WO2012013850A2 - Composé destiné à traiter des maladies neurodégénératives, des troubles cognitifs et/ou des démences - Google Patents

Composé destiné à traiter des maladies neurodégénératives, des troubles cognitifs et/ou des démences Download PDF

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WO2012013850A2
WO2012013850A2 PCT/ES2011/070539 ES2011070539W WO2012013850A2 WO 2012013850 A2 WO2012013850 A2 WO 2012013850A2 ES 2011070539 W ES2011070539 W ES 2011070539W WO 2012013850 A2 WO2012013850 A2 WO 2012013850A2
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disease
gemifloxacin
diseases
treatment
dementias
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PCT/ES2011/070539
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English (en)
Spanish (es)
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WO2012013850A3 (fr
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Javier Santos Burgos Muñoz
Maria del Carmen Ramos Martín
Saleta SIERRA ÁVILA
Beatriz SUÁREZ GONZÁLEZ
Juan María Alfaro Sánchez
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Neuron Biopharma, S.A.
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Publication of WO2012013850A2 publication Critical patent/WO2012013850A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • AD Alzheimer's disease
  • the search and validation of new neuroprotective compounds that prevent neuronal damage is a necessity.
  • MCI mild cognitive impairment
  • incipient dementia also known as incipient dementia or isolated memory impairment.
  • MCI is recognized as a risk factor for AD, and affects about 30 million people worldwide.
  • the MCI is considered as a previous step to the EA, where between 10 and 15% of individuals with MCI progress to EA each year (Grundman et al. Arch. Neurol 2004; 61 [1]: 59-66).
  • AD Alzheimer's disease
  • WO 2009/149149 the use of one or more antibiotics, alone or in combination with exogenous microflora or with one or more probiotic compounds, is described in order to modify the population of the microflora to provide an autoimmune regulatory effect and thus make possible the treatment of autoimmune diseases
  • gemifloxacin commonly used as an antibiotic, has neuroprotective properties.
  • Gemifloxacin is a fourth generation compound of the fluoroquinolone family, which are bactericidal antimicrobials that inhibit bacterial DNA synthesis by blocking subunit A of DNA gyrase (topoisomerase II).
  • Fluoroquinolones have a high oral bioavailability, an excretion mainly by the renal route, and in general they are well tolerated, although some of them have manifested side effects in the central nervous system such as encephalopathies, seizures, confusion or psychosis.
  • the inventors have revealed the neuroprotective activity of gemifloxacin by studying the protection of neuronal death caused by endoplasmic reticulum stress (Example 1), by disorganization of the cytoskeleton (Example 2) or by mitochondrial damage (such as inhibition of succinate dehydrogenase) (Huntington's model) in human cell lines of inertic origin (Example 3).
  • Neuroprotective activity has been confirmed by studying the protection of neuronal death caused by apoptosis in human cell lines of cholinergic origin (Example 4).
  • gemifloxacin has the ability to inhibit acetylcholinesterase (AChE) (Alzheimer's model) both in vitro and in vivo (Example 5).
  • gemifloxacin crosses the blood-brain barrier efficiently (Example 6), thus making it potentially useful for the treatment of neurodegenerative diseases, cognitive deficits or dementias, and especially for Alzheimer's and Huntington's diseases.
  • These examples show the potential use of gemifloxacin in the prevention and / or treatment of neuronal death associated with neurodegenerative diseases (eg, Alzheimer's disease, mild cognitive impairment, Huntington, Parkinson's, multiple sclerosis, amyotrophic lateral sclerosis, Creutzfeldt-Jakob, cognitive and / or psychomotor deficits, ataxias, dementias, cerebrovascular diseases, Alexander's disease, etc.), cognitive deficits, dementias, diseases associated with aging, pathological processes associated with age and progeria.
  • neurodegenerative diseases eg, Alzheimer's disease, mild cognitive impairment, Huntington, Parkinson's, multiple sclerosis, amyotrophic lateral sclerosis, Creutzfeldt-Jakob, cognitive and / or psychomotor deficits, ataxias,
  • results obtained can be extrapolated for prophylactic or therapeutic purposes for application to the population at risk. Also, because this compound is commonly used as an antibiotic, pharmacovigilance studies have shown that it has a relatively low toxicity profile, so its use as a neuroprotective drug is very appropriate and does not require complex clinical trials as occurs with others. Candidates for drugs whose toxicity and safety are unknown.
  • the invention relates to the use of gemifloxacin in the preparation of a pharmaceutical composition for the prevention and / or treatment of neurodegenerative diseases, mild cognitive impairment, cognitive deficits, dementias or diseases associated with aging, processes pathological associated with age and progeria.
  • the invention relates to the use of a pharmaceutical composition of gemifloxacin for the prevention and / or treatment of neurodegenerative diseases, mild cognitive impairment, cognitive deficits, dementias or diseases associated with aging, pathological processes associated with age and progeria takes place by neuroprotection, in particular by direct inhibition of neuronal death.
  • the invention relates to the use of a pharmaceutical composition of gemifloxacin for the prevention and / or treatment of neurodegenerative diseases, mild cognitive impairment, cognitive deficits, dementias or diseases associated with aging, pathological processes associated with age and progeria, by modulating the neurotransmitter acetylcholine.
  • the invention relates to a method for the prevention and / or treatment of neurodegenerative diseases, cognitive deficits, dementias or diseases associated with aging, in a subject in need of treatment, which comprises administering to said subject a therapeutically efficient amount of gemifloxacin, or a pharmaceutically acceptable salt, prodrug and / or solvate thereof.
  • Figure 1 is a bar graph depicting the protective effect of gemifloxacin on neuronal death caused by tunicamycin (TM). The figure shows the percentage of cell death (taking 100% of that produced by TM) of the cultures treated with 23 ⁇ TM and gemifloxacin at different concentrations, representing the means ⁇ SD of 3 independent experiments per triplicate. * Significant difference with respect to treatments with TM alone, according to the Student test (p ⁇ 0.05).
  • Figure 2 is a bar graph depicting the protective effect of gemifloxacin on neuronal death caused by okadaic acid (AO).
  • the figure shows the percentage of cell death (taking 100% of that produced by AO) of the cultures treated with 20 nM AO and gemifloxacin at different concentrations, representing the means ⁇ SD of 3 independent experiments per triplicate. * Significant difference compared to treatments with AO alone, according to the Student test (p ⁇ 0.05).
  • Figure 3 is a bar graph depicting the protective effect of gemifloxacin on neuronal death caused by 3-nitropropionic acid (3-NP). The figure shows the percentage of cell death (taking 100% of that produced by 3-NP) of the cultures treated with 30 ⁇ 3-NP and gemifloxacin at different concentrations, representing the means ⁇ SD of 3 independent experiments in triplicate.
  • Figure 4 is a bar graph depicting the protective effect of gemifloxacin on neuronal death caused by camptothecin (CPT).
  • CPT camptothecin
  • the figure shows the percentage of cell death (taking 100% of that produced by CPT) of the cultures treated with 20 nM CPT and gemifloxacin at different concentrations, representing the means ⁇ SD of 3 experiments triplicate independent. * Significant difference with respect to treatments with CPT alone, according to the Student test (p ⁇ 0.05).
  • Figure 5 is a bar graph depicting the neuroprotective effect of gemifloxacin [encoded in the figure as GFX] (compared to the specific cashase inhibitor Z-VAD-fmk) of caspase activation 3/7 by camptothecin (CPT).
  • the figure shows the percentage of inhibition of caspase activity 3/7 of the cultures treated with 50 ⁇ CPT and gemifloxacin at different concentrations and Z-VAD-fmk (in the presence or absence of CPT), representing the means ⁇ SD of 2 experiments triplicate independent.
  • * Significant difference with respect to treatments with CPT alone, according to the Student test (p ⁇ 0.05).
  • Figure 6 is a bar graph depicting the neuroprotective effect of gemifloxacin [encoded in the figure as GFX] (in comparison to the specific cashase inhibitor Z-VAD-fmk) of camptothecin apoptosis (CPT) and determined by flow cytometry.
  • the figure shows the percentage of apoptotic cells determined by DNA fragmentation (taking 100% activity produced by CPT) of cultures treated with 50 ⁇ con CPT and gemifloxacin at different concentrations and Z-VAD-fmk (in the presence or absence of CPT), representing the means ⁇ SD of 2 independent experiments in triplicate.
  • * Significant difference with respect to treatments with CPT alone, according to the Student test (p ⁇ 0.05).
  • Figure 7 is a bar graph depicting the in vitro inhibitory effect on gemifloxacin acetylcholinesterase (AChE) (as compared to the specific inhibitor of BW284c51).
  • the figure shows the percentage of AChE activity and its modification by gemifloxacin at different concentrations and BW284c51, represents the mean ⁇ SD of 2 independent experiments in triplicate. * Significant difference with respect to the control (without treatment), according to the Student test (p ⁇ 0.05).
  • Figure 8 is a bar graph depicting the inhibitory effect in vivo on gemifloxacin acetylcholinesterase (AChE) (as compared to the specific inhibitor of BW284c51).
  • the figure shows the percentage of AChE cellular activity normalized by the amount of protein and its inhibition by gemifloxacin at different concentrations and BW284c51, representing the means ⁇ SD of 2 independent experiments in triplicate. * Significant difference with respect to the control (without treatment), according to the Student test (p ⁇ 0.05).
  • Figure 9 is an XY scatter plot depicting the passage of blood-brain barrier by passive diffusion of gemifloxacin compared to a drug that crosses the barrier easily (verapamil) and another that does not cross the barrier (theophylline).
  • the percentage of hematoencephalic barrier passage of the compounds is represented on the X axis, and the effective permeability parameter (P e ) of the compounds is represented on the Y axis.
  • Figure 10 is an XY scatter plot depicting the blood-brain barrier passage of gemifloxacin [encoded in the figure as GFX] in adult zebrafish (means ⁇ SEM).
  • the fish were administered with 1 000 mg / kg of gemifloxacin in the water and the amount of compound was determined in the animals' brains after 15 min, 30 min, 1 h, 4 h and 24 h post-treatment by UPLC / MS.
  • On the X axis the post-treatment time of gemifloxacin is represented, and on the Y axis the amount of gemifloxacin in the brain (weight / weight) is represented.
  • a "neurotoxic substance” as used herein is chemical substances that produce functional, structural and biochemical alterations of the central nervous system. These adverse effects involve changes that cause deregulation or alteration of the nervous system. The nature of such change may be neurochemical, morphological, or behaviorally related and may manifest itself temporarily or permanently.
  • the term "neurodegenerative disease”, as used herein, includes diseases that result from degeneration or deterioration of nerve tissue, in particular neurons, which leads, over time, to dysfunction or disability; The term degeneration includes loss of cell viability, loss of cell function and / or loss of the number of cells (n eu ronas and others).
  • neurodegenerative diseases include Alzheimer's disease, mild cognitive impairment, Huntingon's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Alexander's disease, cognitive and / or psychomotor deficits, ataxias, dementias, cerebrovascular diseases, Amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), etc.
  • said neurodegenerative disease is a disease related to neuronal death caused by a neurotoxic substance, for example, a substance that produces endoplasmic reticulum stress, apoptosis, cytoskeleton disorganization, degeneration of the basal ganglia or mitochondrial damage.
  • neuronal degeneration and “neuroprotector”, as used herein, refer to the attenuation of the effects of neuronal degeneration or death by any known mechanism or by knowing for example, necrosis, apoptosis, autophagy, excitotoxicity, damage oxidative, mitochondrial damage, endoplasmic reticulum damage, byproduct deposition, loss of cellular architecture, etc., or the disappearance of the effects of neuronal degeneration or death by any known mechanism or by knowing for example, necrosis, apoptosis, autophagy, excitotoxicity, oxidative damage, mitochondrial damage, endoplasmic reticulum damage, byproduct deposition, loss of cellular architecture, etc., or the decrease or disappearance of its side effects.
  • subject refers to a member of a mammalian species, and includes, but is not limited to, pets, primates and humans; preferably, the subject is a human being, male or female, of any age or race. In a particular embodiment, said subject is a mammal who suffers, or is susceptible to disease.
  • neurodegenerative such as a chronic neurodegenerative disease or a disease associated with aging.
  • salt should be understood as meaning any form of gemifloxacin in which the compound assumes an ionic form, or is charged and is coupled with a counterion (a cation or anion) or is in solution.
  • a counterion a cation or anion
  • solvate should be understood as meaning any form of the gemifloxacin compound that has another molecule (more likely a solvent) attached through a non-covalent bond.
  • prodrug or “prodrug” is used in its broadest sense and encompasses those derivatives that are converted in vivo into the compounds of the invention. Such derivatives will readily occur to those skilled in the art, and include, depending on the functional groups present in the molecule. Examples of well known methods for producing a prodrug of a given acting compound are known to those skilled in the art and can be found, for example, in Krogsgaard-Larsen et al., "Textbook of Drugdesign and Discovery” Taylor & Francis (April 2002).
  • Particularly favorable derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient (for example, allowing a compound administered orally to be more easily absorbed in the blood) or those that increase the administration of the original compound a biological compartment (for example, the brain or lymphatic system) in relation to the original species.
  • pharmaceutically acceptable refers to molecular compositions and entities that are physiologically tolerable and do not normally produce allergic reactions or similar unfavorable reactions such as gastric disorders, dizziness, and reactions of the same style, when administered in humans or animals.
  • pharmaceutically acceptable means that it is approved by an agency. regulatory, or that is included in a pharmacopoeia for use in animals, and particularly in humans.
  • the invention relates to the use of gemifloxacin of formula (I), salts, prodrugs and / or solvates thereof, in the elaboration of a pharmaceutical composition for the prevention and / or treatment of death.
  • gemifloxacin of formula (I) in particular that associated with neurodegenerative diseases, mild cognitive impairment, cognitive deficits, dementias, diseases associated with aging and / or pathological processes associated with age and progeria.
  • the results of the research carried out by the inventors demonstrate that the prevention and / or treatment of neurodegenerative diseases, mild cognitive impairment, cognitive deficits, dementias, diseases associated with aging and / or pathological processes associated with age and progeria with the gemifloxacin compound takes place, at least partially, by neuroprotection, in particular by direct inhibition of neuronal death, that is, by inhibiting the death of neuronal cells of the nervous system once said compound has crossed the blood brain barrier (BHE). Therefore, this mechanism of action would take place without the involvement of the immune system.
  • Numerous trials carried out by the inventors have revealed both the neuroprotective effect of gemifloxacin against the action of different neurotoxic substances, and its antiapoptotic effect in cholinergic neurons of human origin.
  • Example 1 The neuroprotective effect of gemifloxacin against the action of a substance causing endoplasmic reticulum stress (tunicamycin) in human cholinergic cells is described in Example 1.
  • gemifloxacin is able to quantitatively and significantly reduce the neural death caused by endoplasmic reticulum stress, which demonstrates the neuroprotective capacity of said compound ( Figure 1).
  • Example 2 the inventors analyzed in Example 2 in more detail the neurodegenerative process by analyzing the neuronal death caused by the action of okadaic acid (AO), which is a causative substance cell death due to disorganization of the cytoskeleton.
  • the AO is used to pharmacologically model one of the histopathological marks observed in the brains of Alzheimer's patients, such as neurofibrillary tangles or tangles, a consequence of the hyperphosphorylation of the Tau protein.
  • gemifloxacin is able to quantitatively and significantly reduce neuronal death caused by disorganization of the cytoskeleton, which demonstrates the neuroprotective capacity of said compound (Figure 2).
  • Example 3 the inventors analyzed in Example 3 in more detail the neurodegenerative process by analyzing neuronal death caused by treatment with 3-nitropropionic acid (3-NP) , which is a mitochondrial toxin that interferes with the syntheses of ATP, in which the enzyme is succinate dehydrogenase, which causes oxidative stress and cell death.
  • 3-NP is used to pharmacologically model the neurodegeneration characteristic of Huntington's disease.
  • gemifloxacin is able to quantitatively reduce neuronal death caused by damage mitochondrial, which shows the neuroprotective capacity of said compound (Figure 3).
  • Example 4 the inventors analyzed in Example 4 in more detail the neurodegenerative process by analyzing the neuronal death caused by the action of a substance causing apoptosis (camptothecin [CPT] ), determining that gemifloxacin is able to quantitatively and significantly reduce neuronal death caused by apoptosis, which demonstrates the neuroprotective capacity of said compound ( Figure 4).
  • CPT camptothecin
  • the inventors analyzed the neurodegenerative process with more analysis by analyzing the activity of caspase 3/7 determining that gemifloxacin It is able to quantitatively and significantly reduce the activation of caspase 3/7, compared to a specific inhibitor of neuronal death by apoptosis, Z-VAD-fmk, which shows the neuroprotective capacity of said compound ( Figure 5).
  • the inventors analyzed in more detail the neurodegenerative process by means of flow cytometry of neuronal death caused by apoptosis and its inhibition by gemifloxacin, compared with a specific inhibitor of Neural death by apoptosis, Z-VAD-fmk, determining that gemifloxacin is able to quantitatively and significantly inhibit neuronal death caused by apoptosis (Figure 6).
  • Example 5 the inventors analyzed in Example 5 the ability to inhibit the enzyme acetylcholinesterase (AChE) in vitro, since this enzyme degrades the neurotransmitter acetylcholine (ACh), forming choline and acetate and preventing ACh activity in the nervous synapse, which has been related to, among others, Alzheimer's disease (AD).
  • AChE acetylcholinesterase
  • gemifloxacin is able to inhibit AChE in vitro, compared to a specific AChE inhibitor, which shows the neuroprotective capacity of said compound (Figure 7).
  • gemifloxacin or a pharmaceutical formulation thereof is claimed for use in the modulation of the neurotransm isor acetilcol ina and for the prevention and / or treatment of diseases related to the modulation of the neurotransmitter acetylcholine.
  • Example 6 the inventors analyzed in Example 6 the ability to cross the blood-brain barrier (BHE) of gemifloxacin.
  • BHE blood-brain barrier
  • gemifloxacin is able to pass through BHE by passive diffusion with greater efficiency than a known compound (theophylline) that does not cross the barrier, and close to a known compound that does cross it (verapamil), which shows the therapeutic utility of said compound in neuroprotection ( Figure 9).
  • the researchers determined that gemifloxacin is capable of crossing BH E in an vertebrate animal model such as zebrafish, confirming its therapeutic utility (Figure 10).
  • neurodegenerative diseases are due to autoimmune processes such as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS).
  • the pharmaceutical composition provided by this invention comprising gemifloxacin, is used for the prevention and / or treatment of amyotrophic lateral sclerosis and multiple sclerosis, such that the active substance gemifloxacin acts by neuroprotection, in particular by direct inhibition. of neuronal death.
  • gemifloxacin For its ad mini stration in the prevention and / or treatment of neurodegenerative diseases, cognitive deficits, dementias or diseases associated with aging, gemifloxacin will be formulated in a pharmaceutical composition, in a therapeutically effective amount, together with one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition provided by this invention may contain gemifloxacin or one or more other drugs together with one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical position comprises only gemifloxacin.
  • Said pharmaceutical composition is useful for the treatment of neurodegenerative diseases, cognitive deficits, dementias or diseases associated with aging.
  • compositions comprising gemifloxacin may be formulated in any pharmaceutical form of administration suitable for administration by the route of administration chosen.
  • the pharmaceutical compositions provided by this invention may be formulated in a solid pharmaceutical form for oral administration (eg, granules, tablets, capsules, etc.), in a liquid pharmaceutical form for oral administration. (eg, solutions, suspensions, emulsions, etc.), in a pharmaceutical form for parenteral administration (eg, solutions, suspensions, emulsions, etc.).
  • the pharmaceutically acceptable carriers and excipients appropriate for the chosen pharmaceutical form and route of administration will be chosen, for example, binders, diluents, disintegrants, lubricants, huctants, etc. , for the formation of solid farm administration forms, and buffers, surfactants, etc., for the formulation of liquid administration pharmaceutical forms.
  • Such vehicles and excipients must be pharmaceutically acceptable and pharmacologically tolerable and must be able to be combined with other components of the formulation without exerting any adverse effect on the treated subject.
  • Information on said vehicles and excipients, as well as on said pharmaceutical forms of administration of said active ingredient can be found in galenic pharmacy treaties.
  • the pharmaceutical composition provided by this invention comprises at least gemifloxacin in a therapeutically efficient amount.
  • therapeutically efficient amount refers to the amount of drug calculated to produce the desired effect.
  • the dose of drug to be administered to a subject may vary within a wide range depending on numerous factors, including the characteristics of the drug used, eg, its activity and biological half-life, the concentration of the drug in the composition. pharmaceutical, the clinical situation of the subject, the severity of the pathology, the pharmaceutical form of administration chosen, etc.
  • the pharmaceutical composition provided by this invention can be administered one or more times a day for preventive or therapeutic purposes or with other administration guidelines, not necessarily daily but also on a timely, weekly basis, etc.
  • the dose of active ingredient administered to a subject in need of treatment for the treatment and / or prevention of the aforementioned conditions is in the range of 0.1 to 20 mg / kg of body weight, usually between 0.2 and 15 mg / kg body weight and preferably between 1 and 13 mg / kg body weight.
  • the invention relates to a method for the prevention or treatment of neurodegenerative diseases, cognitive deficits, dementias or diseases associated with aging, in a subject in need of treatment, which comprises the administration to said subject of a pharmaceutical composition. comprising a therapeutically efficient amount of gemifloxacin, or a pharmaceutically acceptable salt, prodrug and / or solvate thereof.
  • this method of prevention or treatment acts by neuroprotection, in particular by direct inhibition of neuronal death.
  • Another aspect of the invention concerns a method in which the pharmaceutical composition provided by this invention, if desired, can be used together with other drugs, for example, drugs useful in the treatment of neurodegenerative diseases, cognitive deficits, dementias or diseases associated with aging, in order to increase the efficiency of the pharmaceutical composition provided by this invention, thereby generating a combination therapy.
  • additional drugs may be part of the same pharmaceutical composition or, alternatively, may be provided as a separate pharmaceutical composition for administration at the same time (simultaneous administration) as the pharmaceutical composition provided by this invention or at different times (sequential administration) with respect to the administration of the pharmaceutical composition provided by this invention.
  • examples of additional drugs that may be part of the same therapy or pharmaceutical composition with gemifloxacin are: drugs for the treatment of Alzheimer's (tacrine, rivastigm ina, memantine, donepezil, galantam ina.
  • parkinson's carbidopa, levodopa, bromocriptine, pramipexole, ropinirole, amantadine, rasagiline
  • antipsychotics such as haloperidol
  • antidepressants such as amitriptyline
  • anti-inflammatory agents such as lorazepam
  • anti-inflammatory agents such as aspirin
  • dietary supplements Vitamins E, C, B, folate or Ginkgo biloba extract or drugs against the rest of neurodegeneratives indicated in the patent.
  • the invention relates to gemifloxacin for the treatment and / or prevention of neurodegenerative diseases, cognitive deficits, dementias or diseases associated with aging.
  • the characteristics of the drug as well as those of said diseases have already been mentioned previously.
  • the invention in another aspect, relates to a method for the prevention and / or treatment of neurodegenerative diseases, cognitive deficits, dementias or diseases associated with aging, which comprises administering to a subject in need of treatment a therapeutically efficient amount of gemifloxacin. or of a pharmaceutical composition provided by this invention which includes gemifloxacin.
  • a method for the prevention and / or treatment of neurodegenerative diseases, cognitive deficits, dementias or diseases associated with aging which comprises administering to a subject in need of treatment a therapeutically efficient amount of gemifloxacin. or of a pharmaceutical composition provided by this invention which includes gemifloxacin.
  • the characteristics of the drug as well as those Pharmaceutical diseases and compositions comprising a drug have already been mentioned previously.
  • the assay was performed on cells in SK-N-MC human neuroblastoma culture from "American Type Culture Collection (ATCC)". In all cases, strict sterility standards were followed and handling was carried out in class II biological safety cabins that follow the European standard EN 12469. The cells were kept in the culture medium "Minimun Essential Medium Eagle “supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.05 mg / ml gentamicin and 10% fetal bovine serum.
  • ATCC American Type Culture Collection
  • TM tunicamycin
  • TM Tunicamycin
  • TM plus gemifloxacin mesylate medium with TM (23 ⁇ ) plus gemifloxacin mesylate at 1, 4, 10, 40 or 100 ⁇ .
  • the cells were incubated (at 37 ° C and 5% CO 2 ) with these treatments for 22 h, after which the WST-1 reagent was added.
  • the WST-1 test is based on the measurement of metabolic activity.
  • the cellular damage causes the loss of the ability of the cells to obtain the energy necessary to maintain their metabolic functions and cell growth, so that metabolically active (living) cells reduce the salt of tetrazol iu ma formazan through the succinate system -tetrazolium reductase (from the mitochondrial respiratory chain).
  • the formazan formed can be detected colorimetrically, since it has an absorbance of 440nm.
  • the reading was done on a plate reader at 440 nm.
  • the assay was performed on cells in SK-N-MC human neuroblastoma culture from "American Type Culture Collection (ATCC)". In all cases, strict sterility standards were followed and handling was carried out in class II biological safety cabins that follow the European standard EN 12469. The cells were kept in the culture medium "Minimun Essential Medium Eagle” supplemented with pyruvate. 1 mM sodium, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.05 mg / ml gentamicin and 10% fetal bovine serum. The inhibition produced by gemifloxacin mesylate from cell death caused by treatment with okadaic acid (AO) was analyzed.
  • AO okadaic acid
  • the AO is an inhibitor of protein phosphatase 1 (PP1) and causes cell death by disorganization of the cytoskeleton.
  • the AO is one of the histopathological marks observed in the brains of Alzheimer patients, such as neurofibrillary tangles or tangles, as a result of hyperiosphorylation of the Tau protein.
  • AO - Okadaic Acid
  • the cells were incubated (at 37 ° C and 5% CO2) with these treatments for 22 h, after which the WST-1 reagent was added.
  • the WST-1 test is based on the measurement of metabolic activity.
  • the cellular damage causes the loss of the ability of the cells to obtain the energy necessary to maintain their metabolic functions and cell growth, so that metabolically active (living) cells reduce the salt of tetrazolium to formazan through the succinate-tetrazolium system reductase (from the m itochondrial respiratory chain).
  • the formazan formed can be detected colorimetrically, since it has an absorbance of 440nm.
  • the reading was carried out on a plate reader at 440 nm.
  • the assay was performed on cells in SK-N-MC human neuroblastoma culture from "American Type Culture Collection (ATCC)". In all cases, strict sterility standards were followed and handling was carried out in class II biological safety cabins that follow the European standard EN 12469. The cells were kept in the culture medium "Minimun Essential Méd ium Eagle "(MEM) supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.05 mg / ml gentamicin and 10% fetal bovine serum.
  • MEM Minimum Essential Méd ium Eagle
  • 3-NP 3-nitropropionic acid
  • 3-NP is a mitochondrial toxin that interferes with the synthesis of ATP, since it is an inhibitor of the enzyme succinate dehydrogenase, producing oxidative stress and cell death.
  • 3-NP causes degeneration of the basal ganglia and movement functions such as dystonia, chorea and hypokinesia, mimicking some aspects of Huntington's disease such as neuroanatomic, physiological and chemical changes.
  • Control culture medium (medium)
  • 3-Nitropropionic (3-NP) medium with 3-NP 30 ⁇ , which causes the death of 50% of the cells.
  • the cells were incubated (at 37 ° C and 5% CO 2 ) with these treatments for 22 h, after which the WST-1 reagent was added.
  • the WST-1 test is based on the measurement of metabolic activity.
  • the cellular damage causes the loss of the ability of the cells to obtain the energy necessary to maintain their metabolic functions and cell growth, so that metabolically active (living) cells reduce the salt of tetrazolium to formazan through the succinate-tetrazolium system reductase (from the m itochondrial respiratory chain).
  • the formed formazan can be detected colorimetrically, since it has an absorbance of 440nm.
  • the reading was done on a plate reader at 440 nm.
  • CPT camptothecin
  • SK-N-MC cells in pass not exceeding 1 5, were seeded on 96 well plates treated for adherent cells with a cell concentration of 5x10 4 cells / well; 3 wells of the plate were seeded for each test condition.
  • CPT Camptotecina
  • CPT plus gemifloxacin mesylate medium with CPT (20 nM) plus gemifloxacin mesylate at 1, 4, 10, 40 or 100 ⁇ .
  • the cells were incubated (at 37 ° C and 5% CO 2 ) with these treatments for 22 h, after which the WST-1 reagent was added.
  • the WST-1 test is based on the measurement of metabolic activity.
  • the cellular damage causes the loss of the ability of the cells to obtain the energy necessary to maintain their metabolic functions and cell growth, so that metabolically active (living) cells reduce the salt of tetrazolium to formazan through the succinate-tetrazolium system reductase (from the respiratory chain m itocond rial).
  • the formed formazan can be detected colorimetrically, since it has an absorbance of 440nm.
  • the reading was done on a plate reader at 440 nm.
  • Active caspase 3/7 as a method of quantification of apoptosis, was analyzed using the Apo-ONE® Homogeneous Caspase-3/7 kit (Promega).
  • Caspase 3 and caspase 7 are cysteine proteases characterized by mediating the breakdown of other proteins, being effector caspases that trigger apoptotic signaling. Caspase 3/7 activation was quantified by treatment with CPT with a pre-treatment for 24 hours with gemifloxacin mesylate (GFX).
  • GFX gemifloxacin mesylate
  • SK-N-MC cells in pass not exceeding 1 5, were seeded on 96 well plates treated for adherent cells with a cell concentration of 4x10 4 cells / well; 3 wells of the plate were seeded for each test condition.
  • cell pre-treatments (with gemifloxacin mesylate at 4 or 10 ⁇ ) were carried out for 24 h with 100 ⁇ of total volume. After 24 hours of pre-treatment, cell treatments were carried out for 6 hours with 100 ⁇ of total volume for the following conditions:
  • the measurement of active caspase 3/7 was performed following the manufacturer's instructions.
  • the lysis buffer was added to the cultured cells, which smooths and permeates the cells, and the substrate Z-DEVD-R1 10 of the effector caspases 3 and 7.
  • the active caspase 3 or 7 causes the breakdown of the peptides DEVD of the substrate and emits fluorescence at 499/521 nm (emission / excitation), which is determined by the Infinite M200 fluorometer (Tecan).
  • cell pre-treatments (with gemifloxacin mesylate at 4 or 10 ⁇ ) were carried out for 24 hours with 2 ml of total volume for the following conditions. After 24 hours of Pretreatment proceeded to the cell treatments for 6 h with 2 ml of total volume for the following conditions:
  • the cells were collected together with their culture medium and centrifuged at 300 xg for 5 min. The medium was removed, a PBS wash was performed and fixed for 2 minutes with 500 ⁇ of 70% ethanol at -20 ° C. Once fixed, they were centrifuged at 400 xg for 5 min, washed with PBS and propidium iodide was added at 0.05 mg / ml, diluted in a cycle buffer (0.1% sodium citrate, 0.3% nonidet P-40 and 0.02 mg RNAse / ml) and incubated 1 hour at 37 ° C. They were analyzed by flow cytometry, comparing the fluorescence of propidium iodide against the amount of DNA. The percentage of apoptosis was measured on the sub-G1 region (fragmented DNA) of each of the conditions.
  • Acetylcholinesterase is the enzyme that degrades the neurotransmitter acetylcholine (ACh), forming choline and acetate and preventing ACh activity at the nervous synapse.
  • the activity of this enzyme has been related to Alzheimer's disease (AD), since: (i) it is detected less ACh in the brains of patients with AD than in healthy controls, (ii) ACh is a fundamental neurotransmitter for the creation of memories, learning and other intellectual activities that are compromised in AD, and (iii) AChE degrades ACh, so AChE inhibition is a therapeutic target of AD.
  • the cell AChE assay was performed using the SK-N-MC neuronal cells, which have acetylcholinesterase activity so they are a good cellular model.
  • the treatments were carried out on the cells and after 24 h they were lysed and the AChE activity measurement test based on the Ellman method was performed, in the same way as in vitro.
  • the iso-OMPA tetraisopropyl pyrophosphoramide
  • the results obtained are shown in Figure 8, as the percentage of cellular AChE activity of each treatment referred to the control and normalized by the amount of total protein.
  • PBL Porcine Polar Brain Lipid
  • This compound was stored at -20 ° C dissolved in dodecane at 1 00 g / mL in glass vials.
  • the gemifloxacin, verapamil and theophylline mesylate compounds were stored at -20 ° C and used at 100 ⁇ in a phosphate buffer at pH 7.4 containing monobasic sodium phosphate (0.41 M) and dibasic potassium phosphate (0.287M) in 1% DMSO .
  • a 96-well filter plate with a pore size of 45 ⁇ (MAIPN4550) 5 ⁇ ⁇ of PBL was added at 20 pg / mL, and after two minutes, 300 ⁇ of the phosphate buffer was added. This plate was considered the acceptor plate and was placed on the top of the sandwich.
  • 300 ⁇ of gemifloxacin, verapamil or theophylline mesylate was added, at 100 ⁇ trip and in triplicate.
  • a target that included only 1% of DMSO was included in the phosphate buffer used. This plate was called the donor plate. The acceptor plate was placed on the donor plate forming the sandwich system.
  • the compounds under study spread from the wells of the donor plate to the corresponding wells of the acceptor plate during 18h in which the system remained intact.
  • the remaining compound prepared was kept in the same conditions of humidity, temperature and darkness as the sandwich system constituted by the plates.
  • 100 L of the wells of the donor and acceptor plates were transferred to a special 96-well plate for UV reading.
  • 1 00 ⁇ L of the compounds prepared for the performance of the test were transferred, while retaining the plates (basal wells).
  • the UV plate was introduced into a spectrophotometer in which a UV scanner was carried out from 230 to 498 nm, with readings every 4 nm.
  • the percentage of barrier passage was calculated as well as the effective permeability (P e ) as described in the literature (Wexler et al. J. Biomol Screen 2005; 10 [4]: 383-90).
  • P e effective permeability
  • the appropriate wavelength was selected in each case and the data referring to said wavelength were taken for the wells of the acceptor plate, donor or the basal wells.
  • the percentage of BHE passage was calculated, estimating 1 00% with the absorbance of the average of the basal wells, so that the percentage of compound existing in the acceptor and donor wells could be calculated after the time of plate contact.
  • the following formula was applied:
  • the aim of the trial was to determine whether gemifloxacin (GFX) was able to cross the blood brain barrier (BHE), determining the bioavailability of the compound in the brain of an adult zebrafish.
  • GFX gemifloxacin
  • BHE blood brain barrier
  • the adult zebrafish were treated by immersion of the compound without renewal frequency (static test) at a dose of 1,000 mg / kg following a kinetics of treatment times that ended with the humanitarian sacrifice and extraction of the brains of the animals to 15 min, 30 min, 1 h, 4 h and 24 h post-treatment. In the handling processes, strict precautions were taken to prevent contamination. Four animals were used for each experimental group, and the trial had a total of 24 zebrafish divided into the following administration guidelines:
  • the BHE passage of gemifloxacin in the brains of the fish was determined by the UPLC / MS technique, as shown in Figure 10.
  • the equipment used for this purpose was an Acquity / Quatro Premier XE (Waters) with Quatro Premier XE (Waters) mass detector.
  • the mobile phase consisted of methanol (solvent A) and 0.1% formic acid (solvent B).
  • the elution gradient was: 0-4 min 90% A and 10% B.
  • the column temperature was 35 ° C with a flow of 0.3 mL / min and a sample injection volume of 5 ⁇ .
  • the MS conditions set were: capillary voltage of 4 kV, cone voltage of 20 V in positive mode and a temperature of 1 20 ° C.
  • the measurement range selected in SCAN mode was 160-1000 m / z.

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Abstract

L'invention concerne l'utilisation du composé gémifloxacine pour le traitement de maladies neurodégénératives, d'une altération cognitive légère, de troubles cognitifs, de démences, de maladies associées au vieillissement et de processus pathologiques associés à l'âge et à la progérie, et notamment de la maladie d'Alzheimer, compte tenu de la découverte de nouvelles propriétés intrinsèques à ce composé.
PCT/ES2011/070539 2010-07-26 2011-07-22 Composé destiné à traiter des maladies neurodégénératives, des troubles cognitifs et/ou des démences WO2012013850A2 (fr)

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Cited By (1)

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EP2680842A1 (fr) * 2011-03-02 2014-01-08 Bionomics Limited Méthodes de traitement d'une maladie ou d'un état pathologique du système nerveux central

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6262071B1 (en) * 1999-06-29 2001-07-17 Smithkline Beecham Corporation Methods of use of antimicrobial compounds against pathogenic amycoplasma bacteria
US20010014670A1 (en) * 1998-01-09 2001-08-16 Brian J. Balin Treatment and diagnosis of alzheimer's disease

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010014670A1 (en) * 1998-01-09 2001-08-16 Brian J. Balin Treatment and diagnosis of alzheimer's disease
US6262071B1 (en) * 1999-06-29 2001-07-17 Smithkline Beecham Corporation Methods of use of antimicrobial compounds against pathogenic amycoplasma bacteria

Non-Patent Citations (1)

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Title
AMARANTE, G.W. ET AL.: 'Mechanism and synthesis of pharmacologically active quinolones from Morita-Baylis-Hillman adducts' TETRAHEDRON vol. 66, 2010, pages 4370 - 4376 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2680842A1 (fr) * 2011-03-02 2014-01-08 Bionomics Limited Méthodes de traitement d'une maladie ou d'un état pathologique du système nerveux central
EP2680842A4 (fr) * 2011-03-02 2014-10-29 Bionomics Ltd Méthodes de traitement d'une maladie ou d'un état pathologique du système nerveux central

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