Compounds, synthetic intermediates, uses, pharmaceutical compositions and neuromodulation therapy methods
阅读说明：本技术 化合物、合成中间体、用途、药物组合物以及神经调节治疗方法 (Compounds, synthetic intermediates, uses, pharmaceutical compositions and neuromodulation therapy methods ) 是由 R·A·雷默 A·S·海曼 于 2018-05-14 设计创作，主要内容包括：本发明属于药学、医学、化学和生物技术领域。本发明的化合物是一种肽,并且与最相似的肽类化合物相比,显示出令人吃惊的稳定性且易于处理。本发明的药物组合物包含所述肽类化合物,并且即使当口服给药时,也显示出令人吃惊的治疗结果。在一些实施方案中,与血加压素和大麻二酚的效果相比,本发明组合物的给药提供了优异的治疗结果。在一些实施方案中,在惊厥的治疗性和预防性治疗、疼痛阈值的调节和重要的神经保护中,本发明组合物的口服给药在神经调节方面提供了显著的且令人吃惊的结果,还大大减少了多发性硬化的临床症状。(The present invention belongs to the fields of pharmacy, medicine, chemistry and biotechnology. The compounds of the present invention are peptides and exhibit surprising stability and ease of handling compared to the most similar peptidic compounds. The pharmaceutical compositions of the present invention comprise said peptide compounds and show surprising therapeutic results even when administered orally. In some embodiments, administration of the compositions of the present invention provides superior therapeutic results compared to the effects of vasopressin and cannabidiol. In some embodiments, oral administration of the compositions of the present invention provides significant and surprising results in terms of neuromodulation, as well as substantially reducing the clinical symptoms of multiple sclerosis, in the therapeutic and prophylactic treatment of convulsions, modulation of pain thresholds, and important neuroprotection.)
1. A compound of the formula, and/or modified forms thereof, cyclic, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms thereof, forms modified with other functional groups, forms modified with amino acids or peptides including unnatural amino acids, D-amino acids, salts thereof, and/or combinations thereof:
R1-N-AA1-K-AA2-R2
wherein:
AA1is an amino acid selected from F, W, L, I, V, P, G;
AA2is hydrogen or an amino acid selected from F, W, L, I, V, P, G;
when R is2When it is an amino acid L, R1Is absent, or R1Is hydrogen or amino acid V; and is
When AA2When it is hydrogen, R2Is absent, or when R1When it is hydrogen, R2Is hydrogen or an amino acid L.
2. The compound of claim 1, modified, cyclized, modified with amide, alkyl, alkoxy, halogen, hydroxy, or polyethylene glycol groups, or modified with other functional groups, modified with amino acids or peptides including unnatural amino acids and D-amino acids, salts thereof; and/or combinations thereof.
3. Synthetic intermediates useful for preparing pharmaceutical compounds characterized by comprising a peptide compound of the formula and/or modified forms thereof, cyclic, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms thereof, modified forms with other functional groups, modified forms with amino acids or peptides including unnatural amino acids, D-amino acids, salts thereof, and/or combinations thereof:
R1-N-AA1-K-AA2-R2
wherein:
AA1is an amino acid selected from F, W, L, I, V, P, G;
AA2is hydrogen or an amino acid selected from F, W, L, I, V, P, G;
when R is2In the case of the amino acid L,R1is absent, or R1Is hydrogen or amino acid V; and is
When AA2When it is hydrogen, R2Is absent when R1When it is hydrogen, R2Is hydrogen or an amino acid L.
4. Use of a peptide compound of the formula and/or modified forms thereof, cyclic, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms thereof, forms modified with other functional groups, forms modified with amino acids or peptides including unnatural amino acids, D-amino acids, salts thereof, and/or combinations thereof:
R1-N-AA1-K-AA2-R2
wherein:
AA1is an amino acid selected from F, W, L, I, V, P, G;
AA2is hydrogen or an amino acid selected from F, W, L, I, V, P, G;
when R is2When it is an amino acid L, R1Is absent, or R1Is hydrogen or amino acid V; and is
When AA2When it is hydrogen, R2Is absent, or when R1When it is hydrogen, R2Is hydrogen or an amino acid L,
characterized in that the use is for the preparation of a product of pharmaceutical value selected from ligands for diagnostic use and therapeutic or prophylactic medicaments for mammals.
5. Use according to claim 4, characterized in that said compound is selected from: NFK, NWK, NLK, NFKF, NWKF, NLKF, NFKW, NWKW, NLKW, NFKL, NWKL, NLKL, VNFK, VNWK, VNLK, modified forms thereof including cyclic, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms, forms modified with other functional groups, or forms modified with amino acids or peptides including unnatural amino acids, D-amino acids, salts thereof; and/or combinations thereof.
6. Use according to claim 4, for the preparation of a medicament for the treatment of: disorders of energy and/or lipid metabolism; hypertension, intestinal motility disorders; the immune system; balancing calcium circulation; thyroid disorders, reproductive organ disorders, obesity, diabetes, immune system/inflammatory diseases or disorders, osteopenia, osteoporosis, cancer.
7. Use according to claim 4, for the preparation of a medicament for the analgesia and/or treatment of migraine, pain, neuropathic pain in a mammal.
8. Use according to claim 4, for the preparation of a medicament for the therapeutic or prophylactic treatment of convulsions in a mammal.
9. Use according to claim 4, for the preparation of a neuromodulation or neuroprotection medicament for the treatment of psychotic disorders, anxiety, schizophrenia or bipolar disorder, Alzheimer's disease, Parkinson's disease, autism, epilepsy, multiple sclerosis.
10. A pharmaceutical composition for modulating metabolic function in a mammal, characterized by comprising a pharmaceutically acceptable carrier; and a compound of claim 1 as an active ingredient.
11. A pharmaceutical composition for analgesia in a mammal, characterized by comprising a pharmaceutically acceptable carrier; and a compound of claim 1 as an active ingredient.
12. A pharmaceutical composition for the therapeutic or prophylactic treatment of convulsions in a mammal, characterized by comprising a pharmaceutically acceptable carrier; and a compound of claim 1 as an active ingredient.
13. A pharmaceutical composition for therapeutic or prophylactic neuromodulation and/or neuroprotection in a mammal, characterized by comprising a pharmaceutically acceptable carrier; and a compound of claim 1 as an active ingredient.
14. A pharmaceutical composition for the therapeutic or prophylactic treatment of multiple sclerosis in a mammal, characterized by comprising a pharmaceutically acceptable carrier; and a compound of claim 1 as an active ingredient.
15. Pharmaceutical composition according to any one of claims 10 to 14, in the form of tablets, gels, oral liquids or syrups, capsules, suppositories, injectable solutions, inhalable forms or adhesive forms.
16. Pharmaceutical composition according to claim 15, characterized in that it is a dosage form for human use comprising from 4 to 800 μ g/kg of active ingredient.
17. Method for in vitro diagnosis of the presence, number and/or location of cannabinoid and/or muscarinic receptors, as well as for assessing the binding of other compounds to said receptors, characterized in that it comprises: contacting a compound of claim 1 with a biological sample containing one or more of said receptors; the binding is then detected by visual, optical, spectroscopic, chemical and/or radioactive signals.
Technical Field
The present invention belongs to the field of medicine science, medicine, chemistry and biotechnology. More specifically, the invention describes a compound and its use for the preparation of ligands of diagnostic and/or therapeutic value, synthetic intermediates in the preparation of compounds of pharmaceutical value, the use of compounds for the preparation of medicaments, pharmaceutical compositions containing said compounds and methods of treatment. The compounds of the present invention are peptidic and exhibit surprising stability and ease of handling compared to more closely related peptidic compounds. The pharmaceutical compositions of the present invention comprise said peptide compounds and show surprising therapeutic results even when administered orally. In some embodiments, administration of the compositions of the present invention provides superior therapeutic results compared to the effects of vasopressin (heppressin) and cannabidiol. In some embodiments, oral administration of the compositions of the present invention provides important and surprising results in the therapeutic or prophylactic treatment of seizures, modulation of pain thresholds, and important neuroprotection, with greatly reduced clinical symptoms of multiple sclerosis.
Background
The peptide compounds of the invention are surprisingly stable at extreme temperatures, do not cause fibrillation problems, and are easy to handle in the preparation of products of pharmaceutical value, including ligands and pharmaceutical compositions for use in diagnostics.
The peptide compounds most closely related to the invention are vasopressin and its biologically active, large-size variants. However, vasopressin presents technical difficulties such as instability and a tendency to fibrillate, which limit its use in pharmaceutical formulations (Bomar MG & Galane AK, Modulation of the cannabinoid receptors by pressor peptides.520-524). These limitations have reduced the initial enthusiasm for vasopressin: its natural tendency to aggregate/fibril formation greatly limits the range of concentrations possible in synthetic, pharmaceutical preparation and therapeutic applications. The same literature (Bomar & Galande) even speculates that vasopressin self-assembly may be physiologically relevant and may have potential pathogenicity or neurotoxicity, similar to the amyloid peptide fibrils associated with Alzheimer's disease, Parkinson's disease and type II diabetes.
The results of the present invention are surprising in view of the closest background and the inventors have not found in the literature any report or suggestion that the peptidic compounds of the present invention would be more stable and easier to handle than vasopressin and therefore in this respect the present invention has unexpected improvements and unexpected importance.
The cannabinoid system, which comprises CB1 and CB2 receptors and their endogenous ligands, acts on a variety of metabolic functions including controlling food intake, energy and/or lipid metabolism, regulating intestinal motility, immune system, calcium circulation balance, etc. Cannabinoid receptors are widely expressed in the brain, including the cortex, hippocampus, amygdala, pituitary and hypothalamus. CB receptors, particularly CB1, have been identified in many peripheral organs and tissues, including thyroid, adrenal gland, reproductive organs, adipose tissue, liver, muscle and gastrointestinal tract.
The Bomar & Gallande article also shows that the N-extended form of vasopressin (e.g., RVD-Hp, VD-Hp) has shown agonist effects at the CB1 receptor, and thus, differences in only two or three amino acids interfere with the peptide's ability to exhibit either antagonistic or agonistic effects, thereby producing the opposite effect on CB 1. Furthermore, the signaling pathways of different known peptides differ from the classical pathway mediated by the G protein, making extrapolation according to the literature reports available today unreliable. Similar collision effects may also occur with the CB2 receptor.
The literature also clearly shows that, although vasopressin and the known extended-size variants are therapeutically active, peptide compounds with a size of less than 6 amino acids do not provide a therapeutic effect (Szlavicz et al, 2015; Dvor a csk pu et al, 2016; Bomar & Galande, 2013; Bauer et al, 2012).
In contrast, the present invention surprisingly demonstrates that: in addition to providing a more potent therapeutic effect in the known uses of vasopressin and the extended-size variants, it also provides other uses not previously known or suggested. In addition, small changes in peptide size may not only result in substantial and unexpected changes in therapeutic effect, but may also result in no effect at all.
It should also be noted that studies using peptides similar to vasopressin (pephans) have faced several challenges. For example, manipulation of the larger peptides is hindered by fibril aggregation/formation. Furthermore, the metabolic stability of the peptides in vivo is limited and the data of each pharmacological experiment are difficult to interpret without pharmacokinetic analysis, leading to confusion in the literature. Although studies have been directed to the administration of vasopressin (Hp) and RV-Hp, endogenous peptides naturally occurring at physiological levels appear to be only RVD-Hp (pepcan 12) and pepcan 23.
In this context, a variety of compounds have been detected in the art to have cannabinoid receptor modulating activity. Among others, some are aimed at developing drugs for weight loss and waist thinning, such as rimonabant (rimonabant). However, this compound was subsequently associated with an increased occurrence of human mental illness and was removed from the world market.
The compounds of the present invention also provide advantages as good candidates for alternative cannabinoid compounds, such as cannabidiol. Although cannabidiol has proven to be effective, regulatory issues have been faced due to its source (Cannabis sativa) plants. The present invention provides an additional therapeutic approach for patients with seizures and difficult to obtain medication, based on a peptidic compound and without the use of derivatives of cannabis. The results show that the compounds of the invention provide other surprising technical advantages in use when used as anticonvulsants, including increased therapeutic efficacy, oral use, lower doses, fewer occurrences of side effects (such as collapse and epistaxis), and other technical advantages.
Pilocarpine (often referred to as "Pilo") is an alkaloid extracted from the leaves of the pilocarpus plant (pilocarpus jaborandi), a plant used for centuries by indians living in brazil, the bark of the tree, the garden balsam (Tupi-Guarani), which utilize its properties to produce sweat and saliva. Pilocarpine is a non-specific muscarinic agonist, slowly degrades, has no effect on nicotine receptors, and is administered by Brazilian doctorsCoutinoh was introduced into clinical practice in 1874 by extracts of pilocarpus leaves to obtain diaphoretic (producing sweat) and sialagogue (producing saliva) effects.
Although pilocarpine has pharmaceutical properties, it induces the occurrence of convulsions at high concentrations and is therefore used as a relevant experimental model. Pilocarpine-induced seizures cause neurotoxicity at the cellular level and may be associated with increased brain oxidative stress and changes in certain amino acid concentrations (Santos et al, 2011).
Administration of pilocarpine in this experimental model results in severe brain damage, neurotoxicity, and often ends up in death of the animal. However, prior to this finding pilocarpine elicits cholinergic changes that can induce Status Epilepticus (SE) associated with the action of convulsive stereotypes. Pilocarpine can induce status epilepticus by administering it directly to the brain or by intraperitoneal administration. In some experiments described in this patent application, which used the substance, its detrimental neuronal/intracranial effects have been inhibited or prevented by the compositions of the invention: a significant proportion of the animals subjected to these experiments had no symptoms associated with brain damage and survived without significant damage compared to the group of animals treated with other known substances.
Much understanding of the mechanisms of epilepsy has been derived from studies in animal experimental models, particularly in rats and mice. In this context, pilocarpine is administered to rodents that mimic human Epilepsy (ELT), and is commonly referred to as the "pilocarpine model". This model was developed by Turski et al in 1983, and is currently one of the most widely used models of epilepsy, since its histological, biochemical, pharmacological, electrophysiological and behavioral characteristics (Turski et al, 1983) all similarly reproduce those found in human ELT carriers.
The pilocarpine model can also be used to demonstrate changes in muscarinic receptors, such as the occurrence of salivation. Acetylcholine plays an important role in cognitive functions (e.g., learning and memory) through its muscarinic receptors (machrs). machrs are receptors that form complexes with G protein receptors in the cell membranes of certain neurons and other cells. They play a variety of roles, including as terminal receptors in the parasympathetic nervous system that are stimulated by acetylcholine released from the postganglionic fibers.
One of the muscarinic receptors is known as a muscarinic receptor because it is more sensitive to muscarinic receptors than to nicotine. Its counterpart is the nicotinic acetylcholine receptor (nAChR), an ion channel receptor that is also important in the autonomic nervous system. Many drugs and other substances, such as pilocarpine and scopolamine (escopolamine), manipulate these two different receptors as selective agonists or antagonists.
machrs are best characterized in transmembrane receptors (7TM) and are widely expressed in the Central Nervous System (CNS). 5mAChR subtypes (M1, M2, M3, M4 and M5) were cloned and are generally divided into two distinct classes based on signal transduction. mAChRM1, M3, and M5 are subtypes that signal and activate phospholipase C and mobilize intracellular calcium through the Gq/11 protein. However, mAChRM2 and M4 inhibit adenylate cyclase and reduce intracellular concentration of cAMP mainly through Gi/o protein. The major mAChR in the CNS is the M1 subtype, which is located in the cortex, hippocampus, striatum, and thalamus, where it is present postsynaptic. M2 machrs are located primarily in the brainstem and thalamus, although also in the cortex, hippocampus and striatum, where they control acetylcholine release. The levels of machrs of M3 and M5 were expressed in the CNS much lower than M1 or M2 machrs, but M3 machrs are present in the cortex and hippocampus, while the location of M5 machrs in the substantia nigra (substantia nigra) is very discrete. M4 machrs are present in many regions of the brain, including the cortex and hippocampus, but are more prominent in the striatum, where they are thought to play a role in controlling dopamine release and regulating spontaneous activity (nocosor activity).
Given the wide and diverse distribution of machrs in the CNS, it is not surprising that all subtypes have been evaluated as potential drug targets. Some of these targets are widely recognized in the literature as M1 being involved in alzheimer's disease, while others are relatively new, such as M5, which has been linked to drug abuse dependence and addiction.
Multiple sclerosis is a neuroinflammatory disease of the central nervous system, has a strong neurodegenerative component, and has a great influence on the health of patients. There are still few satisfactory treatment options available for this disease and there is a great need to develop alternatives, as is the case with the present invention.
Multiple sclerosis is characterized by the destruction of oligodendrocytes and neurons, resulting in heterogeneous and cumulative clinical symptoms. Currently available treatments are only partially effective and are primarily directed at the inflammatory phase of the disease. However, the neurodegenerative component of this disease remains the greatest challenge for new therapeutic approaches. The present invention is directed to fill this important gap.
Although the exact cause of the disease is not clear, it is postulated that autoreactive T lymphocytes play a central role in their pathophysiology. The most common animal model of multiple sclerosis is Experimental Autoimmune Encephalomyelitis (EAE) because they share many physiological and clinical features. There are several models of EAE that reflect different clinical, immunological and histological aspects of human multiple sclerosis. The actively induced model of EAE in mice is robust (robust) and provides reproducible results, particularly useful for finding therapeutic agents by using transgenic mice challenged by autoimmune neuroinflammation. The EAE model is often used as a "proof of principle" of the efficacy of new treatment strategies for multiple sclerosis.
Disclosure of Invention
The present invention solves many of the known problems in the prior art, such as providing: peptide compounds that are more stable and easier to handle than vasopressin; useful as synthetic intermediates for the preparation of compounds of pharmaceutical value; the use of said compounds for the preparation of ligands of diagnostic value for the cannabinoid and/or muscarinic system; the use of said compounds for the preparation of a medicament improved compared to those containing vasopressin or cannabidiol; a pharmaceutical composition for regulating metabolic function and/or for improving analgesia compared to a composition containing vasopressin; an immunomodulatory pharmaceutical composition; a neuromodulatory, neuroprotective pharmaceutical composition for the therapeutic or prophylactic treatment of convulsions and/or multiple sclerosis; a method of treatment; a molecular entity providing these and/or other technical effects without the disadvantages resulting from the use of vasopressin, such as instability, formation of aggregates and/or lower therapeutic effect, and without the inconveniences resulting from the use of cannabinoid substances, such as collapse and epistaxis, etc.
It is a further object of the present invention to provide a peptidic compound which has high stability at extreme temperatures and/or is easy to handle, and which is particularly suitable for use in pharmaceutical formulations and medicaments. The peptidic compounds of the invention are particularly advantageous in terms of administration, bioavailability and/or therapeutic effect in animals compared to other peptides, such as vasopressin and known variants, which are generally of larger size (9-15 amino acids). In addition to providing previously unknown uses and being surprising in various aspects, the peptidic compounds of the present invention also provide oral administration to a mammal and provide higher therapeutic effects than vasopressin.
It is another object of the present invention to provide a compound substitute for cannabidiol which is advantageous in terms of preparation, handling, use and safety and which can replace cannabidiol in all or almost all of the applications already described.
It is another object of the present invention to provide a peptide compound useful as a receptor ligand for muscarinic and/or cannabinoid systems. Thus, the peptidic compounds of the present invention are useful for diagnostic applications. Another object of the invention is an in vitro method for diagnosing the presence, location and/or number of cannabinoid and/or muscarinic receptors as well as assessing the binding of other compounds to such receptors.
The compounds of the invention are also useful for modulating muscarinic and/or cannabinoid receptors by: by modulating the CB1 receptor, the CB2 receptor, both, by modulating the binding or action of other substances that interact in the cannabinoid system, by modulating proteases or peptidases that lead to the production or degradation of active peptides in the cannabinoid system, or combinations thereof.
Another object of the present invention is a synthetic intermediate in the preparation of compounds of pharmaceutical and/or diagnostic value, comprising the peptidic compounds of the invention, which may also comprise chemical modifications, substitutions, comprising other functional groups.
Another object of the present invention is the use of the peptidic compounds of the present invention for the preparation of improved pharmaceutical compositions for modulating metabolic function in a mammal.
Another object of the present invention is the use of the peptide compounds of the present invention for the preparation of improved analgesic pharmaceutical compositions.
Another object of the present invention is the use of the peptide compounds of the present invention for the preparation of neuromodulatory substances and/or neuroprotective medicaments in mammals.
Another object of the present invention is the use of the peptidic compounds of the present invention for the preparation of a medicament for the therapeutic or prophylactic treatment of epileptic seizures in a mammal. Furthermore, the administration of the peptide compounds of the present invention to animals provides important and surprising technical advantages, including superior anticonvulsant activity compared to cannabidiol and vasopressin, the use of which (vasopressin) as an anticonvulsant is the subject of co-pending patent applications by the same inventors.
Another object of the present invention is the use of the peptidic compounds of the present invention for the preparation of a medicament for the therapeutic or prophylactic treatment of multiple sclerosis in a mammal.
Other objects of the invention are methods of treatment for: therapeutic or prophylactic treatment of metabolic disorders, pain, seizures, multiple sclerosis, and for neuromodulation and/or neuroprotection.
Another object of the present invention is an improved pharmaceutical composition for modulating metabolic function in a mammal.
Another object of the present invention is an improved analgesic pharmaceutical composition.
Another object of the present invention is a neuromodulatory and/or neuroprotective pharmaceutical composition for a mammal.
Another object of the present invention is an improved pharmaceutical composition for the therapeutic or prophylactic treatment of seizures in mammals.
Another object of the invention is a pharmaceutical composition for the therapeutic or prophylactic treatment of multiple sclerosis in a mammal.
The compounds of the present invention are peptidic compounds of the formula and/or modified forms thereof, cyclic, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms thereof, modified forms with other functional groups, modified forms with amino acids or peptides including unnatural amino acids, D-amino acids, salts thereof, and/or combinations thereof:
R1-N-AA1-K-AA2-R2
wherein:
AA1is an amino acid selected from F, W, L, I, V, P, G;
AA2is hydrogen or an amino acid selected from F, W, L, I, V, P, G;
when R is2When it is an amino acid L, R1Is absent, or R1Is hydrogen or amino acid V; and
when AA2When it is hydrogen, R2Is absent, or when R1When it is hydrogen, R2Is hydrogen or an amino acid L.
The compounds of the invention are synthetic and different from the known natural forms and can be used for the preparation of products of pharmaceutical value selected from ligands for diagnostic use and therapeutic or prophylactic medicaments for mammals.
In one embodiment, AA1F, W or L.
In one embodiment, R1And R2Are all hydrogen.
In one embodiment, the compounds of the invention are selected from: NFKF, NWKF, NLKF, NFKW, NWKW, NLKW, NFKL, NWKL, NLKL, VNFK, VNWK, VNLK, and modified or cyclic forms thereof, amidated, alkylated, alkoxylated, halogenated, hydroxylated, or pegylated forms thereof, forms modified with other functional groups and amino acids or peptides including non-natural forms such as the d-amino acid form, salts thereof; and/or combinations thereof.
In one embodiment, the peptidic compounds of the invention are selected from: NFK tripeptide, NFKF tetrapeptide, NFKL tetrapeptide, and modified or cyclic forms thereof, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms, modified with other functional groups and with amino acids or peptides including non-natural forms such as d-amino acid forms, salts thereof; and/or combinations thereof.
The pharmaceutical compositions of the invention comprise the peptide compounds of the invention and a pharmaceutically acceptable carrier, and may be in the form of tablets, gels, oral liquids or syrups, capsules, suppositories, injectable solutions, inhalable forms or adhesive forms (adhesive forms), optionally containing other active ingredients.
These and other objects of the present patent application will be immediately recognized by those skilled in the art and companies interested in this field will be described in sufficient detail in the following specification.
Drawings
The following detailed description and the annexed drawings set forth in detail certain illustrative aspects and implementations of the invention, and are set forth in greater detail to provide further support to those skilled in the art, so that s/he may understand and reproduce the inventive concept of the present invention in any of its embodiments. These details or figures should not be construed as limiting, but merely as illustrating some embodiments of the invention.
The data on the figure show the results of concentration measurements by HPLC on each of these actives after freezing for 24 hours or after heating at 100 ℃ for 10 minutes (n-2), respectively, also shows statistically significant data with asterisks indicating (＊) p <0.05 compared to control, (＊＊＊) p <0.005 compared to control and (＊＊＊＊) p <0.0001 compared to control.
Figure 2 shows the results of testing compounds of the invention in an embodiment of NFKF, both in pilocarpine model, compared to the results of the vasopressin test, giving the time to the first salivation after administration of the following therapeutic doses: control (saline), vasopressin (Hp or PVNFKFLSH, 0.551334 μmol/kg), vasopressin (0.91889 μmol/kg), compound of the invention in an embodiment of NFKF (0.540882 μmol/kg), NFKF (0.901469 μmol/kg), compound PEP-19(DIIADDEPLT, 0.908117 μmol/kg), asterisks indicate statistical significance, (＊) p <0.05 compared to control, (＊＊) p <0.01 compared to control, and # indicates p <0.05 compared to Hp 0.91889 μmol/kg.
Figure 3 shows the results of testing the compounds of the invention in an embodiment of NFKF as anticonvulsants, and comparing them to the results of the vasopressin test as anticonvulsants, both in the pilocarpine model the time percentages of the first seizure after administration are given (relative to control) for control (saline), vasopressin (Hp or PVNFKFLSH, 0.551334 μmol/kg), vasopressin (0.91889 μmol/kg), the compounds of the invention in an embodiment of NFKF (0.540882 μmol/kg), NFKF (0.901469 μmol/kg), the compound PEP-19(DIIADDEPLT, 0.908117 μmol/kg), asterisks indicate statistical significance (＊) for p < 0.05; (＊＊) for p <0.01 for control, and + for p <0.05 for Hp 0.91889 μmol/kg.
FIG. 4 shows the results of testing of compounds of the invention in an embodiment of NFKF in the pilocarpine model, indicating the time to first salivation following administration of control, cannabidiol (30mg/kg) or NFKF (500 μ g/kg). There was no statistical significance between the data between the control and the other test compounds under the conditions tested.
FIG. 5 shows the results of testing compounds of the present invention in an embodiment of NFKF as an anticonvulsant in the pilocarpine model, indicating the time to the first convulsion following administration of cannabidiol (30mg/kg) or NFKF (500 μ g/kg). asterisks indicate statistical significance (＊＊) p <0.02 versus control and (＊＊＊) p <0.002 versus control.
Figure 6 shows the results of a neuroprotective test with a compound of the present invention in an embodiment of NFKF in a pilocarpine model, indicating the survival/death curves of the animals following NFKF administration. Survival curves of animals given to control (saline only) are shown in a); the survival curve of animals given cannabidiol 30mg/kg is shown in B); the survival curve of animals given NFKF500 μ g/kg is shown in C); in D), all curves are shown in one graph. It is noteworthy that only 2 animals died in the group treated with the neuroregulator substance NFKF 500. mu.g/kg. The remaining animals in the NFKF group (3 in total) remained alive for more than one week, while virtually all other animals died in less than 30 minutes. Thus, the mean survival time for this group was significantly different and much higher than the other groups. Furthermore, when the NFKF group (500 μ g/kg) was compared with the group given cannabidiol (30mg/kg), the survival rate of the first group (i.e., the NFKF500 μ g/kg group) was 3 times higher, even when this compound was used at a
Figure 7 shows the results of neuroprotection tests performed on different embodiments of the compounds of the invention (NFKF, NFKL and NFK, all 600 μ g/kg), previously administered to groups of animals (n ═ 5 in each group) followed by pilocarpine, showing the survival/death curves of the animals after 24 hours in the model tested. The vertical axis shows the number of dead animals. The control was saline administration prior to pilocarpine administration.
Figure 8 shows the results of testing the compounds NFKF, NFKL, NFK, FKF, FKL and the dipeptides NF, FK, KF and KL of the present invention in the pilocarpine model, indicating the time to first salivation after administration of the control or test compound ＊ has a p <0.05 compared to the control, the results reflecting the average results of 7 animals in the control group and the average results of 6 animals in the group treated with NFK.
FIG. 9 shows the results of testing the compounds NFKF, NFKL, NFK, FKF, FKL and the dipeptides NF, FK, KF and KL of the present invention in the pilocarpine model, indicating the time at which the first signal occurs following administration of the control or test compound.
FIG. 10 shows the results of testing the compounds NFKF, NFKL, NFK, FKF, FKL and the dipeptides NF, FK, KF and KL of the present invention in the pilocarpine model, indicating the time to onset of seizures after administration of the control or test compound, ＊ p <0.05 compared to control and ＊＊ p <0.001 compared to control.
FIG. 11 shows the results of testing the compounds NFKF, NFKL, NFK, FKF, FKL and the dipeptides NF, FK, KF and KL of the present invention in the pilocarpine model, indicating the time to death after control or test compound administration. ＊ compared to control p < 0.05.
Figure 12 shows the results of the binding assay of various compounds to the CB1 receptor in an assay with an anti-CB 1 antibody sensitive to activation/conformational change of the receptor indicating the percentage value of binding to the CB1 receptor relative to control ＊ versus control p < 0.05.
Figure 13 shows an overview of the three-dimensional structure of a GPCR, in this case a subtype 1 cannabinoid receptor. Reference is made to seven transmembrane helices (I-VII), intracellular loops (ICL1 and ICL2) and extracellular loops (ECL2 and ECL 3).
Fig. 14 shows the overlay of the AM6538 structure at the crystal structure (PDB 5TGZ) and the results obtained after verification by the Goldscore function (when the figure is color, the crystal structure is purple and the results of the Goldscore function are blue/cyan, although for the present application such color is not relevant).
Figure 15 shows the major interaction observed for AM6538 at the CB1 receptor binding site.
Figure 16 shows the major interactions observed for rimonabant at the CB1 receptor binding site.
Figure 17 shows the major interaction observed for cannabidiol at the CB1 receptor binding site.
Figure 18 shows the major interactions observed for peptidic compounds of the invention in the NFKF embodiment at the CB1 receptor binding site.
Figure 19 shows the results of a method to obtain the CB2 receptor structure. In a), the three-dimensional structure of the CB2 receptor obtained is shown; in B), the Ramachandran diagram of the obtained human CB2 model is shown.
Figure 20 shows the major interaction between the CB2 receptor and the AM6538 ligand.
Figure 21 shows the major interaction between the CB2 receptor and rimonabant.
Figure 22 shows the major interaction between the CB2 receptor and cannabidiol.
Figure 23 shows the major interactions between the CB2 receptor and the compounds of the present invention in the NFKF embodiment.
Figure 24 shows pain threshold results applied to the back of the right paw of a rat expressed as force in grams when the animal responded by kicking, the force in grams required to induce the response was the pain threshold, the vertical axis represents the pressure applied at the legs.
FIG. 25 shows the results of a test for controlling multiple sclerosis with a compound of the invention in an embodiment of NFKF A) shows the clinical score curve for Wild Type (WT) mice (i.e., normal) receiving induction of EAE with MOG, B) shows the clinical score curve in an EAE model for mice receiving MOG-induced, knocked-out endopeptidase gene 24.15 (i.e., transgenic and predisposed to having symptoms of multiple sclerosis), C) shows the clinical score curve in an EAE model for mice knocked-out for endopeptidase gene 24.15 (i.e., transgenic and predisposed to having symptoms of multiple sclerosis) after receiving MOG induction and neuroprotection with NFKF ＊ has a P of 24.15KO + NFKF <0.05 compared to WT and a P of 24.15KO <0.05 compared to WT.
Detailed Description
The inventive concepts of several objects of the present invention are peptide compounds and/or modified forms thereof, cyclic, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms thereof, modified forms thereof with other functional groups, with amino acids or peptides including unnatural amino acids, D-amino acids, salts thereof, and/or combinations thereof, of the formula:
R1-N-AA1-K-AA2-R2
wherein:
AA1is an amino acid selected from F, W, L, I, V, P, G;
AA2is hydrogen or an amino acid selected from F, W, L, I, V, P, G;
when R is2Is amino acid L, R1Is absent, or R1Is hydrogen or amino acid V; and
when AA2When it is hydrogen, R2Is absent, or when R1When it is hydrogen, R2Is hydrogen or an amino acid L.
The compounds of the invention are synthetic and different from the known natural forms and can be used for the preparation of products of pharmaceutical value selected from ligands for diagnostic use and therapeutic or prophylactic medicaments for mammals.
In one embodiment, AA1F, W or L.
In one embodiment, R1And R2Are all hydrogen.
In one embodiment, the compounds of the invention are selected from: NFKF, NWKF, NLKF, NFKW, NWKW, NLKW, NFKL, NWKL, NLKL, VNFK, VNWK, VNLK and modified or cyclic forms thereof, amidated, alkylated, alkoxylated, halogenated, hydroxylated or pegylated forms thereof, forms modified with other functional groups and with amino acids or peptides including non-natural forms such as the d-amino acid form, salts thereof; and/or combinations thereof.
In one embodiment, the peptidic compounds of the invention are selected from: NFK tripeptide, NFKF tetrapeptide, NFKL tetrapeptide, and modified or cyclic forms thereof, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms, modified with other functional groups and with amino acids or peptides including unnatural amino acids such as d-amino acid forms, salts thereof; and/or combinations thereof.
The peptide compounds have surprisingly high stability at extreme temperatures and are easy to handle, and are particularly suitable for pharmaceutical formulations and medicaments. The peptidic compounds of the invention are particularly advantageous in terms of administration, bioavailability and/or therapeutic effect in animals compared to other peptides, such as vasopressin and known variants, which are generally of larger size (9 to 23 amino acids). The peptide compounds of the present invention also provide for oral administration to mammals and provide therapeutic effects that are greater than the known therapeutic effects of vasopressin and can advantageously replace vasopressin in all or almost all of the applications already described.
The compounds of the invention are also synthetic intermediates in the preparation of compounds of pharmaceutical value, comprising the peptidic compounds of the invention, and comprising chemical modifications, substitutions, inclusion of other functional groups.
The compounds of the present invention are advantageous alternatives to cannabidiol, are advantageous in terms of preparation, handling, use and safety, and are useful in replacing cannabidiol in all or almost all of the applications already described.
The compounds of the invention are ligands for muscarinic and/or cannabinoid system receptors and may be used in diagnostic applications or for modulating muscarinic and/or cannabinoid receptors by: by modulating the CB1 receptor, the CB2 receptor, both, by modulating the binding or action of other substances that interact in the cannabinoid system, by modulating proteases or peptidases that result in the production or degradation of active peptides in the cannabinoid system, or combinations thereof. The test results presented in this patent application indicate that the compounds of the invention interact with and/or modulate the activity of cannabinoids and/or muscarinic receptors.
The compounds of the present invention are useful in improved pharmaceutical compositions for modulating metabolic function in a mammal.
The compounds of the present invention are useful in improved analgesic pharmaceutical compositions.
The compounds of the present invention are useful in pharmaceutical compositions for the therapeutic or prophylactic treatment of seizures in mammals. Administration of the peptide compounds of the present invention to animals provides important and surprising technical advantages, including superior anticonvulsant activity over cannabidiol and vasopressin, the use of vasopressin as an anticonvulsant is the subject of co-pending patent application PCT/BR2017/050313 by the same inventors.
The compounds of the present invention are useful in neuromodulation and/or neuroprotective pharmaceutical compositions in mammals. The test results presented in this patent application indicate that the compositions of the invention modulate neuronal effects and are also neuroprotective, anticonvulsant and useful in the treatment of multiple sclerosis.
For the purposes of the present invention, the following definitions are used.
Products of pharmaceutical value
In the context of the present application, "compound of pharmaceutical value" means any molecular entity, including the compounds described as being common inventive concepts of the present application, and also including molecular entities obtained by: chemically modified/derivatized or containing other functional groups, linear or branched side chains, altered hydrophilicity or hydrophobicity, etc., provided that they contain as core the entity R1-N-AA1-K-AA2-R2 as defined above, with the exception of natural and known entities.
Pharmaceutical composition
In the context of the present patent application, a "pharmaceutical composition" is understood to be any and all compositions containing active principles which act in a manner to maintain and/or restore homeostasis for prophylactic, palliative and/or therapeutic purposes, and which can be administered orally, topically, parenterally, enterally and/or intrathecally.
Pharmaceutically acceptable formulations
In the context of this application, "pharmaceutically acceptable formulation" means a formulation containing pharmaceutically acceptable excipients and carriers well known to those skilled in the art, such as convenient dosages and treatments developed for a particular composition that can be described in a number of treatment regimens, including oral, parenteral, intravenous, intranasal, intravitreal and intramuscular, intracerebral, intracerebroventricular and intraocular treatments and administrations and/or formulations.
Modified peptides
In the context of the present application, "modified peptide" is to be understood as meaning non-naturally occurring, artificially modified or synthetic peptides, including halide, cyclized, amidated, alkylated, alkoxylated, hydroxylated, pegylated forms, forms having further functional groups on any amino acid, or salt forms thereof, as well as amino acids or peptides in including non-natural forms such as d-amino acid forms. Peptidic compounds can be pegylated using techniques known to those skilled in the art, for example, with a reagent containing a succinimide group that preferentially reacts with a primary amine present in the N-terminal region of the peptide. The peptidic compounds of the invention may be alkylated on any amino acid using techniques known to those skilled in the art, including, for example, the Mitsuobu reaction described in Reichwein & Liskamp (Reichwein JF & Liskamp RMJ, Site-specific N-alkylation of peptides on the soluble phase, Tetrahedron Letters, Volume 39,
Cyclic or cyclic peptides
In the context of the present application, "cyclic peptide, cyclized peptide or cyclic peptide" is understood to be a peptide having a covalent bond between the two ends of a linear peptide molecule by any method known in the art, in particular by enzymatic activity. A cyclic peptide can be used instead of a linear peptide because it is more difficult to degrade because its ends or regions attacked by hydrolytic enzymes are not exposed as much as the linear peptide.
Agonists
In the context of the present application, "agonist" is understood to be an agent, drug, hormone, neurotransmitter or other signaling molecule which forms a complex with a receptor site, thereby triggering an active response of the cell.
Inverse agonists/antagonists
In the context of this application, an "inverse agonist or antagonist" is understood to be one or more agents (e.g., agents, drugs, hormones or enzymes) that bind to an agonist receptor and produce a pharmacological effect opposite to that of the agonist such that the effect of one agent partially or fully inhibits the effect of another agent. Specifically, when a compound acts in the presence of an agonist, but decreases its activity, it is an inverse agonist; antagonists are compounds that completely block agonist activity.
Equivalent dose for human body
In the present invention, the concept of "human equivalent Dose" is the Dose expected to produce an effect in humans of the same magnitude as the effect in animals at a given Dose, as described in "guidelines for industry Estimating the Maximum Safe Starting Dose in Initial Clinical trials for Therapeutics in addition health volumes", published by the central food and drug administration drug evaluation and research (CDER) 2005-7 months in Pharmacology and Toxicology. In the guidelines, conversion of observed animal dose (mg/kg) to human equivalent dose (mg/kg) requires dividing the results obtained in rats by 6.2 and dividing the results obtained in mice by 12.3. These values apply to a person having a standard weight of 60 kg. For other species or weights outside the standard weight range, the human equivalent Dose (DEH) can be calculated by the following formula: DEH-animal dose x in mg/kg (animal weight in kg/human weight in kg)0.33. The present guideline considersA safety range of 10 times the tested concentration limit is sufficient.
CB receptor ligands
In the context of the present patent application, "CB receptor ligand" is understood as a compound or molecule that interacts with a CB system and/or with a CB1 or CB2 receptor.
Modulating CB receptor function or cannabinoid system
In the context of the present patent application, "modulation of the function of a CB receptor" refers to an interaction which leads to an alteration of the biochemical activity of the CB receptor, in particular CB1 or CB 2. It will be appreciated that the change is positive when an antagonist or inverse agonist action occurs at the CB receptor and negative when an agonist action occurs at the CB receptor. The test results presented in the present patent application indicate that the compounds of the invention may interact with the CB1 receptor and/or the CB2 receptor and/or modulate the CB1 receptor and/or the CB2 receptor as allosteric modulators of the CB1 and/or CB2 receptor. Thus, the compounds of the present invention are useful for modulating the cannabinoid system by: the improvement of the natural system by modulating the CB1 receptor or the CB2 receptor, both, by modulating the binding or action of other substances that interact in the cannabinoid system, by modulating proteases or peptidases that lead to the production or degradation of active peptides in the cannabinoid system, or by protecting the natural molecule with the compounds of the invention, i.e., the compounds of the invention may also protect the natural system independently of binding to the receptor, or a combination of such effects.
Modulating the function of muscarinic receptors
In the context of the present application, the term "modulating the function of muscarinic receptors" is to be understood as an interaction leading to a modification of the muscarinic acetylcholine receptors (machrs) which play an important role in cognitive functions, such as learning and memory, controlling dopamine release, modulating spontaneous activity, which modulation may also be used to control alzheimer's disease and/or to control dependence on or addiction to drugs of abuse. It is understood that a change is positive when an antagonist or inverse agonist action occurs at a muscarinic receptor, and negative when an agonist action occurs at a muscarinic receptor. The tests presented in this patent application show that the compounds of the invention interact with and/or modulate muscarinic receptors.
Regulating metabolic function
In the context of the present application, the term is understood to include the regulation: energy and/or lipid metabolism; arterial hypertension, intestinal motility regulation; the immune system; the balance of calcium circulation, disorders associated with thyroid, peripheral organs and tissues including reproductive organs, adipose tissue, liver, muscle and gastrointestinal tract, can be used to treat obesity, diabetes, disease or immune/inflammatory disorders, osteopenia, osteoporosis, cancer. In this case, the peptidic compounds of the invention may also be used in pharmaceutical compositions for the treatment of metabolic disorders, including the prevention of overweight; regulating appetite; inducing satiety; prevention of weight gain following successful weight loss; increase energy consumption; aesthetic weight loss; or binge eating disorder.
Nerve-regulating substance
In the context of the present application, the term "neuromodulatory substance" or "neuromodulatory substance" is intended to modulate neuronal/neural function, including modulating brain, cortex, hippocampus, amygdala, pituitary, hypothalamic activity; adrenal gland. Neuromodulation includes beneficial modulation of neuroprotection against agents or disorders that lead to pathophysiological processes. The neuroprotective agent or compound is preferably used prior to (or during) the prodromal phase of the disease, which usually begins many years before the onset of symptoms. In the present invention, neuromodulatory substances may potentially be used for the therapeutic or prophylactic treatment of various neurological conditions or diseases, including essential tremor, migraine, pain, neuropathic pain, multiple sclerosis, amyotrophic lateral sclerosis, psychiatric diseases such as anxiety, schizophrenia or bipolar disorder, congenital diseases such as dementia, alzheimer's disease, parkinson's disease, autism, and may potentially be used to ameliorate the pathophysiological processes involved in the occurrence of seizures and/or epilepsy, as well as other clinical conditions associated with neuronal excitatory disorders or neuronal damage due to ischemia, hypoxia or other harmful conditions.
Although it is demonstrated in the present patent application that the compounds of the present invention bind to and/or modulate cannabinoid receptors, the surprising pharmaceutical effects of the present invention may be associated with an effect on CB1 and/or CB2 and/or muscarinic receptors, or may be associated with modulation of adenosine uptake, GGPR55, PPAR γ receptors, intracellular calcium levels, modulation of opioid receptors where the cannabinoid receptors form heterodimers, or a combination thereof. Thus, any therapeutic indication associated with these goals may benefit from the present invention.
The invention is further defined by the following clauses.
The peptide compounds are described above.
The use of the above-mentioned peptide compounds for the preparation of a product of pharmaceutical value selected from ligands for diagnostic use and therapeutic or prophylactic medicaments for mammals.
The use as described above, wherein AA1 and/or AA2 is F, W or L.
The use as described above, wherein the peptidic compound is selected from the group consisting of: NFKF, NWKF, NLKF, NFKW, NWKW, NLKW, NKKL, NWKL, NLKL, VNFK, VNWK, VNLK, as well as modified, cyclic forms, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms, forms modified with other functional groups, and forms modified with amino acids or peptides including non-natural forms such as the d-amino acid form, salts thereof; and/or combinations thereof.
The use as described above, wherein the peptidic compound is selected from the group consisting of: NFK tripeptide, NFKF tetrapeptide, NFKL tetrapeptide, and modified, cyclic forms, amidated, alkylated, alkoxylated, halogenated, hydroxylated, pegylated forms thereof, modified forms with other functional groups, and modified forms with amino acids or peptides, including non-natural forms such as d-amino acid forms, salts thereof; and/or combinations thereof.
Synthetic intermediates in the preparation of pharmaceutically valuable compounds comprising the above compounds.
The use of the above compounds as synthetic intermediates in the preparation of other compounds of pharmaceutical and/or diagnostic value.
The above compound, which is modified.
The use of the above compounds for the preparation of cannabinoid receptor ligands and/or muscarinic receptors.
The use of the above-mentioned compounds for the preparation of conjugates (binders) of diagnostic value in mammals.
The use of the above compounds for the preparation of a pharmaceutical composition for the regulation of metabolic function.
The application of the compound in preparing analgesic medicine composition.
The use of the above compounds for the preparation of an immunomodulating pharmaceutical composition.
The use of the above compounds for the preparation of neuromodulatory and/or neuroprotective medicaments in mammals.
The use of the above compounds for the preparation of a therapeutic or prophylactic anticonvulsant for mammals.
The use of the above compounds for the preparation of a medicament for the therapeutic or prophylactic treatment of multiple sclerosis in a mammal.
A pharmaceutical composition for modulating metabolic function in a mammal comprising a pharmaceutically acceptable carrier; and the above compound as an active ingredient.
A mammalian analgesic pharmaceutical composition comprising a pharmaceutically acceptable carrier; and the above compound as an active ingredient.
A neuromodulatory and/or neuroprotective pharmaceutical composition comprising a pharmaceutically acceptable carrier; and the above compound as an active ingredient.
A pharmaceutical composition for the therapeutic or prophylactic treatment of a seizure in a mammal comprising a pharmaceutically acceptable carrier; and the above compound as an active ingredient.
A pharmaceutical composition for the therapeutic or prophylactic treatment of multiple sclerosis in a mammal comprising a pharmaceutically acceptable carrier; and the above compound as an active ingredient.
A pharmaceutical composition comprising an NFK tripeptide, an NFKF tetrapeptide, an NFKL tetrapeptide, or a combination thereof.
The pharmaceutical composition as described above, in the form of a tablet (tablete), a tablet (compomid), a gel, an oral liquid or syrup, a capsule, a suppository, a solution for injection, an inhalable form or an adhesive form.
A therapeutic modulator of metabolic function in an animal comprising administering the peptide. In particular, for this application, the peptides of the invention are preferably modified so that their molecular weight is greater, thereby minimizing or preventing their passage through the blood-brain barrier.
A therapeutic method for the therapeutic or prophylactic treatment of pain comprising administering to an animal a compound as described above.
A therapeutic or prophylactic neuromodulatory and/or neuroprotective treatment method comprising administering to an animal the above compound.
A therapeutic method for the therapeutic or prophylactic treatment of seizures which comprises administering to an animal a compound as described above.
A therapeutic method for the therapeutic or prophylactic treatment of multiple sclerosis comprising administering to an animal a compound as described above.
The results of in vitro and in silico tests have shown that the compounds of the invention have advantages in the preparation of pharmaceutical compositions, their stability and therapeutic effect.
The compounds of the invention have proven to be much more stable than vasopressin, which, and the known variants thereof, also cause technical problems of fibrogenesis or fibrillation.
The results of in vivo testing in mammals show excellent therapeutic efficacy at low doses with no evidence of significant side effects.
The peptidic compounds of the invention have also proven to be suitable synthetic intermediates for the preparation of other compounds useful in diagnostic and/or therapeutic applications. The tests carried out in the present invention show that the compounds of the invention can be suitably modified with biotin known to the person skilled in the art, which biotin can be used in diagnostic formulations or kits. The peptidic compounds of the invention also show binding to the CB1 receptor in assays with anti-CB 1 antibodies sensitive to activation/conformational change of the receptor when modified/protected with biotin. Another object of the invention is an in vitro method for diagnosing the presence, number and/or location of cannabinoid, opioid and/or muscarinic receptors, as well as for assessing the binding of other compounds to these receptors.
The neuromodulatory/neuroprotective effects produced by administration of the compounds of the present invention in mammals can be evidenced by a reduction and/or absence of symptoms, brain damage, and death associated with administration of known harmful substances, such as pilocarpine. Tests carried out with the EAE/MOG model (the most common model for replicating multiple sclerosis) also show that administration of the peptide compounds of the invention to animals greatly reduces the clinical symptoms of multiple sclerosis, a disease essentially associated with neuronal damage.
In one embodiment, the invention provides the use of said compounds for the preparation of a neuromodulatory, neuroprotective medicament and/or for the therapeutic or prophylactic treatment of seizures in a mammal. Administration of a compound of the invention to an animal provides neuromodulatory, neuroprotective, anticonvulsant, and/or symptomatic multiple sclerosis (symptomatomatic multiple sclerosis) inhibitory activity; make oral administration feasible; does not have or does not involve the disadvantages resulting from the preparation, storage, transport and use of cannabinoid materials and provides additional advantages in the preparation of therapeutic products for mammals, their administration and/or effects. Furthermore, administration of the compounds of the present invention to animals provides important and surprising technical advantages, including superior anticonvulsant activity over vasopressin, the use of vasopressin as an anticonvulsant is the subject of co-pending applications by the same inventors.
The present invention describes the use of compounds for the preparation of pharmaceutical compositions useful for a variety of medical conditions, including those associated with the central nervous system. Surprisingly, although the active compounds of the present invention are peptides or primarily peptides, oral administration provides a brain effect in animals.
In one embodiment, in vivo testing with the compositions of the present invention shows surprising results regarding their neuroprotective activity. The compositions of the present invention provide surprising, effective and long-term protection against damage caused by subsequently administered substances known to be harmful to neurons when pre-administered to an animal. Thus, the neuroprotection provided by the compounds of the present invention is particularly useful as a treatment option for a variety of medical conditions, including those associated with neuronal excitability disorders, such as seizures.
In various embodiments, in vivo tests with the compounds of the present invention demonstrate surprising effects with respect to their anticonvulsant activity. When used as anticonvulsants, the compounds of the present invention also offer advantages as good candidates to replace cannabinoid compounds (e.g., cannabidiol) known to be useful as anticonvulsants. While cannabidiol has been shown to have an effect as an anticonvulsant, regulatory issues are faced due to its source (the cannabis plant). The present invention provides an additional treatment for patients with seizures and having difficulty in obtaining a drug, which is based on peptides, i.e. without the use of derivatives of cannabis. Furthermore, the results show that the compounds of the present invention provide other surprising technical advantages in use when used as anticonvulsants, including higher therapeutic efficacy, oral use, lower dosages, fewer side effects (such as collapse and epistaxis), and other technical advantages.
The molecular mechanism of how Cannabidiol (CBD) arrests seizures is not fully understood. The known CBD: inhibition of adenosine reuptake; is a 5-HT 1A agonist; is an antagonist of GPR55(CB 3); is a PPAR gamma receptor agonist; in addition to interacting with CB1 and CB2, intracellular Ca2+ was also increased. On the other hand, vasopressin has a major role through pERK1/2 and AKT. It is therefore surprising and difficult to explain why both the peptidic compounds of the invention and the CBD have an anticonvulsant effect.
In experimental convulsive model testing, administration of the pharmaceutical composition of the present invention to a mammal produces superior therapeutic effects at low doses compared to the dose of the reference compound cannabidiol. Furthermore, the test results show that the compositions of the present invention provide surprising technical advantages in use, including higher therapeutic efficacy, feasibility of oral use, absence of carrier oils (which in many cases can cause side effects), lower dosage and less occurrence of side effects (such as collapse and epistaxis).
In vivo comparative tests have demonstrated that the compounds of the present invention have superior activity and/or require fewer doses than vasopressin, the use of which as an anticonvulsant is the subject of a co-pending patent application by the same inventors.
The pharmaceutical compositions of the present invention are also useful for the treatment of diseases associated with modulation of the activity of the cannabinoid system, Cannabinoids (CB) and/or muscarinic receptors, which have several technical advantages and which do not have the known adverse effects of congeners (conners) available in the prior art.
Furthermore, as will be demonstrated in the examples below, the use of the compounds of the present invention in the preparation of neuromodulation, neuroprotection and/or anticonvulsant drugs provides for the delivery of drugs that can be orally administered to mammals. The test results show a significant cerebral effect, indicating that administration of the compounds of the invention allows the active ingredient to cross the blood brain barrier. Thus, the results demonstrate/support the utility of the compounds of the invention, whether they are active substances that act directly on the target (i.e. do not degrade during oral ingestion), or whether they are precursors that act on the target after chemical modification (in this case, they are characterized as prodrugs). The property of providing important effects even by oral administration is particularly desirable because mammalian native enzymes usually degrade peptides and proteins in the digestive tract and few drugs with peptide activity have proven to be viable. Surprisingly, however, the compounds of the present invention provide strong therapeutic effects (even when administered orally), in which case, even more surprisingly, they act in the brain.
Thus, regardless of the mechanism of action (which is not the subject of the present application), oral administration of the pharmaceutical composition of the invention provides important neuromodulation, neuroprotection, anticonvulsant, pain modulation and symptomatic multiple sclerosis, even avoiding death, clearly demonstrating the surprising degree and relevance of the technical problem solved.
The pharmaceutical composition of the present invention comprises the above compound and a pharmaceutically acceptable carrier, optionally further comprising other pharmaceutically acceptable active substances and/or salts thereof. The pharmaceutical compositions of the present invention may be administered in the form of tablets, gels, capsules, oral liquids or syrups, suppositories, injections or other suitable forms of administration for pharmaceutical and medical purposes.
The following examples are intended to illustrate some of the various methods of practicing the invention, but not to limit the scope thereof.
Comparative tests show that the compounds of the invention have superior in vitro stability at extreme temperatures compared to vasopressin.
In some embodiments, the neuromodulatory/neuroprotective/anticonvulsant effects of the compositions of the present invention have been evaluated in vivo by oral administration to animals. The pharmaceutical compositions of the invention are administered to mammals (Mus musculus or mice) at oral therapeutic doses with different embodiments of the compounds of the invention and compared to other compounds or saline controls. In these experiments, the test compound was administered orally 10 minutes prior to pilocarpine (intraperitoneal) administration. Pilocarpine hydrochloride (320mg/kg, Merck) dissolved in 0.9% sterile saline was administered intraperitoneally to induce SE (status epilepticus) (Turski et al, 1983). In the Turski model, neurotoxic effects begin approximately 15-25 minutes after Pilo injection, with the onset of motor and marginal seizures, the animals gradually progressed to persistent (clonic) seizure states that are characteristic of SE (Sanabria and Cavalheiro, 2000).
Comparative tests have also shown that the compounds of the present invention show superior in vivo therapeutic activity compared to vasopressin, the use of vasopressin as an anticonvulsant is the subject of co-pending patent application PCT/BR/2017050313 by the same inventors.
The compounds of the invention are important GPCR (cannabinoid receptor) ligands/modulators useful for modulating GPCR receptor activity under pathological conditions, as well as for modulating target GPCRs, and as vectors for other targeted therapeutic molecular entities for cells expressing GPCRs that dimerize with cannabinoid receptors. The compounds of the invention are also useful in therapy in combination with antibodies (especially monoclonal antibodies) selective for the activated/modulated conformation of the receptor. Such therapies offer the advantage of conferring high specificity, and potentially also reduce the administered dose.
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