 14465-77). Furthermore, Nox4 has a crucial involvement in PD dementia, as demonstrated in the document The Role of NOX4 in Parkinson's Disease with Dementia (Choi et al. International Journal of Molecular Sciences 2019; 20 (3): 696) showing that increased expression of Nox4 in the hippocampal dentate gyrus in PD context induces A expression and oligomer A11 production, and thereby reduces cognitive function. These documents validated Nox1 and Nox4 as important targets for the development of new therapeutic approaches to the disease. In this context, the present disclosure is a benefit for its possible application to the reformulation of Nox1-Nox4-specific inhibitors, which are currently available, but with poor solubility and so low pharmacological applicability. Increasing they solubility, makes them more bioavailable and so with higher therapeutic efficacy for PD.
Example 2: Stroke
(80) In an embodiment, neurovascular diseases are the leading cause of death in the world. In terms of costs, stroke alone totals 64.1 billion euros (Europe) and 44.2 billion euros (United States of America) of expenses per year. Stroke occurs because of suppressing blood flow to the brain, caused by ischemia or hemorrhage. While approximately 20% of patients die within the first month after a stroke, survivors often develop severe neurological dysfunctions and chronic disabilities, which imposes a significant socioeconomic burden. Current therapies only affect a small number of patients and can cause significant side effects.
(81) The administration of anticoagulant drugs, such as aspirin, has a limited preventive power, while thrombolytic therapies, such as the recombinant tissue plasminogenic activator (rtPA), have a narrow therapeutic window and may induce cerebral hemorrhage, edema and ischaemic cell death as described by Suzuki et al. (Novel situations of endothelial injury in strokemechanisms of stroke and strategy of drug development: intracranial bleeding associated with the treatment of ischemic stroke: thrombolytic treatment of ischemia-affected endothelial cells with tissue-type plasminogen activator. Journal of pharmacological sciences 2011, 116, 25-29). The beneficial effects of surgical procedures, such as angioplasty, are still unclear, highlighting the importance and need to develop more efficient and safe therapies to treat these patients.
(82) The document Oxidative stress and pathophysiology of ischemic stroke: novel therapeutic opportunities (CNS & neurological disorders drug targets 2013, 12, 698-714), demonstrates that increases in oxidative stress levels are associated with brain damage that occur after an ischaemic stroke. Furthermore, the document Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system (Circulation research 2012, 110 (10), 1364-1390), reveals that different isoforms of Nox's, namely isoforms 1, 2, 4 and 5, are involved. In stroke, ROS derived from Nox's may have protective or deleterious function, depending on the isoform involved as proposed (Kleikers et al. NADPH oxidases as a source of oxidative stress and molecular target in ischemia/reperfusion injury. Journal of molecular medicine 2012, 90 (12), 1391-1406. Gray et al. Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling. Arteriosclerosis, thrombosis, and vascular biology 2016 36 (2), 295-307). Considering this and making the specific inhibition of the isoform that participates in the pathological process associated with stroke, the development of antioxidant therapies for this disease, by inhibition of Nox, presents itself as having a great therapeutic potential to deal with this pathology. Thus, there is another potential application for the present disclosure.
Example 3: Amyotrophic Lateral Sclerosis (ALS)
(83) Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons and subsequent activation of glial cells leading to the development of muscle weakness and disability, and eventually fatal respiratory and cardiac deficits, between 3 to 5 years after its diagnosis. Despite the intensive research done so far, there is only one drug that has been approved for the treatment of ALS (riluzole), and its effect on survival is modest (de Jongh A D et al. Evidence for a multimodal effect of riluzole in patients with ALS? Journal of Neurology, Neurosurgery & Psychiatry 2019; 90:1183-1184). The pathological mechanisms underlying the development of this pathology are still largely unknown; however, the contribution of oxidative stress is once again a factor that promotes the development of the disease, and the participation of Nox's in this process has also been documented (Harraz et al. SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model. The Journal of clinical investigation 2008, 118 (2), 659-670).
(84) Previous studies investigating the role of Nox's in ALS patients found that those with lower isoform 2 activity in peripheral blood showed significant increases in survival time (Marrali et al. NADPH oxidase (NOX2) activity is a modifier of survival in ALS. Journal of neurology 2014, 261 (11), 2178-2183). The same was observed by Marden et al. (Redox modifier genes in ALS in mice. The Journal of clinical investigation 2007, 117 (10), 2913-2919), in mice with ALS. However, knockouts for Nox's isoforms 1 and 2 showed to increase survival and delay in the onset of the development of the disease. On the other hand, treatment with the broad inhibitor apocynin (Apo) in an in vitro model of ALS improved the survival of motor neurons when co-cultured with astrocytes carrying a mutation associated with the development of the disease (Marrali et al. NADPH oxidase (NOX2) activity is a modifier of survival in ALS. Journal of neurology 2014, 261 (11), 2178-2183), further reinforcing that inhibition of Nox's may have a neuroprotective role in ALS. The pharmacological inhibition of Nox's also showed benefits in animal models of ALS. Increasing survival time by almost 50%, as well as leading to an increased number of motor neurons in the spinal cord (Harraz et al. SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model. The Journal of clinical investigation 2008, 118 (2), 659-670). In this case, the potential application of our disclosure becomes even stronger, since the reformulations of Nox's inhibitors can lead to an increase in its effectiveness and expand the neuroprotective effect already described so far with Apo.
(85) Table 1 depicts the samples used in the assays of the present disclosure.
(86) TABLE-US-00001 TABLE 1 identification of the samples Sample 1 Control cells Sample 2 Cells exposed to Cholinium chloride (20 M) Sample 3 Cells exposed [Chol].sub.2[N1(4)inh] + N1(4)inh(20 M) Sample 4 Cells exposed to MPP+ (10 M) Sample 4 Cells exposed to MPP+ (10 M) and [Chol].sub.2[N1(4)inh] + N1(4)inh (20 M) Sample 6 Cells exposed to MPP+ (10 M) and Cholinium cloride (20 M) Sample 7 Cells exposed to 6OHDA (50 M) Sample 8 Cells exposed to 6OHDA (50 M) and [Chol].sub.2[N1(4)inh] N1(4)inh (20 M) Sample 9 Cells exposed to 6OHDA (50 M) and Cholinium cloride (20 M) Sample 10 Substantia nigra exposed to saline Sample 11 Substantia nigra exposed to [Chol].sub.2[N1(4)inh] + N1(4)inh (0.2 mg/kg/day) Sample 12 Substantia nigra exposed to 6OHDA (10 g/2 l) Sample 13 Substantia nigra exposed to 6OHDA (10 g/2 l) and [Chol].sub.2[N1(4)inh] + N1(4)inh(0.2 mg/kg/day) Sample 14 All mice organs exposed to 0.02; 0.04; 0.06; 0.16 mg/kg/day of [Chol].sub.2[N1(4)inh] + N1(4)inh Sample 15 All mice organs exposed to 0.16 mg/kg/day of [Chol].sub.2[N1(4)inh] + N1(4)inh Sample 16 All rats organs exposed to 0.007 mg/kg/day of [Chol]2[N1(4)inh] + N1(4)inh Sample 17 All rats organs exposed to 0.062 mg/kg/day of [Chol]2[N1(4)inh] + N1(4)inh Sample 18 All rats organs exposed to 2.5 mg/kg/day of PQ Sample 19 All rats organs exposed to 2.5 mg/kg/day of PQ and to 0.007 mg/kg/day of [Chol]2[N1(4)inh] + N1(4)inh Sample 20 All rats organs exposed to 2.5 mg/kg/day of PQ and to 0.062 mg/kg/day of [Chol]2[N1(4)inh] + N1(4)inh
(87) In an embodiment, for the synthesis of [Chol].sub.2[N1(4)inh], a cholinium salt was used as the cation source and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) as the anion source. The [Chol].sub.2[N1(4)inh] was synthesized by metathesis reaction with 1:1.05 mole ratio of cholinium bicarbonate (80% in water, m/v) and N1(4)inh solution prepared in a minimum amount of absolute ethanol. The N1(4)inh was mixed by drop-wise addition into the cholinium bicarbonate solution under dark and cold conditions (using an ice-bath at 5 C.) and with continuous stirring. The reaction mixture was further kept at 5 C. under continuous stirring for 2 h. The excess solvent and water were removed under continuous flow of nitrogen gas until complete drying (2-3 h of nitrogen gas flow with the IL containing vial maintained in the ice-cold water bath (10 C.)). The synthesized [Chol].sub.2[N1(4)inh] and N1(4)inh mixture (1:1, mol:mol) was finally collected from the ice-cold water bath and stored under dried, cool and dark conditions.
(88) In an embodiment,
(89) The purity of [Chol].sub.2[N1(4)inh]+N1(4)inh was evaluated by .sup.1H NMR.
(90) In an embodiment,
(91) The purity of [Chol].sub.2[N1(4)inh]+N1(4)inh and N1(4)inh was also evaluated by .sup.13C NMR.
(92) In an embodiment,
(93) In an embodiment, solutes (N1(4)inh and [Chol].sub.2[N1(4)inh]+N1(4)inh) were added in excess to a fixed volume (500 L) of water and PBS solution. These mixtures were incubated at 37 C., under constant agitation 1150 rpm and for a minimum of 72 h, using an Eppendorf Thermomixer Comfort equipment. Throughout this process, solute was added to the mixture, whenever necessary, i.e., until achieving the solution saturation. All samples were filtered using syringe filters (0.45 m) to remove possible suspended solid particles. The quantification of N1(4)inh and [Chol].sub.2[N1(4)inh]+N1(4)inh was carried by UV-spectroscopy, using a UV-spectrophotometry (SYNERGY|HT microplate reader, BioTek) at a wavelength of 416 nm and 480 nm, respectively. The interference of the PBS with the quantification method was also ascertained and blank control samples were always used. The obtained results disclose that the solubility of the IL-based formulation in water increased 36 fold whereas the solubility in PBS increased 21 fold.
(94) In an embodiment,
(95) In an embodiment, solutes (N1(4)inh and [Chol]2[N1(4)inh]+N1(4)inh) were added in excess to a fixed volume (500 L) of water and PBS solution. These mixtures were incubated at 37 C., under constant agitation 1150 rpm and for a minimum of 72 h, using an Eppendorf Thermomixer Comfort equipment. Throughout this process, solute was added to the mixture, whenever necessary, i.e., until achieving the solution saturation. All samples were filtered using syringe filters (0.45 m) to remove possible suspended solid particles. The quantification of N1(4)inh and [Chol]2[N1(4)inh]+N1(4)inh was carried by UV-spectroscopy, using a UV-spectrophotometry (SYNERGY|HT microplate reader, BioTek) at a wavelength of 416 nm and 480 nm, respectively. The interference of the PBS with the quantification method was also ascertained and blank control samples were always used. The obtained results disclose that the solubility of the IL-based formulation in water increased 28 fold whereas the solubility in PBS increased 16 fold.
(96) In an embodiment,
(97) In an embodiment, the decomposition temperature was determined by thermogravimetric analysis (TGA). The TGA curve reveals a sharp decrease in the degradation temperature at around 200 C., demonstrating that [Chol]2[N1(4)inh]+N1(4)inh decomposition starts occurring at 200 C., and as also shown by the precursor.
(98) In an embodiment,
(99) In an embodiment, the potential toxicity of [Chol]2[N1(4)inh]+N1(4)inh towards N27 dopaminergic neurons was evaluated after 24 h of exposure to 20 M or 30 M of [Chol]2[N1(4)inh]+N1(4)inh. No statistically significant differences were observed between treatments and control cells (CTR/untreated cells), demonstrating that the IL-based formulation of [Chol]2[N1(4)inh]+N1(4)inh is not toxic to dopaminergic neurons. The data are expressed as a percentage of CTR and shown as the meanSEM of at least five repetitions of three independent experiments (n=3). Statistical analysis was performed using one-way ANOVA (non-parametric analysis), followed by the Kruskal-Wallis test followed by Dunn's multiple comparison test. {circle around (1)} Control cells; {circle around (2)} Cells exposed to 20 M of [Chol].sub.2[N1(4)inh]+N1(4)inh; {circle around (3)} Cells exposed to 30 M of [Chol].sub.2[N1(4)inh]+N1(4)inh.
(100) In an embodiment,
(101) In an embodiment,
(102) In an embodiment,
(103) In an embodiment, changes in the number of tyrosine hydroxylase (TH)-immunoreactive neurons in mice SN, 7 days after brain intraventricular infusion of 0.2 mg/kg/day of [Chol].sub.2[N1(4)inh]+N1(4)inh. {circle around (1)} Control group (saline only); {circle around (2)} [Chol].sub.2[N1(4)inh]+N1(4)inh treated group. The number of TH-positive neurons in the SN of mice treated with [Chol].sub.2[N1(4)inh]+N1(4)inh, is not statistically different from the ones quantified in the SN of control group ({circle around (2)} compared with {circle around (1)}), demonstrating that the IL-based formulation of [Chol].sub.2[N1(4)inh]+N1(4)inh is not toxic to dopaminergic neurons in vivo. The results are expressed as a percentage of Saline (Group {circle around (1)} which are animals exposed only to saline solution) and represented as the meanSEM of at least 3 independent experiments (n=3-4). Statistical analysis performed using one-way ANOVA, followed by a multiple comparison analysis using the Bonferroni test. No statistical differences were found between the two groups.
(104) In an embodiment,
(105) In an embodiment
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(117) The term comprising whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
(118) Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice versa, if not specifically excluded. For example, the term an ionic liquid or the ionic liquid also includes the plural forms ionic liquids or the ionic liquids, and vice versa. In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
(119) Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
(120) The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.
(121) The embodiments described above are combinable.
(122) The following claims further set out particular embodiments of the disclosure.