• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    The present invention provides a drug that allows effective treatment of motor neuron diseases. In particular, the present invention provides a composition comprising: A) a compound or any one of its pharmaceutically acceptable salts, wherein said compound has the formula (I): {image-01} ; i B) a pyrimidine antagonist derived from the compound of formula i or any of its pharmaceutically acceptable salts, capable of blocking the methylation reaction of deoxyuridyl acid to convert it to thymidylic acid by inhibiting the enzyme thymidylate synthase; For use in the treatment of a motor neuron disease. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2577003A1
    申请号:ES201431825
    申请日:2014-12-11
    公开日:2016-07-12
    发明作者:Rosario Osta Pinzolas；Amaya RANDO ZALDUENDO；Janne TOIVONEN；Pilar ZARAGOZA；Antonio MUSARÓ
    申请人:Universidad de Zaragoza；Universita degli Studi di Roma La Sapienza；
    IPC主号:
    专利说明:

    Compositions for the treatment of motor neuron diseases. Technical field
    The present invention is within the field of Medicine and Pharmacy, and refers to a pharmaceutical composition and / or a combined pharmaceutical preparation comprising a compound capable of blocking the methylation reaction of deoxyuridyl acid to convert it to thymidyl acid by inhibition of the enzyme thymidylate synthase such as tegafur, carmofur or 5-fluorouracil for the treatment of motor neuron diseases. Particularly, for the treatment of amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, Kennedy disease or spinobulbar progressive muscular atrophy and / or spinal muscular atrophy (SMA). Prior art
    Motor neurons are a type of nervous system cells that are located in the brain and spinal cord and have the function of producing stimuli that cause the contraction of different muscle groups in the body. They are therefore essential for daily activities that require muscle contraction: walking, talking, moving hands and in general all body movements. Two types of motor neurons can be distinguished:
    • First motor neuron or upper motor neuron: They are found in the cerebral cortex and emit nerve endings that form the so-called pyramidal pathway that connects to the spinal cord.
    • Second motor neuron or lower motor neuron: They are located in the anterior horn of the spinal cord and emit nerve endings that reach the body's muscles directly and cause their voluntary contraction.
    Motor neuron diseases cause a deficiency in the ability to perform voluntary movements (motor involvement) and fasciculations. Fasciculations are small involuntary contractions that can be easily perceived as small movements under the skin or detected by diagnostic tests such as electromyogram. Higher mental abilities such as intelligence or memory are not affected. Within this type of diseases there are two syndromes that occur when the motor pathways called upper motor neuron syndrome and lower motor neuron syndrome are injured.
    Upper motor neuron syndrome occurs when the descending motor pathways are injured from the cerebral cortex to the spinal cord. This syndrome has the following characteristics:
    •  Weakness distribution: the upper motor neuron syndrome affects large muscle groups and never affects individual muscles, because they affect upper fibers that connect with many lower neurons, which in turn innervate and contract different muscles.
    •  If the lesion is unilateral it can lead to monoparesis or hemiplegia (which is the most frequent). If the lesion is above the accusation, in the brain, hemiplegia is contralateral. But if instead the lesion is below the accusation hemiplegia is ipsilateral
    •  If the lesion is bilateral the patient presents tetraplegia or paraplegia depending on the level of the lesion.
    •  If the facial muscles are affected, we will only appreciate clear weakness in the lower portion (deviation of the mouth) because the lower facial muscles are innervated by the contralateral hemisphere, and the upper facial muscles are innervated on both sides (bihemispheric ) so that if there is an injury to any of the hemispheres, it is compensated with the other hemisphere. The muscles of the neck, trunk, mandibular, extra-ocular are hardly going to be affected unilaterally by this hemispheric innervation.
    •  In the extremities there is usually greater involvement of the distal musculature and fine and precision movements are affected.
    •  Atrophy. The marked atrophy is not characteristic, but moderate and secondary to disuse.
    •  Muscle tone and muscle reflexes. In the acute phase there may be a transient episode of hypotonia (flaccidity) and decreased reflexes, but subsequently the tone increases (spasticity) and the reflexes increase (hyperreflexia). This rise in tone is due to the fact that the reflex arc loses influences that it normally receives from the upper nervous structures, the reflex arc being uninhibited. Spasticity is a type of hypertonia.
    •  The resistance noted by the scanner depends on the speed of the movement.
    •  Phenomenon phenomenon. The resistance is greater at the beginning until it yields.
    •  It does not homogeneously affect the agonist and antagonist muscles: in the MMII they predominate in the extensor muscles and in the MMSS it predominates in the flexor muscles, which justifies the posture of these patients: flexion and pronation of the MMSS and in the MMII extension and adduction and with the foot in plantar flexion and inversion (equine foot varus). It also justifies the hemiplegic march in scythe or reaper (circumference of a lower limb, elevation of the pelvis).
    •  When the pyramidal pathway is injured, the plantar cutaneous reflex becomes extensor (Babinski's sign) and abolition of the abdominal reflex on the affected side.
    •  In the Upper Motor neuron syndrome there are no fasciculations. These are going to be characteristic of lower motor neuron syndrome.
    Lower motor neuron syndrome occurs when the lesion is located in the anterior horn of the spinal cord. This syndrome has the following characteristics:
    •  The distribution of weakness depends on the injured structure and can affect a single muscle or a restricted group of muscles.
    •  Presence of fasciculations. They are spontaneous contractions of one or more motor units. They are observed, by inspection, as "jumps" in the muscle belly and also by electromyography (EMG). In addition to denervation, fibrillations may occur, which are contractions of isolated muscle fibers detected by the electromyogram.
    The main diseases of motor neurons are:
    10 • Amyotrophic lateral sclerosis. It is the most frequent variety and is known by the acronym ELA.
    • Primary lateral sclerosis. 15 • Progressive muscular atrophy.
    • Kennedy disease or progressive spinobulbar muscle atrophy.
    • Spinal muscular atrophy (SMA):
    or AME I. Severe childhood form or Werdnig-Hoffman disease.
    or AME II. Intermediate form.
    25 or AME III. Juvenile form, mild in nature. Also called Kugelberg-Welander disease.
    or AME IV. Mild form of the adult.
    Next we associate each of the motor neuron diseases with the type of degeneration they produce:
    The present invention faces the problem that a drug, or a combination of drugs, that allows an effective treatment of motor neuron diseases, in particular amyotrophic lateral sclerosis, has not yet been found, allowing to increase the efficacy of riluzole and increase hope and
    10 quality of life of patients. It is therefore necessary to find alternative treatments for this type of disease. Brief Description of the Invention
    The present invention provides a drug that allows an effective treatment of motor neuron diseases. In particular, the present invention provides a composition comprising:
    a) a compound or any of its pharmaceutically acceptable salts, wherein said compound has the formula (I):
    ; I
    b) a pyrimidine antagonist derived from the compound of formula I or any of its pharmaceutically acceptable salts, capable of blocking the methylation reaction of deoxyuridyl acid to convert it to thymidyl acid by inhibiting the enzyme thymidylate synthase;
    for use in the treatment of motor neuron disease. Preferably, for use in the treatment of amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, Kennedy disease or spinobulbar progressive muscular atrophy and spinal muscular atrophy (SMA). More preferably, for use in the treatment of amyotrophic lateral sclerosis. Brief description of the figures
    Figure 1: Survival curve. To elaborate Figure 1, the point was established as the moment when the animal was no longer able to turn when it was placed supine and the age at which each mouse reached that point was recorded. The survival curve was determined using the Kaplan-Meier estimator and the differences were analyzed using the Log-Rank Test. A significant increase (p = 0.0003) was observed in the survival of the animals that had received the treatment.
    Figure 2: Evaluation of motor activity through the Rotarod test. To carry out Figure 2, the motor capacity was assessed by the Rotarod test. To do this, the mouse was placed on a cylinder that rotates on its own axis at a constant speed of 12 rpm and the time that the animal is able to remain on said cylinder is recorded. It is considered that the motor capacity is not affected when the animal is able to remain 180 seconds. The values were recorded weekly and represented in an XY distribution (age of the animal versus the time it is able to remain in the rotarod). The differences were established by Student's t-test, these being significant when p <0.05. A delay of one week was observed in the appearance of symptoms (96 to 103 days) and a slowdown in the progression of the disease from that age.
    Figure 3: Evaluation of motor activity and muscle strength through the Grid test. To perform figure 3, muscle strength was assessed by the grid test. This test consists of placing the animal on a grid and rotating said grid 180º. The animal is therefore suspended from the grid to which it is held with its limbs. The time it took for the mice to fall was recorded, up to a maximum of 180 seconds. The test was performed weekly and the values were represented in an XY distribution (age-time in the grid). The differences between the means were analyzed using the Student's t-test. A discrete effect of the motor force treatment was observed at the age of 124 days.
    Figure 4: Evolution of the weight in grams. To carry out Figure 2, it was determined that weight is a good parameter of the evolution of ALS disease in the animal model. Therefore, the weight of the animals was recorded weekly and it was observed that the treatment slowed the weight loss, observing significant differences between the treated and untreated animals at the age of 124 days.
    Figure 5: Evolution of the weight with respect to the initial weight. In order to correct the intragroup variability of the animals' weight before starting the study, the relative weight loss of the animal based on the initial weight was determined before receiving the treatment or the placebo. To obtain this value, the weight of each week was divided by the weight at the beginning of the experiment. From 96 days of age it was observed that the treatment tended to reduce weight loss, the relative weight of the treated animal being significantly greater from 110 days of age.
    Figure 6: Percentage of Hematopoietic Stem Cells (CMHs) in peripheral blood. The percentage of Hematopoietic Stem Cells in blood was determined by flow cytometry of peripheral blood samples taken from the tail vein. WT animals that did not receive the treatment and treated and untreated transgenic animals were analyzed. Extractions were performed before treatment and 4 post-treatment, at 75, 90 and 105 days of age and at sacrifice. It was observed that none of the administrations of the treatment significantly affected the percentage of hemapotoietic stem cells in peripheral blood if we compare the transgenic animals treated against those not treated.
    Figure 7 Percentage of Lymphocyte Precursor Cells (CPLs) in peripheral blood By flow cytometry, peripheral blood samples taken from the tail vein determined the percentage of Hematopoietic Stem Cells in blood. Wildtype (WT) animals that did not receive the treatment and treated and untreated transgenic animals were analyzed. Extractions were performed before treatment and 4 post-treatment, at 75, 90 and 105 days of age and at sacrifice. An increase in the percentage of CPLs in peripheral blood was observed after the third administration of the treatment, of the transgenic animals treated with respect to the untreated ones, bringing the values of the transgenic animals treated closer to the values of the WT animal. This effect of the treatment on the CPLs was also observed at the time of sacrifice, since as can be seen in the graph, the increase in CPLs in peripheral blood of the untreated transgenic animals is corrected as an effect of the treatment.
    Figure 8 Percentage of Monocyte Precursor Cells in peripheral blood. By flow cytometry, peripheral blood samples taken from the tail vein determined the percentage of Hematopoietic Stem Cells in blood. Wildtype (WT) animals that did not receive the treatment and treated and untreated transgenic animals were analyzed. Extractions were performed before treatment and 4 post-treatment, at 75, 90 and 105 days of age and at sacrifice. A reduction in the percentage of CPMs in peripheral blood was observed with respect to untreated transgenic animals after the first administration of the treatment at 75 days of age. And as already happened in the CPLs, it was observed that the treatment blocks the increase in CPMs that occurs in untreated transgenic animals at the time of slaughter.
    Figure 9 Percentage of lymphocytes in peripheral blood. By flow cytometry, peripheral blood samples taken from the tail vein determined the percentage of Hematopoietic Stem Cells in blood. Wildtype (WT) animals that did not receive the treatment and treated and untreated transgenic animals were analyzed. Extractions were performed before treatment and 4 post-treatment, at 75, 90 and 105 days of age and at sacrifice. It was observed that none of the administrations of the treatment significantly affected the percentage of hemapotoietic stem cells in peripheral blood if we compare the transgenic animals treated against those not treated. Yes, significant differences were observed at the age of 105 days in the percentage of lymphocytes of WT animals with respect to transgenic animals.
    Figure 10 Percentage of monocytes in peripheral blood. By flow cytometry, peripheral blood samples taken from the tail vein determined the percentage of Hematopoietic Stem Cells in blood. Wildtype (WT) animals that did not receive the treatment and treated and untreated transgenic animals were analyzed. The 5 extractions were performed before treatment and 4 post-treatment, at 75, 90 and 105 days of age and at sacrifice. A reduction in the percentage of monocytes was observed in the transgenic animals treated against those not treated after the first administration of the treatment. From 90 days of age, an increase in the percentage of monocytes in peripheral blood of transgenic animals with respect to animals was observed.
    10 Wildtype. Detailed description of the invention
    The authors of the present invention have discovered how surprisingly the
    15 anti-metabolite 5-fluoracil, to date used as an antineoplastic treatment, is able to prolong the survival of the mouse SOD1G93A, animal model of Amyotrophic Lateral Sclerosis. In this sense, the authors of the present invention have established how the treatment with 5-fluorouracil delays the onset of the symptoms of Amyotrophic Lateral Sclerosis, improves the motor capacity of said disease and
    20 prolongs the survival of those subjects suffering from amyotrophic lateral sclerosis.
    Thus, the present invention provides a composition and a combination therapy that is useful in the treatment of Amyotrophic Lateral Sclerosis as well as of
    25 other related motor neuron diseases such as primary lateral sclerosis, progressive muscular atrophy, Kennedy disease or spinobulbar progressive muscular atrophy and / or spinal muscular atrophy (SMA), avoiding or minimizing the side effects of other treatments.
    Therefore, a first aspect refers to the use of a composition, hereafter referred to as the composition of the invention, comprising:
    a) a compound or any of its pharmaceutically acceptable salts or prodrugs, wherein said compound has the formula (I):
    ; I
    B) a pyrimidine antagonist derived from the compound of formula I or any of its pharmaceutically acceptable salts, capable of blocking the methylation reaction of deoxyuridyl acid to convert it to thymidyl acid by inhibiting the enzyme thymidylate synthase;
    for use in the preparation of a medicament for the treatment of motor neuron disease. Alternatively, the present invention relates to a composition comprising:
    a) a compound or any of its pharmaceutically acceptable salts, wherein said compound has the formula (I):
    ; I
    10 b) a pyrimidine antagonist derived from the compound of formula I or any of its pharmaceutically acceptable salts, capable of blocking the methylation reaction of deoxyuridyl acid to convert it to thymidyl acid by inhibiting the enzyme thymidylate synthase;
    15 for use in the treatment of motor neuron disease. Preferably, said composition is used in the treatment of a human subject, more preferably in the treatment of a human subject that does not suffer from cancer concurrently with motor neuron disease.
    In a preferred embodiment of the first aspect of the invention, the derivative is selected from the list consisting of capecitabine, carmofur, doxifluridine, tegafur, alpha-fluoro-betaalanine, floxuridine, ftorafur, and oligomers thereof.
    In another preferred embodiment of the first aspect of the invention, motor neuron disease is selected from the list consisting of amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, Kennedy disease or spinobulbar progressive muscular atrophy and spinal muscular atrophy ( AME). Preferably, the disease is amyotrophic lateral sclerosis.
    In a preferred embodiment of the first aspect of the invention, the composition comprises a compound of formula I and the motor neuron disease is amyotrophic lateral sclerosis. Preferably, the composition comprises a compound of formula I, the motor neuron disease is amyotrophic lateral sclerosis and the
    Treatment is performed on a human subject who does not suffer from cancer concurrently with amyotrophic lateral sclerosis.
    A second aspect of the invention relates to the use of components a) and b) as described in the first aspect of the invention, for use in the preparation of a
    Combined preparation comprising said components juxtaposed for the treatment of any of the diseases defined in claim 1 or 3. Alternatively, the second aspect of the invention relates to a combined preparation comprising components a) and b) as described in the first aspect of the invention, juxtaposed for use in the treatment of any of the diseases defined in claim 1 or 3. Preferably, said preparation is used in the treatment of a human subject, more preferably in the treatment of a human subject who does not suffer from cancer concurrently with motor neuron disease.
    It should be emphasized that the term "combined preparation" or also called "juxtaposition", herein, means that the components of the combined preparation need not be present as a joint, for example in a true composition, in order to be available for combined application. , separate or sequential. In this way, the expression "juxtaposed" implies that it is not necessarily a true combination, in view of the physical separation of the components.
    In a preferred embodiment of the second aspect of the invention, the derivative is selected from the list consisting of capecitabine, carmofur, doxifluridine, tegafur, alphafluoro-beta-alanine floxuridine, ftorafur, and oligomers thereof.
    In another preferred embodiment of the second aspect of the invention, motor neuron disease is selected from the list consisting of amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, Kennedy disease or spinobulbar progressive muscular atrophy and spinal muscular atrophy (SMA). ). Preferably, the disease is amyotrophic lateral sclerosis.
    All compounds of the present invention (therefore including any of components a) or b) of the present invention, described above, include pharmaceutically acceptable salts, esters, amides and prodrugs, including but not limited to carboxylic salts, addition salts. of amino acids, esters, amides, and prodrugs of the compounds of the present invention that are, within the scope of medical judgment, suitable for use in patients without excessive toxicity, irritation, allergic response, and the like, with a benefit / reasonable risk, and effective for its intended use, as well as hybrid ion forms (zwitterionic forms, in English), where possible, of the compounds of the present invention. The term "salts" refers to the relatively harmless, inorganic and organic acid addition salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by reacting separately the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative examples of such salts include the salts hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobsonate, lauryl sulphonate, and the like. These may include cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and ammonium, as well as safe ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium , methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like (see for example, Berge SM, et al, "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, the content of which is incorporated in this application by reference).
    The term "prodrug" refers to compounds that are rapidly transformed in vivo to produce the parent compound of the above formulas, for example, by blood hydrolysis. A detailed discussion of prodrugs is provided in T. Higuchi and
    V. Stella, "Pro-drugs as Novel Delivery Systems," Val. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both incorporated herein by reference. In a preferred embodiment of any aspect of the invention, the composition to the combined preparation of the present invention is a pharmaceutical composition. In another preferred embodiment, the composition to the preparation further comprises another active principle.
    Another aspect of the invention relates to a pharmaceutical form, hereinafter pharmaceutical form of the invention, comprising the composition of the invention, the combined preparation of the invention, any of the components a) to b) of the combined preparation of the invention, a simultaneously, components a) and b) of the combined preparation of the invention for use in the treatments specified in the first aspect of the invention.
    According to the present invention, the pharmaceutical form is the individualized arrangement to which drugs (active ingredients) and excipients (pharmacologically inactive matter) are adapted to constitute a medicament. Examples of liquid pharmaceutical forms can be solutions (oral solutions), aromatic waters, syrups, elixirs, mouthwashes, mouthwashes, potions, mucilages, emulsions, suspensions, eye drops, lotions, tinctures, fluid extracts, injections, gargles, etc ...
    Accordingly, a further aspect of the present invention includes pharmaceutical compositions comprising one or more compounds of the invention described above together with a pharmaceutically acceptable carrier. For administration, the compounds are usually combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be mixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia, gelatin, sodium alginate , polyvinylpyrrolidone, and / or polyvinyl alcohol. Alternatively, the compounds of this invention can be dissolved in saline, water, polyethylene glycol, propylene glycol, colloidal solutions of carboxymethyl cells, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and / or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include temporary delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
    On the other hand, the dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, the age, weight, sex, tolerance, ... of the mammal. In the sense, the therapeutically effective amount of the components of the invention, in general, will be determined, among other causes, by the characteristics of said prodrugs, derivatives or the like and the therapeutic effect to be achieved. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are the vehicles known to those skilled in the art.
    As used herein, the term "active ingredient" means any component that potentially provides a pharmacological activity or other effect different in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. Examples
    The invention will now be illustrated by tests carried out by the inventors.
    Example 1: Administration of 5-fluorouracil delays the onset of symptoms of amyotrophic lateral sclerosis and prolongs survival in SOD1G93A mice.
    For the conduct of this experiment, SOD1G93A transgenic mice were used. These mice are high-copy classical transgenic mice for the mutated human SOD1 (G93A) gene with a genetic background of strain B6SJL. These transgenic mice were obtained in "The Jackson Laboratory (Bar Harbor, ME) (B6SJL-Tg (SOD1G93A) 1Gur / J)" and housed in the facilities of the Animal Experimentation Service of the University of Zaragoza under cycles of 12 hours of light and 12 hours of darkness and with a relative humidity of 55%. Food and water were supplied ad libitum. All procedures were approved by the Ethical Committee of the University of Zaragoza.
    The identification of transgenic and wild-type animals ("wildtype") was performed using the PCR technique on DNA from the tail of mice. DNA extraction and subsequent amplification were performed following the protocol described by "The Jackson Laboratory". At 70 days of age, the 5-fluorouracil product was injected intraperitoneally. The dose administered was 150 mg / kg mouse weight diluted in physiological serum. Three injections were given 15 days apart between them. Control mice received physiological serum. 80 animals balanced by sex and treatment were used.
    Next, the rotarod test was performed, which is a grid and survival test. The grid test was used to determine muscle strength and the onset of ALS symptoms. The animals performed this test once a week from the ninth week. Each mouse was placed on a grid that was subsequently turned, the mouse being suspended from its extremities. The time it took for each mouse to fall was timed. Each of the mice had three attempts and the longest period of time was recorded up to a maximum of 180 seconds. The rotarod test was used to assess motor coordination, strength and balance. The animals were placed on the rotating bar of the apparatus and the time that the animal was able to remain on the bar at a constant speed of 12 rpm was recorded. Each animal had three attempts taking a limit of 180 seconds. It was established as a point of euthanasia the moment in which the animal was not able to turn on its own within 30 seconds when it was placed supine.
    To avoid the variability in the weight that each mouse presented at the beginning of the treatment, the weight referred to the initial weight of each animal.
    As a result of the treatment with 5-fluorouracil 150 mg / kg IP 3 times every 15 days the following results could be obtained. First, the delay in the appearance of symptoms, secondly the improvement in the motor capacity of the mice and finally the prolongation in the survival of the mice. The onset of symptoms was established as the week in which the animals began to lose weight (such as the first day that the animals could not be held in the inverted grid for three minutes). Maximum survival was achieved in the group treated with 5fluorouracil, which reached an average of 136 days, 8 days longer than the controls that lived an average of 128 days (p = 0.0003) (see Figure 2 and Table 1). Table 1.
    Between week 13 and 14 a marked decrease in activity on rotarod was observed in the control group, while in the group of treated animals these deficiencies were observed from week 15. In addition, mice in the control group began to lose weight from week 12, while the mice in the treated group showed a slight weight loss just after the first injection, but this weight loss was temporary and the mice recovered their initial weight and even exceeded it, reaching their maximum weight in week 14.
    Example 2: The administration of 5-fluorouracil intraperitoneally modifies the number of blood stem cells detected in peripheral blood.
    For the conduct of this experiment, SOD1G93A transgenic mice were used. These mice are high-copy classical transgenic mice for the mutated human SOD1 (G93A) gene with a genetic background of strain B6SJL. These transgenic mice were obtained in "The Jackson Laboratory (Bar Harbar, ME) (B6SJL-Tg (SOD1-G93A) 1Gur / J)" and housed in the facilities of the Animal Experimentation Service of the University of Zaragoza under 12-hour cycles of light and 12 hours of darkness and with a relative humidity of 55%. Food and water were supplied ad libitum. All procedures were approved by the Ethical Committee of the University of Zaragoza.
    The identification of transgenic and wild-type animals ("wildtype") was performed using the PCR technique on DNA from the tail of mice. DNA extraction and subsequent amplification were performed following the protocol described by "The Jackson Laboratory". At 70 days of age, the 5-fluorouracil product was injected intraperitoneally. The dose administered was 150 mg / kg mouse weight diluted in physiological serum. Three injections were given 15 days apart between them. Control mice received physiological serum.
    From 4 days after the administration of 5-fluorouracil, at which time the reduction of both white and red series cells in peripheral blood is greater; The presence of hematopoietic stem cells, lymphatic and myeloid precursors, as well as lymphocytes and monocytes was studied. 20 animals balanced by sex and treatment were used. Thus, at 75, 90 and 105 days of age blood was drawn by puncturing the tail vein. The blood was collected in a capillary tube with EDTA. The blood was centrifuged to obtain the cell fraction and the cells were resuspended in 50 microliters of PBS. The presence of hematopoietic stem cells in peripheral blood was evaluated. For this, approximately 40 ul of blood was diluted in 1 ml PBS (1% BSA and 0.1% Sodium Azide). After a centrifuge pulse, the plasma was removed and the cell fraction re-suspended in 50 ml of PBS (1% BSA and 0.1% Sodium Azide) containing 1 ul of the following antibodies: Anti-Mouse Ly-6A / E (Sca-1) PE (12-5981 eBioscience), Anti-Mouse CD117 (e-Kit) APC (17-1171 eBioscience), Mouse Hematopoietic Lineage eFluor® 450 Cocktail (88-7772 eBioscience) and PE-Cy ™ 7 Rat Anti-Mouse CD127 (560733 BD Biosciences). After a 30 minute incubation, the erythrocytes were lysed with a homemade lysis buffer consisting of 8.99 g / 1 of NH4CI, 1 g / l KHCO3, 0.372 g / l of EDTA, pH = 3 for 15 minutes in the dark. Then, by centrifugation at 400g for 5 minutes, the labeled leukocyte pellet was collected, washed with ml of PBS (1% BSA and 0.1% Sodium Azide) and centrifuged again for 1 minute at maximum speed. Finally, the cells were diluted in 500 ul of ml PBS (1% BSA and 0.1% Sodium Azide) and analyzed by flow cytometry in a Gallios Flow Cytometer (Beckman Coulter) for the following subpopulations based on their immunophenotype: Hematopoietic stem cells (CMHs) (lin-, Sca-1 +, e-kit +); Myeloid Precursor Cells (CPMs) (lin-, Sca-1 -, e-kit +); and Lymphoid Precursor Cells (CPLs) (Lin-, CD127 +). Subpopulations of monocytes and lymphocytes were positive for the cocktail of blood lineage antibodies and separated according to their complexity. The data were analyzed with the Kaluza cytometric analysis software and the data were shown as the percentage of cells of each subpopulation with respect to the total of selected events.
    The results obtained indicate that after the first injection of 5-fluorouracil, the percentage of myeloid precursor cells in peripheral blood of the treated SOD animals decreases. This decrease was also observed at the time of sacrifice, when transgenic mice show an increase in monocytes in their peripheral blood compared to wild-type animals of the same age. As a result of the decrease in myeloid precursors, the percentage of monocytes was also reduced at 75 days in the treated group with respect to the control group. From 90 days of age, the percentage of monocytes in peripheral blood was increased in transgenic animals compared to wildtype animals, but no differences were observed between treated and untreated SOD mice.
    In the case of lymphoid precursor cells, treatment with 5-fluorouracil increased the percentage of said cells in peripheral blood at 105 days of age, observing this trend from 90 days of age. However, at the time of slaughter in the untreated SOD animals an increase in the percentage of the progenitor cells was observed while the untreated SOD animals was not observed.
    权利要求:
    Claims (11)
    [1]
    1. Use of a composition comprising:
    a) a compound or any of its pharmaceutically acceptable salts, wherein said compound has the formula (1):
    ; I
    B) a pyrimidine antagonist derived from the compound of formula I or any of its pharmaceutically acceptable salts, capable of blocking the methylation reaction of deoxyuridyl acid to convert it to thymidyl acid by inhibiting the enzyme thymidylate synthase;
    20 in the development of a medicine for the treatment of motor neuron disease.
    [2]
    2. Use of the composition according to claim 1, wherein said derivative is selected
    from the list consisting of capecitabine, carmofur, doxifluridine, tegafur and alpha-fluoro-beta25 alanine, floxuridine, ftorafur, and oligomers thereof.
    [3]
    3. Use of the composition according to any of claims 1-2, wherein the motor neuron disease is selected from the list consisting of amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, disease of
    30 Kennedy or progressive spinobulbar muscular atrophy and spinal muscular atrophy (SMA).
    [4]
    4. Use of components a) and b) as described in claim 1 or 2, for the preparation of a combined preparation comprising said components juxtaposed for the treatment of any of the diseases defined in the
    Claim 1 or 3.
    [5]
    5. Use according to any of claims 1-4, wherein said motor neuron disease is amyotrophic lateral sclerosis.
    6. Use of a composition comprising a compound or any of its pharmaceutically acceptable salts, wherein said compound has the formula (I):
    in the development of a medication for the treatment of amyotrophic lateral sclerosis.
    [7]
    7. Use of a composition comprising:
    a) a compound or any of its pharmaceutically acceptable salts, wherein said compound has the formula (I):
    ; I
    15 b) a pyrimidine antagonist derived from the compound of formula I or any of its pharmaceutically acceptable salts, capable of blocking the methylation reaction of deoxyuridyl acid to convert it to thymidyl acid by inhibiting the enzyme thymidylate synthase,
    20 in the preparation of a medicament for the treatment in a human subject of a motor neuron disease, where said human subject does not suffer from cancer concurrently with motor neuron disease.
    Use of the composition according to claim 7, wherein said derivative is selected from the list consisting of capecitabine, carmofur, doxifluridine, tegafur, alpha-fluoro-betaalanine, floxuridine, ftorafur, and oligomers thereof
    [9]
    9. Use of the composition according to any of claims 7-8, wherein the
    30 motor neuron disease is selected from the list consisting of amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, Kennedy disease or progressive spinobulbar muscular atrophy and spinal muscular atrophy (SMA).
    [10]
    10. Use of components a) and b) as described in claim 1 or 2, for the preparation of a combined preparation comprising said components juxtaposed for the treatment of any of the diseases defined in claim 1 or 3 in a subject human who does not suffer from cancer concurrently
    5 to motor neuron disease.
    [11]
    11. Use according to any of claims 7-10, wherein said motor neuron disease is amyotrophic lateral sclerosis.
    12. Use of a composition comprising a compound or any of its pharmaceutically acceptable salts, wherein said compound has the formula (I):
    15 in the preparation of a medication for the treatment of amyotrophic lateral sclerosis in a human subject who does not suffer from cancer concurrently with amyotrophic lateral sclerosis.
    [13]
    13. Use of the combined composition or preparation according to any of the
    Claims 1-12, wherein the combined composition or preparation further comprises another active ingredient.
    [14]
    14. Use of the combined composition or preparation according to any of the
    claims 1-13, wherein said combined composition or preparation is administered intravenously or intra-arterially.
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    同族专利:
    公开号 | 公开日
    WO2016092139A1|2016-06-16|
    ES2577003B1|2017-07-20|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20180369211A1|2015-06-25|2018-12-27|Lysosomal Therapeutics Inc.|Methods and compositions for treating neurodegenerative disorders|
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    ES201431825A|ES2577003B1|2014-12-11|2014-12-11|COMPOSITIONS FOR THE TREATMENT OF MOTORCYCLE DISEASES.|ES201431825A| ES2577003B1|2014-12-11|2014-12-11|COMPOSITIONS FOR THE TREATMENT OF MOTORCYCLE DISEASES.|
    PCT/ES2015/070896| WO2016092139A1|2014-12-11|2015-12-11|Compositions for the treatment of motor neurone diseases|
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