Use of 3-amino-4-methylpyridine for the treatment of demyelinating diseases

Abstract

The present invention describes the methylated analogue of 4-aminopyridine (4-AP), which is the 3-amino-4-methylpyridine (3A-4MetP) compound represented by the formula (I): (I) for use in the treatment of a Central Nervous System (CNS) disease, wherein the CNS disease is selected from the group consisting of a disease associated with alterations in the functioning of voltage-dependent potassium (KV) channels, and a demyelinating disease.

Description

USE OF 3-AMINO-4-METHYLPYRIDINE FORTHE TREATMENT OF DEMYELINATING DISEASESField of the invention

[0001] The present invention is related to the pharmaceutical application of the compound 3-amino-4-methylpyridine (3A-4MetP), belonging to the family of compounds analogous to the 4-aminopyridine (4-AP) molecule, to treat more efficiently the symptoms related to demyelinating diseases in the medical and pharmacological fields.Background of the invention

[0002] Myelin is a type of sheath made up of proteins and fatty substances that coats nerve cells (neurons) of the Central Nervous System (CNS) allowing electrical impulses (action potentials, AP) to be transmitted quickly and efficiently throughout the neurons. A rupture or detachment of this myelinated arrangement gives rise to demyelinating diseases (Figure 1 ). These include multiple sclerosis (MS), leukodystrophy (LEU) and encephalomyelitis (ENC), to name a few.

[0003] In MS, for example, myelin detachment causes an imbalance between the density of voltage-gated sodium (Na+) and potassium (K+) axonal channels (Navand Kv, respectively). This fact translates into an increase in the density of Kvchannels that were originally covered by myelin sheaths through the axon of the neuron. This abnormal redistribution of Kv channels produces an attenuation and slowing down of the propagation rate of the APs causing the neurological deficit (see Figure 1 ). In humans, this disease leads to a series of symptomatic manifestations, such as: fatigue, muscle numbness, partial paralysis, involuntary tremors, sensory loss, among others. The causes of this autoimmune and incurable disease are unknown, and the diagnostic protocols are complex, and generally very expensive. Approximately 2.8 million people in the world are known to suffer MS (World Federation of Neurology, https: / / wfneurology.org / ), of whom 70% are young adults; and it is estimated that in Mexico there areapproximately 20 thousand cases of people diagnosed with this disease (https: / / www.gob.mx / salud / prensa).

[0004] At present there are medications prescribed by specialists to alleviate the symptoms of patients diagnosed with MS. Specifically, the 4-aminopyridine (4-AP, CAS No. 504-24-5), known commercially as Ampyra® (in the US) or Fampyra® (in the European Union), is an established drug for this purpose. Briefly, the excitatory-like effects of 4-AP on the CNS were first described during the 1950s, but it was not until the 1970s that its blocking effect on the ionic current generated by Kvchannels was discovered. By the 1980s, 4-AP was shown to improve AP conduction of demyelinated fibers and thus shown to be of beneficial use in MS patients. However, it was not until 2010 when a sustained-release formulation of 4-AP (Ampyra®) was shown to improve movement in people with MS and it was subsequently approved by the US Food and Drug Administration (FDA) for this purpose.

[0005] Multiple studies on the structure and activity of 4-AP derivatives have been developed; particularly, the drug 3,4-Diaminopyridine (3,4-DAP, CAS No. 54-96- 6) is a structural 4-AP analogue (Figure 2) which has shown a higher blocking efficiency on Kvchannels than the same 4-AP. 3,4-DAP is marketed as Firdapse® and since 2017 it is a drug approved by the FDA for its administration in patients diagnosed with Lambert-Eaton Myasthenic Syndrome (LEMS). LEMS is an autoimmune disease in humans that mistakenly attacks the neurons of the nervous system (NS) making them unable to release a sufficient amount of acetylcholine, causing an interference in the AP of the NS and muscles, causing muscle weakness. Although 3,4-DAP is a molecule that shows higher Kv channel blocking efficiency than 4-AP, it is also known that 3,4-DAP fails to cross the blood-brain barrier (BBB), and therefore, its use in demyelinating diseases of the CNS such as MS is not approved. For this reason, the synthesis and characterization of new Kv channel blockers that show better physicochemical, pharmacological, dosing, and elimination properties in the body than those already known is an urgent need worldwide.

[0006] Blockade of Kv potassium channels in the CNS by 4-AP involves a series of sequential processes that depend on lipophilicity, pH, and cell membrane voltage (Table 1 , Figures 3 to 7). This mechanism implies that a non-protonated(electrically neutral) 4-AP molecule must first cross the BBB barrier until it reaches the intracellular vestibule of a neuron. Subsequently, a protonated form of 4-AP (positively charged) can bind to its binding site in the channel pore. Therefore, this type of process implies that certain features and specific properties of Kvchannel blockers are fulfilled, such as p7favalues being close to physiological pH (7.1 -7.4) and the partition coefficient (log D) is within the range of -2<log D<2, mostly.

[0007] Although the use of 4-AP derivatives has been documented, as disclosed in the document Rodriguez-Rangel S. et al. (2020) “Structure-activity relationship studies of four novel 4-aminopyridine K+channel blockers”, in which four new 4- aminopyridine (4-AP) derivatives containing methyl (-CH3), methoxy (-OCH3) and trifluoromethyl (-CF3) in positions 2 and 3 were characterized as potential candidates to be used in positron emission tomography (PET) and / or therapy, describing the physicochemical properties of these compounds (basicity and lipophilicity) as well as the analysis of its ability to block the Shaker Kv channel under different voltage and pH conditions. The results in this paper demonstrate that three of the four derivatives can efficiently block voltage-gated potassium channels. Specifically, 3-methyl-4-aminopyridine (3Met-4AP) was found to be approximately ~7 times more potent than 4-AP and 3-fluoro-4-aminopyridine (3F4AP); it was also shown that 3-methoxy- and 3-trifluoromethyl-4- aminopyridine (3MeO4AP and 3CF34AP) were about 3 and 4 times less potent than 4-AP; and 2-trifluoromethyl-4-AP (2CF34AP) was found to be about 60 times less active. These results suggested that these novel derivatives are potential candidates for therapy and PET imaging. However, the efficacy of any of said derivatives in the treatment of symptoms related to demyelinating diseases has not been demonstrated in vivo.

[0008] As a consequence of the above, it has been sought to eliminate the drawbacks involved in the administration of drugs useful in the treatment of demyelinating diseases in a subject in need thereof, which in turn provides an improved biological activity with a lower dose and faster release rate, which could be obtained by a similar 4-aminopyridine (4-AP) molecule: 3-amino-4- methylpyridine (3A-4MetP); whose synthesis and production cost is practicallythe same as that of other 4-AP derivatives, making its use affordable for anyone suffering from a demyelinating disease.Objects of the invention

[0009] Taking into account the shortcomings of the prior art, it is an object of the present invention to provide the compound 3-amino-4-methylpyridine, which is represented by formula (I):with the ability to block voltage-dependent potassium channels (Kv).

[0010] Another object of the invention is to provide a molecule that possesses a high bioavailability in the organism and, in addition, is capable of being distributed to organs and tissues that possess barriers of difficult permeability.

[0011] These and other objects are achieved by the medical use of a compound of formula (I) according to the present invention.Brief description of the invention

[0012] For this, a first aspect of the invention relates to a new use for the compound 3-amino-4-methylpyridine (3A-4MetP) as an improved Kv channel blocker, and, consequently, its use in the treatment of a CNS disease in a subject in need thereof. Said compound is a methylated analogue of 4-aminopyridine (4-AP) and is represented by the formula (I):CHS / k ,NH2o hT(I)Brief description of the figures

[0013] Novel aspects which are considered characteristic of the present invention will be established with particularity in the appended claims. However, some embodiments, features and some objects and advantages thereof will be better understood in the detailed description, when read in connection with the attached drawings, in which:Fig.1

[0014] [Fig.1] Figure 1 shows the detachment of myelin from the axon of the neuron which causes a demyelinating disease, a) Representation of a myelinated neuron. In the Ranvier nodes the density of Nav channels is greater than Kv channels, b) Representation of a demyelinated neuron. The detachment of the myelin sheaths through the axon causes an increase in the density of Kv channels, causing a neurological deficit in the CNS.Fig.2

[0015] [Fig.2] Figure 2 shows the analogues and methylated derivatives of 4- aminopyridine, through structures which integrate group A that represents the methylated molecules derived from 4-AP (i-v) and group B that represents analogous methylated molecules of 4-AP (vi-x).Fig.3

[0016] [Fig.3] Figure 3 shows the blocking of the Sh Kv channel by 4-AP analogues and methylated derivatives. Shown in black are representative recordings of K+current produced by the Sh Kv channel expressed in 10 different Xenopus laevis oocytes in response to a depolarizing voltage stimulus (voltage protocol, upper left panel) just before the application of each compound. Shown in gray is the remaining K+current after adding 1 mM of each 4-AP methylated derivative or analogue.Fig.4

[0017] [Fig.4] Figure 4 shows the relative current (l[x] / lmax) of the Sh Kv ion channel assessed at 40 mV vs the concentration of 4-AP analogues and derivatives ([X]). Blocking potency of the Sh Kv current by 4-AP derivatives and methylatedanalogues were quantified in terms of the IC50. The solid line represents the adjustment with the Hill equation / c50-iog[x])h)) with a Hill coefficient A~1 for all cases.Fig.5

[0018] [Fig.5] Figure 5 shows the graphical representation of the Kv channel blocking mechanism by 4-AP, wherein a non-protonated 4-AP molecule crosses (dotted line) the lipid bilayer of the axon membrane where the Kv channels are embedded. Subsequently, 4-AP is protonated (H+binding) and can bind to its binding site through the channel pore once it is opened by the V stimulation.Fig.6

[0019] [Fig.6] Figure 6 shows the pH dependence of the Sh Kv channel blockade by 3A-4MetP. Relative current of the Sh Kv channel at 40 mV (l[3A-4MetP] / lmax) at different pH values vs 3A-4MetP concentration. The solid line represents the adjustment with the Hill equation with a Hill coefficient h~1 .Fig.7

[0020] [Fig.7] Figure 7 shows the voltage dependence of the Sh Kv channel blockade by 4AP analogues and methylated derivatives. ICso voltage dependence of the 4-AP, 3Met-4AP and 3A-4MetP molecules in the range of 20 to 60 mV with pH =7.4. The dotted line represents the adjustment with a voltage-dependent one-step binding model (log ICso (V) = log ICso(y=o) + (1 / 2.303)- zF8V / RT)) using RT / zF = 25.69 mV.Fig.8

[0021] [Fig.8] Figure 8 shows the UV-vis spectroscopy of 3A-4MetP, where a) shows the absorbance spectrum of 3A-4MetP in the presence of 1 -octanol (black line) and PBS solution (grey line). The maximum absorbance in 1 -octanol and PBS solution occurs at 242 nm and 230 nm, respectively; and wherein b) shows the 3A-4MetP absorbance vs. concentration curve. The solid lines correspond to the adjustment with the Beer-Lambert equation (A=s L [3A-4MetP]) for L=1 cm. The molar extinction coefficient (E) of the 3A-4MetP in 1 -octanol or PBS solution was 6440 and 5310 mol’1-cm’1, respectively.Fig.9

[0022] [Fig.9] Figure 9 shows the in silico determination of the binding site in the Sh Kv channel pore of the 4-AP, 3Met-4AP, 3,4-DAP and 3A-4MetP molecules. The occupancy in the Sh Kv channel pore of 4-AP (panel a), 3Met-4AP (panel b), 3,4- DAP (panel c) and 3A-4MetP (panel d). Panels a and b suggest that a protonated form of 4-AP or 3Met-4AP can establish two hydrogen bonds with pore residues of the Sh Kv channel. The first occurs between its nitrogen at position 1 (N1 ) with one of the two threonines T441 or T442 while the second bond can be established between the valine V467 and the NH2 group at position 4. Panels c and d indicate that the 3,4-DAP and 3A-4MetP molecules can be placed in the pore of the channel rotated 180sforming three hydrogen bonds with the residues of the pore. On the one hand, 3,4-DAP can equiprobably form two hydrogen bonds between its NH2 groups in positions 3 and 4 with the threonine T441 and / or T442 and a third between its N1 and V467. In this scenario the 3A-4MetP molecule can only form two hydrogen bonds with the channel pore; one between its NH2 group at position 3 with T441 and / or T442 and another between its N1 and the V467 of the channel.Fig.10

[0023] [Fig.10] Figure 10 corresponds to the pharmacokinetics of 4-AP evaluated in the BALB / c murine model. The dotted line represents the fit of the data during the 4-AP elimination step (t>5 min) with a single-compartment pharmacokinetic model.Fig.11

[0024] [Fig.11] Figure 11 shows the pharmacokinetics of 3A-4MetP evaluated in the BALB / c murine model. The dotted line represents the fit of the data during the distribution and elimination stage of 3A-4MetP with a bi-compartmental model.Fig.12

[0025] [Fig.12] Figure 12 represents the results of blockade of Kv 1.2 channel by 4- AP, 3Met-4AP and 3A-4MetP. a. Representative recordings of K+current generated by Kv 1.2 channel expressed in three different Xenopus oocytes in response at 40 mV voltage stimulus before (gray recording) and after (blackrecording) adding 1 mM in extracellular solution of 4-AP, 3Met-4AP and 3A- 4MetP. The dashed line represents the 0 pA level of current, b. Relative current vs. concentration of 4-AP, 3Met-4AP or 3A-4MetP. The solid line represents the fit of the data with the Hill equation.Fig.13

[0026] [Fig.13] Figure 13 shows the results of blockade of Kv 2.1 channel and Kv 2.1 / Kv 6.4 co-expression by 4-AP, 3Met-4AP and 3A-4MetP. a. Representative recordings of K+current generated by Kv 2.1 channel (upper panel) and Kv 2.1 / Kv 6. 4 (lower panel) expressed in six different Xenopus oocytes in response to a voltage stimulus at 40 mV before (gray recording) and after (black recording) adding 1 mM in extracellular solution of 4-AP, 3Met-4AP or 3A-4MetP. The dotted line represents the zero level of current, b. Relative current vs. concentration of 4- AP, 3Met-4AP and 3A-4MetP evaluated in the Kv 2.1 channel, c. Relative current vs. concentration of 4-AP, 3Met-4AP or 3A-4MetP evaluated in the co-expression (3:1 stoichiometry) Kv 2.1 / Kv 6.4.Fig.14

[0027] [Fig.14] Figure 14 represents the results of Blockade of Kv 7.2, Kv 7.3 channels and Kv 7.2 / Kv 7.3 co-expression by 4-AP, 3Met-4AP and 3A-4MetP. a. Representative K+current recordings obtained in Xenopus laevis oocytes expressing the Kv 7. 2 (top panel), Kv 7.3 (middle panel) and co-expression (1 :1 stoichiometry) of Kv 7.2 / Kv 7.3 (bottom panel) channels before (gray recording) and after (black recording) adding 1 mM in extracellular solution of 4-AP, 3Met- 4AP or 3A-4MetP. The dashed line represents the zero level of current, b. Relative current vs. concentration of 4-AP, 3Met-4AP or 3A-4MetP evaluated in the Kv 7.2 channel. The value of ICso was not possible to determine in the range of the concentrations of the compounds in which evaluated c. Relative current vs. concentration of 4-AP, 3Met-4AP and 3A-4MetP evaluated in the Kv 7.3 channel. The ICso for 4-AP and 3Met-4AP is not determined over the range of concentrations evaluated, while IC50 for 3A-4MetP was 160 pM. d. Relative current vs concentration of 4-AP, 3Met-4AP and 3A-4MetP evaluated in the Kv 7.2 / Kv 7.3 co-expression. Continuous lines in panel c and d represent the fit of the data with the Hill model.Detailed description of the invention

[0028] The present invention relates to a new use for the compound 3-amino-4- methylpyridine (3A-4MetP) in the treatment of CNS diseases in a subject in need thereof. Said compound is a 4-aminopyridine (4-AP) analogue methylated and is represented by the formula (I):

[0029] As used in the present invention, the term "channelopathy" refers to diseases or conditions caused by dysfunctions in voltage-dependent potassium (Kv) channels.

[0030] The term "treatment" is defined as the set of means, actions or interventions applied to cure, alleviate or control a disease, medical condition or health problem. This term includes the relief of symptoms, as well as reducing the complications of the disease or condition.

[0031] Previous studies have shown that the conjugation of a small functional group at position 3 of the 4-AP molecule, such as methyl (CH3), methoxy (OCH3) and fluoromethyl (CF3), do not substantially modify its blocking effect on Kv channels. From those studies, conjugation of the CH3 group at position 3 of 4-AP (3-methyl- 4-aminopyridine, 3Met-4AP) was shown to produce a blocking effect on the Sh Kv channel that is ~7 times greater than that caused by 4-AP. Surprisingly, the p7faand logD determined for the 3Met-4AP molecule were 9.82 and - 1 .232, respectively, and unfortunately they did not show better physicochemical characteristics with regards to the same parameters that were determined for 4- AP, 9.58 and -1 .478, respectively.

[0032] It is known from the prior art that the amino (NH2) and CH3 functional groups are small and have very different physicochemical properties. For example, the NH2 group is a polar and water-soluble molecule that confers the molecules with which it conjugates the possibility of forming intermolecular hydrogen bonds. On the other hand, the CH3 functional group is a nonpolar, hydrophobic, andelectrically neutral molecule whose tetrahedral and symmetrical geometry distributes its electronic charge density almost homogeneously, which contributes to a dipole moment that is practically negligible. For these reasons, interest arose to investigate the Kvchannel blocking effect of methylated analogue or derivative molecules of 4-AP. It was then hypothesized that the simultaneous conjugation of the NH2 and CH3 groups in 4-AP would provide better physicochemical properties (p7faand logD) to the molecule without altering its blocking capacity. To do this, these functional groups were exchanged or substituted in positions 2, 3 and 4 of 4-AP. The molecules proposed for this study were classified into two groups: group A of 4-AP methylated derivatives and group B of 4-AP methylated analogues (see Figure 2). Group A was made up of the molecules: i) 4-AP, ii) 2,4-Diaminopyridine (2,4-DAP), iii) 3,4-DAP, iv) 2-Methyl-4-Aminopyridine (2Met- 4AP) and v) 3Met-4AP, while group B was made up of: vi) 4-Methylpyridine (4- MetP), vii) 2,4-Dimethylpyridine (2,4-DMetP), viii) 3,4-Dimethylpyridine (3,4- DMetP), ix) 2-Amino-4-Methylpyridine (2A-4MetP), and x) 3-Amino-4- Methylpyridine (3A-4MetP). These molecules are commercially distributed and were acquired through the Merck company (Merck KGaA, Darmstadt, Germany), except for 2,4-DAP, which was acquired through Toronto Research Chemicals Inc. (20 Martin Ross Avenue Toronto, ON Canada).

[0033] The blocking efficiency of the molecules that constitute groups A and B (Figure 2) was experimentally determined on the Sh Kv channel expressed in Xenopus laevis oocytes using the COVC methodology and quantified in terms of IC50. The studies carried out demonstrated that the conjugation of the CH3 group in position 2 of 4-AP (2Met-4AP) does not produce a significant blockage on the current of the Sh Kv channel as expected. As previously shown, the conjugation of the CH3 group at position 3 of 4-AP results in a blocking effect that is ~7 times greater than that produced by 4-AP. Consistently, the blocking effect of the 3,4- DAP molecule was greater (~2 times) than that produced by 4-AP (Figure 4, Table 2). Referring to the blocking effect produced by the molecules that constitute group B of Figure 2, it was found that the conjugation of the CH3 functional group in position 4 of the pyridine ring (4-MetP) or the ligation of the NH2 group in position 2 of 4-MetP (2Met-4AP) does not produce relevant effects on the blocking of the Sh Kv channel current. Interestingly, when the NH2 group isconjugated to position 3 of 4MetP, its blocking potency was twice greater than that produced by 4-AP and was similar to that produced by 3,4-DAP (Figure 4), these results motivated the inventors to carry out an exhaustive study on the biophysical (Figures 6 and 7, Tables 3 and 4), biochemical (Table 1 ), pharmacological (Figures 3 and 4) and spectroscopic properties (Figure 8) of the 3A-4MetP analogue.Table 1 . Biochemical properties: p7faand logD values determined for the molecules 4-AP, 3Met-4AP, 3,4-DAP, and 3A-4MetP.

[0034] Table 1 shows the p7fafor each molecule, which was determined from a potentiometric titration curve by adding 50 pL of 0.01 M HCI in a dilution of 15 mg of each compound in 15 mL of type I water. The pH was plotted as a function of the added volume of HCI and subsequently the p7fawas calculated. The logD was measured by mixing (shaking) in a 15 mL volume falcon-type tube 3 / z L of each compound at 5 mg / mL with 1.5 mL of 1 -octanol and PBS solution at pH=7.4. Subsequently, the concentration of each molecule in each phase was determined in terms of the Beer-Lambert law by UV-VIS spectroscopy procedures. A calibration curve of absorbance vs. concentration was determined for each compound. The p7faor logD values shown in the table represent the average of n independent measurements ± s. d.Table 2. Values of the concentration of each 4AP analogue or methylated derivative to inhibit 50% of the Sh Kv channel current (ICso).

[0035] It should be noted that the IC50 values shown in Table 2 were determined by adjusting the l[x] / lmax curves of panel a with the Hill equation. Table 2 shows the average IC50 value of n independent experiments and its 95% of confidence interval. The parameters shown were measured at pH= 7.4 and V= 40mV.Table 3. IC50 values at different pH values.

[0036] Table 3 shows the average IC50 value of n independent experiments and the 95% of confidence interval. The IC50 for each pH condition was determined by adjusting the I[3A-4Metp] / Imax curves with the Hill equation.Table 4. Parameters of the adjustment with a voltage-dependent one-step binding model at pH 7.4.

[0037] Table 4 shows the determined value of IC5o (v=o) and the fraction of electrical distance 8 that each 4-AP analogue or methylated derivative must cross in the electrical field of the membrane to bind at its binding site through the channel pore and block it. The value of <5-0.4 for these three molecules indicates that they can bind at the same binding site. Table 4 shows the average of the adjustment parameters of n independent measurements ± s. d.

[0038] In this sense, the blocking effect of the 3-amino-4-methylpyridine (3A-4MetP, CAS No. 3430-27-1 ) molecule on the Kv channel named Shaker (Sh Kv), a functional model of the KCNA protein family has been elucidated which includesthe channels from Kv 1 .1 to Kv1 .8, which play a fundamental role in certain CNS pathologies such as MS (Kv 1 .1 and Kv 1 .2).

[0039] The blocking efficiency of 3A-4MetP on the Sh Kv channel was determined in terms of the concentration necessary to inhibit 50% of the current generated by the Sh Kv channel (ICso). For this purpose, the experimental cut-open oocyte voltage-clamp (COVC) technique was implemented. The ICso for 3A-4MetP was found to be ~2.5 times lower than that determined for 4-AP and was similar for 3,4-DAP (Figure 4). This fact implies that the blocking potency of 3A-4MetP is as efficient as that of 3,4-DAP and better than that produced by 4-AP (Figure 7). Interestingly, the logD and p7fadetermined for 3A-4MetP were 0.07 and 6. 74, respectively, while for 4-AP it was -1.34 and 9.44, respectively (Table 1 ). These results demonstrate that 3A-4MetP has a higher probability to cross the BBB than 4-AP. These data, moreover, were consistent with the in silico analysis regarding the calculation of the apparent affinity of 3A-4MetP at its binding site (Figure 9 and Table 5) and additionally the need for the application of compound 4-AP for clinical use to be slow and prolonged was deduced therefrom.Table 5. Apparent affinity parameters of the 4-AP, 3Met-4AP, 3,4-DAP and 3A- 4MetP molecules in the Sh Kv channel pore determined by molecular docking.

[0040] The molecular docking assays shown in Table 5 were performed with the AutoDock Tools program, Avogadro V1.2 and UCSF Chimera, free to use programs. The pdb code for the structure of the Sh Kv in its open conformation used for this study was 7SIP. Table 5 shows the mean values of binding energy and inhibition constant obtained for n = 2000 independent trials ± s. d...

[0041] Likewise, in the present invention the blocking capacity of the 3A-4MetP molecule has been elucidated (Figure 2) on the Shaker Kv (Sh Kv) channel expressed in living cells (see Figure 3). The Sh Kv channel cloned from the larvae of the Drosophila melanogaster fly is the functional model of the humanKCNA protein family that includes the Kv1 .1 through Kv 1 .8 channels. Specifically, the Kv 1.1 and Kv 1.2 channels play a fundamental role in certain demyelinating pathologies of the central nervous system, such as multiple sclerosis (MS).

[0042] The experimental cut-open oocyte voltage-clamp (COVC) technique was implemented as an experimental methodology for the present invention to determine the blocking efficiency of 3A-4MetP in terms of the concentration necessary to inhibit 50% of the ionic current (ICso) generated by the Sh Kv channel expressed in Xenopus laevis oocytes. The ICso was found to be 2-fold higher than that determined for 4-aminopyridine (4-AP) and similar to that of 3,4- diaminopyridine (3,4-DAP) Kv channel specific blockers (see Figures 3 and 4).

[0043] The biophysical analysis of the blocking mechanism of the Sh Kv channel by 3A-4MetP indicates that this new inhibitory molecule canonically blocks the ionic current generated by Kv like channels, just as the 4-AP and 3,4-DAP molecules do. That is, this mechanism is a sequential process that depends on pH and membrane voltage (V) (see Figures 5, 6).

[0044] Interestingly, the inventors found that the 3A-4MetP molecule can block CNS- active Kv channels, such as Kv 1.2, 2.1 , 2.1 / 6.4, 7.2 and 7.2 / 7.3; the therapeutic significance of these channels is associated with epileptic disorders, migraine, neurological disorders, seizures and alterations in cognition. Given the effectiveness of the molecule on various Kv channels active in the CNS, it is of great utility in the treatment of CNS diseases, such as a disease associated with alterations in the functioning of voltage-dependent potassium (Kv) channels, as well as a demyelinating disease in a subject in need thereof.

[0045] In a non-limiting embodiment, the subject is a human.

[0046] Regarding the alterations in the functioning of Kv channels, these may be due to dysfunctions resulting from genetic mutations or they may be acquired; together the alterations are called channelopathies.

[0047] Such channelopathies may include epileptic disorders and neurological disorders.

[0048] With respect to the demyelinating disease, it is selected from the group consisting of multiple sclerosis (MS), leukodystrophy (LEU) and encephalomyelitis (ENC).

[0049] 3A-4MetP is an enhanced Kv channel blocker, such that it possesses anticonvulsant, antiepileptic and even local anesthetic and analgesic effects.

[0050] With respect to the problems associated with physicochemical, pharmacological, dosage and elimination properties in the body, the 3A-4MetP molecule represents a solution to the problems of the state of the art. In the first instance, it was surprisingly found that the 3A-4MetP molecule has a higher absorption capacity in the organism and higher bioavailability than the 4-AP molecule, so that the molecule object of the present invention has a higher therapeutic potency.

[0051] On the other hand, according to the pharmacokinetic curve of 3A-4MetP, it can be deduced that once 3A-4MetP is administered to the organism, it is rapidly distributed through the blood circulation and highly perfused organs. The 3A- 4MetP is subsequently redistributed to a peripheral compartment hypothetically formed by tissues, lipids and specific organs that have difficult permeability barriers, such as the blood-brain barrier. Hence, the 3A-4MetP molecule is suitable for the treatment of diseases affecting the Central Nervous System (CNS).

[0052] The above is reinforced by the lipophilicity of 3A-4MetP in conjunction with its p7favalues and the octanol / water partition coefficient (logD) of 3A-4MetP. In this regard, as showed in Table 1 , the p7favalues and the octanol / water partition coefficient (logD) of 3A-4MetP were determined. These parameters were compared with the values determined for 4-AP and 3,4-DAP. These results show that the 3A-4MetP molecule has the lowest p7favalue compared with the molecules 4-AP, 3Met-4AP and 3,4-DAP; thus, this indicates that the 3A-4MetP has a greater facility to cross the blood-brain barrier (BBB) as compared to the other molecules in the physiological pH range (7.1 -7.4). Furthermore, it is known that the 3,4-DAP molecule does not cross the BBB barrier, even though its Kvchannel blocking efficiency is greater than that produced by 4-AP.

[0053] It should also be noted that in silica studies using the molecular coupling methodology (“Molecular Docking”) between the 3D homology model of the Sh Kv channel and the 3A-4MetP, 4-AP and 3,4-DAP molecules indicate these molecules bind to the same binding site through the channel pore to block it (Figure 9). These results imply that the protonated (positively charged) form of 4- AP can establish two hydrogen bonds with the Sh Kv pore; the first with one of the two conserved threonine (T) at positions 441 or 442 (T441 or T442) and the other with the conserved valine (V) at position 467 (V467). Interestingly, the 3,4- DAP and 3A-4MetP molecules are placed rotated 180° in the same region of the Sh Kv channel pore. These molecules can form three hydrogen bonds in the pore region; two with T441 and T442 and one with V467. This fact would explain why the apparent affinity of the 3,4-DAP and 3A-4MetP molecules is greater than that shown by 4-AP.

[0054] Therefore, the 3A-4MetP molecule is a new Kv channel blocker that shows better physicochemical and pharmacological properties than 4-AP and 3,4-DAP and therefore it has great potential for use in the symptomatic treatment of demyelinating diseases in humans, such as multiple sclerosis (MS), leukodystrophy (LEU) and encephalomyelitis (ENC), as well as other diseases such as those exemplified in Table 6 below.Table 6. Pharmacological uses of 4-AP in humans.■"Information obtained from the PubChem portal: https: / / pubchem.ncbi.nlm.nih.gov / compound / 1727

[0055] Based on the foregoing, a method of treating CNS diseases in a subject in need thereof is provided in the present invention.

[0056] Since 3A-4MetP has the potential to cross the blood-brain barrier, its use is preferred in CNS diseases, including diseases associated with alternations in Kv channel function, as well as in demyelinating diseases. However, given its properties as a Kv channel blocker, 3A-4MetP can additionally be employed in the treatment of diseases that do not necessarily develop in the CNS. Such diseases include, but are not limited to, cardiac arrhythmias, dyspnea and hypotension.

[0057] The method comprises administering the molecule 3-amino-4-methylpyridine (3A-4MetP) to a patient in need of the treatment.

[0058] As indicated above, 3A-4MetP advantageously has a higher absorption and bioavailability in the body, increasing the potency of the treatment. Furthermore, the molecule is able to cross the blood-brain barrier, ensuring its therapeutic effect in the CNS.

[0059] The present invention will be better understood from the following Examples, which are presented solely for illustrative purposes to allow a full comprehension of the preferred embodiments of the present invention, without implying that there are no other non-illustrated embodiments that may be put into practice based on the detailed description above.Examples

[0060] Example 1 .

[0061] Below are the results on the study of acute toxicity of the 4-AP, 3Met-4AP and 3A-4MetP molecules carried out in the mouse model of the BALB / c strain orally (see Table 7). During the study it was observed that the effects produced by the administration of the three compounds in the model were similar, increasing their manifestation and intensity as the dose increased. The median lethal dose (LDso (in mg / kg)) was determined for each compound. The LDso was 12.7, 13.23, and 29.3 for 4-AP, 3Met-4AP, and 3A-4MetP, respectively.

[0062] From this study it is concluded that the LDso of compounds 4-AP and 3Met- 4AP is very similar while the 3A-4MetP molecule turned out to have a higher LDso, which indicates that this molecule is less toxic in the BALB / c mouse model. This result implies that the 3A-4MetP molecule has a greater therapeutic margin when compared to the 4-AP and 3Met-4AP compounds.Table 7. Study of the acute toxicity of 4-AP, 3Met-4AP and 3A-4MetP in a murine model*.‘The acute toxicity study was performed as follows: Each dose was evaluated in independent groups of five male specimens of the BALB / c strain between 6 and 8 weeks of age weighing 20-25g. The observation period of the effects produced by each molecule was 180 min. No macroscopic changes were observed in the organs of the mice treated with the different doses of 4-AP, 3Met-4AP and 3A-4MetP. The LD50was calculated by the Bliss method or the Probit model after the administration of each dose. All procedures were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) with project number CUCEI / CINV / CICUAL-02 / 2023.&AII animals showed the indicated symptoms.

[0063] Example 2.

[0064] The pharmacokinetic properties and features of the 3-amino-4-methylpyridine (3A-4MetP) molecule were determined in a murine model. A comparison was made with the pharmacokinetics of 4-aminopyridine (4-AP).

[0065] The parameters analyzed were maximum concentration (Cmax), maximum time (tmax), half-life (ti / 2), elimination constant (Ke), area under the curve (AUC), volume of distribution (Vd) and clearance (CL).

[0066] Animal model:

[0067] All procedures related to the care and handling of experimental animals performed during the development of this study were approved by theInstitutional Committee for the Care and Use of Laboratory Animals (CICUAL) of the University Center for Exact and Engineering Sciences, University of Guadalajara with protocol number CUCEI / CINV / CICUAL-02 / 2023. The pharmacokinetics of 3A-4MetP and 4-AP were evaluated in the murine model of the BALB / c strain with age and weight between 6-8 weeks and 20 to 25 g, respectively. The specimens were acquired through the Biotherium of the National Institute of Public Health and the company Petmaniacs Cuernavaca and stored, after quarantine, in the Biotherium of the Research and Innovation Building of the University Center of Los Lagos of the University of Guadalajara.

[0068] Dosage:

[0069] Single dose of 10 mg / kg of 4-AP or 3A-4MetP diluted in 200 pL of type I water administered orally by gavage method.

[0070] Blood sample extraction and serum preparation:

[0071] The plasma concentration of 4-AP or 3A-4MetP was determined at different times ranging from 0 to 180 min. For this purpose, 10 min before blood sample extraction, each murine specimen was administered subcutaneously with a dose of 30 pL of Heparin Sodium (1000 IU). Subsequently, approximately 1.5 mL of blood sample was drawn by cardiac puncture and under anesthesia. The solid and liquid components of the sample were separated by centrifugation for 10 min at 10,000 rpm and room temperature. The proteins in the serum were precipitated by adding three volume portions of 100% HPLC grade acetonitrile for each volume of serum obtained. The mixture was centrifuged for 10 min at 10,000 rpm at 4 °C. Finally, the sample was filtered and placed in vials for analysis by High Performance Liquid Chromatography (HPLC) procedures.

[0072] High Performance Liquid Chromatography (HPLC): The concentration of 4-AP or 3A-4MetP in each plasma sample was determined by HPLC procedures. The equipment used was the ACQUITY Arc, equipped with the 2998 photodiode array detector (PDA) (Waters Corporation, Milford, Massachusetts, USA). The HPLC method implemented for this purpose is summarized in Table 8:Table 8. HPLC method for quantification of 4-AP and 3A-4MetP in mouse serum.

[0073] Results:

[0074] Studies of the pharmacokinetics of 4-AP and 3A-4MetP molecules in the BALB / c murine model show different properties and characteristics that differ from each other.

[0075] Figure 10 corresponds to the plasma concentration of 4-AP as a function of time. It is observed a semilogarithmic curve of 4-AP concentration. The dashed line represents the fit the data during the 4-AP elimination stage (t> 5 min) with a single-compartment pharmacokinetic model of the form Ce(t) = Co■where Co= Ce(t = 0) = 0.9 ng / gL and Ke= 0.03 min-1.

[0076] On the other hand, the pharmacokinetic curve of 3A-4MetP is shown in Figure 11 . A semilogarithmic curve of 3A-4MetP concentration is observed. The dashed line represents the fit the data during the 3A-4MetP distribution and elimination stage with a bi-compartmental model of the form Ce(t) =where Cx+ C2= Ce(t = 0 ) = 6.5 ng / gL , Kld= 0.18 min’1and K2e= 0.03 min’1. The parameters of pharmacokinetic curves a and b are shown in Table 9.Table 9. Pharmacokinetic parameters of 4-AP and 3A-4MetP.Cmax=maximum concentration; tmax=maximum time; ti / 2=half-life; AUCo-i8o=area under the curve;CL=dose / AUC. determined by the relationship: ti / 2=O.693 / Ke.**Determined by the relation: tdi / 2=O.693 / Kid.***Determined by the relationship: tei / 2=0.693 / K2e.

[0077] Based on the above, it can be assumed that the 4-AP is homogeneously distributed throughout the mouse body with a single distribution and elimination phase. On the contrary, the pharmacokinetic curve of 3A-4MetP indicates that the process of absorption, distribution and elimination occurs in a more complex manner being, apparently; a bi-compartmental pharmacokinetics (Figure 1 1 ). A possible mechanism to explain the trend of the data recorded, is that once 3A- 4MetP is administered to the mouse organism, 3A-4MetP can rapidly distribute to a central compartment consisting mainly of the blood circulation and highly perfused organs and from where 3A-4MetP will subsequently redistribute to a peripheral compartment hypothetically consisting of tissues, lipids and specific organs that possess barriers of difficult permeability.

[0078] These data also indicate the following:

[0079] 1 ) Under similar dosing conditions, the Cmax determined in the pharmacokinetic curve of 3A-4MetP was ~2 times higher than the value measured for 4-AP. This fact implies that 3A-4MetP has a higher absorption capacity by the mouse organism than 4-AP and therefore a higher pharmacological potency.

[0080] 2) tmax of 3A-4MetP was 2 times higher than the value produced by 4-AP. This result implies differences in the absorption and distribution kinetics of these molecules. Moreover, it suggests that the greater therapeutic effect produced by 4-AP is reflected more rapidly in the organism of the animal model used for this study than that produced by the 3A-4MetP molecule.

[0081] 3) ti / 2 determined from the pharmacokinetic curve of 4-AP resulted similar to the slow component of the fit with the bi-exponential function (see Figure 10 a) and b)) associated with the elimination kinetics of 3A-4MetP (tei / 2=23.1 min).

[0082] 4) AUC determined from the pharmacokinetic curve of 3A-MetP was ~2 times higher than the value determined from the curve of 4-AP. This result suggests that the 3A-MetP molecule possesses a higher bioavailability than 4-AP in the mouse organism.

[0083] 5) CL determined for 3A-4MetP was ~2 times lower than the value produced by 4-AP. This observation indicates that 3A-4MetP is metabolized and / or eliminated by the mouse organism in a slower manner compared to 4-AP.

[0084] Given the optimal pharmacokinetic characteristics and properties observed in the BALB / c mouse organism, as well as its improved pharmacological, lipophilicity and p7faproperties, it is concluded that the 3A-4MetP molecule has great potential for pharmaceutical development and therapeutic use in the treatment of demyelinating diseases.

[0085] Example 3.

[0086] A study of the blockade of central nervous system voltage-dependent potassium channels by 3A-4MetP was performed. For this purpose, the blocking potency of 3A-4MetP on voltage-dependent potassium channels (Kv) of the central nervous system (CNS) and its comparison with the blocking potency produced by 4-AP and 3A-4MetP was determined and quantified.

[0087] Measurement parameters were heterologous expression of potassium channels in Xenopus laevis oocytes, ionic current recordings from different CNS Kv channels, concentration required to inhibit 50% ionic current (ICso).

[0088] Methodology.

[0089] In vitro RNA synthesis:Table 10. Information regarding the Kv channel DNA plasmid used in this study.*Functional model of the KCNA channel family.** The cloned rat Kv1.2 shares more than 95% homology with human Kv1.2.

[0090] Heterologous expression of Kv channels in Xenopus oocytes.

[0091] All procedures related to the handling and manipulation of the colony of Xenopus laevis frogs used for this study were previously approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) of the University Center for Exact and Engineering Sciences, University of Guadalajara with protocol number CUCEI / CINV / CICUAL-03 / 2023. For thispurpose, Xenopus oocytes were extracted from each frog by surgery under anesthesia and without the need for sacrifice. Subsequently, the oocytes were isolated by a process of mechanical agitation and enzymatic digestion by the action of collagenase type II (Worthington Biochemical Corp., NJ, USA).

[0092] The oocytes were incubated under an atmosphere at 17 °C and in SOS (Survivor Oocyte Solution) saline comprising 100 mM NaCI, 1 mM MgCh, 10 mM HEPES (4-(hydroxyethyl)-1 -piperazineethanesulfonic acid), 2mM KCI and 1.8 mM CaCh with 50 pg / mL gentamicin and adjusted to a pH of 7.4. Finally, 30-40 ng of RNA coding for each Kv channel used was deposited into each isolated oocyte by nano-volume injection of RNA. Following injection, the oocytes were incubated for 1 to 5 days prior to each experiment.

[0093] Electrophysiology procedures.

[0094] The ionic current generated by each Kv channel expressed in the Xenopus oocyte model was recorded in response to a voltage (V) stimulus using the Cut Open Voltage Clamp (COVC) methodology. For the COVC procedures the internal solution was composed of: 120 mM KOH, 2 mM EGTA (Ethylene glycol- bis(2-aminoethyl ethyl ether)-N,N,N',N'-tetraacetic acid) and 20 mM HEPES. The external solution, in turn, had the following composition: 12 mM KOH, 2mM Ca(OH)2, 105 mM NMDG (N-Methyl-D-Glucaminca)-MES (Methanesulfonic Acid) and 20 mM HEPES. Both solutions were adjusted to pH 7.4 with 1 M MES.

[0095] Ionic currents were recorded, amplified and digitized using the CA-1 A amplifier (Dagan Corp) connected to the USB-1604-HS-2AO multifunction card (AD / DA converter) (Measurement Computing) and controlled by a PC using the GpatchMC64 program (Dept, of Physiology, UCLA).

[0096] Determination of blocking potency of 4-AP, 3Met-4AP and 3A-4MetP.

[0097] The blocking potency of 4-AP, 3Met-4AP and 3A-4MetP on each Kv channel was quantified in terms of the concentration required to inhibit 50% of the ionic potassium (K+) (ICso). For this purpose, dose-response curves were measured for each molecule and in each channel expressed in the oocyte model in the range of 0 to 10,000 pM and under voltage-clamp.

[0098] Data analysis.

[0099] Ionic current recordings were analyzed using Analysis (Dept, of Physiology, UCLA) and Origin Pro 8 (OOriginLab Corporation) software. The IC50 for each molecule was determined by fitting with the Hill equation to the curve Iretvs [X], where / reJis the relative current and [X] is the concentration of each compound. / reZwas quantified by measuring the ionic current of each channel in the same oocyte before and after sequential addition of each [X],

[0100] In the present study it was determined, in terms of the parameter IC50, the ionic current blocking power generated by the Shaker Kv channel, modelfunctional of the KCNA family of proteins that includes the Kv 1.2 and some channels expressed in the CNS such as Kv 2.1 , Kv 2.1 / 6.4, Kv 7.2, Kv 7.3 and Kv 7.2 / 7.3 by the molecules 4-AP, 3Met-4AP and 3A-4MetP. The physiological relevance of these channels is shown in Table 11 .Table 11 . Physiological functions and therapeutic significance of Kv channels.

[0101] Table 12 shows the parameters of the fit of the experimentally measured data shown in Figures 12, 13 and 14 with the Hill equation.Table 12. Values of IC50 determined in different KV channels.* Values determined at 40 mV.** Values of IC50 not determined in the range of concentrations evaluated.

[0102] With reference to Figure 12, the graph shows the trend in the blocking power of 4-AP, 3A-4MetP and 3Met-4AP molecules evaluated in the Kv 1.2 channel (3A-4MetP > 3Met-4AP > 4-AP). It is very reasonable that such a trend prevails in other channels of the KCNA family (Kv 1 .1 to 1 .7).

[0103] Figure 13 shows the blocking effect of these molecules on the Kv 2.1 channel. The results obtained show that the ICso could not be determined in the range of the concentrations of 4-AP, 3A-4MetP and 3Met-4AP evaluated, indicating their low affinity for the Kv 2.1 channel. This result is consistent with what is reported in the literature since it is known that ICso value of 4-AP for this channel is on the order of milli-molar (IO3M). The pattern is repeated in the co-expressed channel Kv 2.1 / 6.4 although compound 3A-4MetP produces an apparent higher blocking potency than 4-AP and 3Met-4AP (Figure 13c).

[0104] On the other hand, when the blocking potency of 4-AP and methylated compounds are evaluated in Kv 7.2, Kv 7.3 and co-expressing Kv 7.2 / 7.3 channels, multivariate effects are observed (Figure 14). ICso of the Kv 7.2 channel could not be determined in the range of the evaluated concentrations as seen in Figure 14b. It is observed that the percentage of current inhibited by those compounds at a fixed concentration of 1 mM was approximately ~50 % for 4-AP and 3A-4MetP and ~30 % for 3Met-4AP.

[0105] Referring to the blockade of the Kv 7.3 channel by those same compounds, Figure 14c shows that IC50 of 4-AP and 3Met-4AP also cannot be determined under the same experimental conditions, while IC50 of 3A-4MetP was, surprisingly, 160 pM. This result should be reasonably interpreted for the purposes oftherapeutic application of 3A-4MetP proposed in the present invention, because although the apparent affinity produced by 3A-4MetP on the Kv 7.3 channel is higher (~1.6) than that observed in the Shaker Kv and Kv 1.2 channels. It is also known in the literature that the density of Kv 1.1 and 1.2 channels expressed in the CNS is higher than the population of Kv 7.3 channels present in this same system.

[0106] Figure 13d shows the ICso determined in the Kv 7.2 / 7.3 co-expression, this generates a trend in blocking potency for such compounds of the form: 3Met-4AP > 4-AP « 3A-4MetP. Kv 7.2 / 7.3 co-expression has been shown to play a critical role in the excitability of neurons as well as to the input response of synaptic stimuli in CNS tissues (Table 11 ).

[0107] The study further demonstrated that both 4-AP and 3A-4MetP can block active Kv channels in the CNS. Therefore, this study represents a remarkable contribution in the analysis of blocking Kv channels: 1.2, 2.1 , 2.1 / 6.4, 7.2, 7.3 and 7.2 / 7.3.

[0108] The above results demonstrated that 3A-4MetP is an active molecule in several Kv channels expressed in the Central Nervous System, being in the KCNA family channels where a higher affinity is observed (based on the results shown in the Shaker Kv and Kv 1 .2 channels). This fact results in the 3A-4MetP molecule being useful in the treatment of the symptomatology of CNS demyelinating diseases and / or diseases involving a mechanism of action related to potassium channel blockade.

[0109] Therefore, the present invention should not be construed as restricted except as required by the prior art and by the scope of the appended claims, i

Claims

Claims1 . A compound of formula (I):for use in the treatment of a Central Nervous System (CNS) disease in a subject in need thereof.

2. The compound for use according to claim 1 , wherein the CNS disease is selected from the group consisting of a disease associated with alterations in the functioning of voltage-dependent potassium (Kv) channels, and a demyelinating disease.

3. The compound for use according to claim 1 or 2, wherein the subject is human.

4. The compound for use according to claim 2 or 3, wherein the demyelinating disease is selected from the group consisting of: multiple sclerosis (MS), leukodystrophy (LEU) and encephalomyelitis (ENC).

5. The compound for use according to claim 4, wherein the demyelinating disease is multiple sclerosis (MS).

6. The compound for use according to claim 1 , wherein the disease associated with alterations in the functioning of voltage-dependent potassium (Kv) channels is an epileptic disorder.

7. A method of treatment of a Central Nervous System (CNS) disease comprising administering a compound of formula (I):in a subject in need thereof.

8. The method according to claim 7, wherein the CNS disease is selected from the group consisting of a disease associated with alterations in the functioning of voltage-dependent potassium (Kv) channels, and a demyelinating disease.

9. The method according to claim 7 or 8, wherein the subject is human.

10. The method according to claim 8, wherein the demyelinating disease is selected from the group consisting of: multiple sclerosis (MS), leukodystrophy (LEU) and encephalomyelitis (ENC).

11. The method according to claim 10, wherein the demyelinating disease is multiple sclerosis (MS).

12. The method according to claim 8, wherein the disease associated with alterations in the functioning of voltage-dependent potassium (Kv) channels is an epileptic disorder.

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