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WO2012009823A1 - Tranquilising/sedating pharmaceutical formulation based on neurosteroids and a phenothiazine derivative for use in mammals - Google Patents

Tranquilising/sedating pharmaceutical formulation based on neurosteroids and a phenothiazine derivative for use in mammals Download PDF

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Publication number
WO2012009823A1
WO2012009823A1 PCT/CL2011/000040 CL2011000040W WO2012009823A1 WO 2012009823 A1 WO2012009823 A1 WO 2012009823A1 CL 2011000040 W CL2011000040 W CL 2011000040W WO 2012009823 A1 WO2012009823 A1 WO 2012009823A1
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WIPO (PCT)
Prior art keywords
pharmaceutical formulation
acepromazine
activity
add
tranquilizer
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PCT/CL2011/000040
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Spanish (es)
French (fr)
Inventor
Luis Aguayo Hernandez
Mario Silva Osorio
José BECERRA ALLENDE
Jorge Fuentealba Arcos
Claudia Perez Manriquez
Antonio Bizama Reyes
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Universidad De Concepcion
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Publication of WO2012009823A1 publication Critical patent/WO2012009823A1/en

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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
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P23/00—Anaesthetics
    • 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
    • A61P25/20—Hypnotics; Sedatives

Definitions

  • the invention corresponds to a pharmaceutical formulation, tranquilizer / sedative, comprising at least one neurosteroid and a tranquilizer of the phenothiazine derivative type, which is used as a pre-anesthetic in mammals.
  • drugs with a calming and sedative effect are used to facilitate patient management in procedures such as: placement of probes, radiography, animal transfers, capture of wild animals, among others.
  • the term tranquilizer and sedative are used as synonyms, however, the former does not produce a depression of the degree of consciousness by increasing the dose, instead the sedatives, by increasing the dose, significantly depress the central nervous system.
  • the researchers define the term sedation / reassurance, as the decrease in the degree of consciousness characterized by a decrease in motor activity and a slower response time to a stimulus. Sedatives generate depression of the central nervous system at high doses, instead high doses of tranquilizers, give rise to extrapyramidal signs such as muscle tremors.
  • sedative doses of propofol mainly reduce the activity of neurons in the cerebral cortex, while hypnotic doses significantly decrease blood flow and metabolism of subcortical structures such as thalamus, midbrain, reticular formation.
  • a significant number of drugs including tranquilizers, sedatives and general anesthetics, modulate GABA ionotropic receptors A) prolonging the opening of chloride channels that mediate rapid synaptic inhibition, generating inhibitory postsynaptic currents. It has also been related to processes such as regulation of wakefulness, anxiety, muscle tension, memory and epileptiform seizures.
  • GABA is one of the main inhibitory neurotransmitters of the central nervous system. In mammals, it has a hyperpolarizing action by increasing the influence of IC " on the postsynaptic neuron; approximately 70% to 90% of the neostriate uses GABA as an inhibitory neurotransmitter and there is a complex and close relationship between GABAergic and dopaminergic neurons, where GABA acts by depressing the dopaminergic pathway.
  • Diazepam is the drug widely used for its sedative, anxiolytic, and hypnotic effects.
  • the sedative action of this benzodiazepine whose molecular target is the GABAA receptor; It is scarce in small animals and its use independently is not useful in performing diagnostic or therapeutic procedures; On the other hand, the muscle relaxation that it produces, makes it the agent of choice in dissociative anesthesia.
  • Xylazine is 2 (2,6-dimethiphenylamino) -4H-5,6-dihydro-1, 3-thiazine hydrochloride and, pharmacologically, is classified as an analgesic and sedative.
  • Xylazine is an adrenergic agonist at 2 , although it has also been observed that it acts on cholinergic, serotorinergic, H2 histamine and opioid receptors.
  • the administration of xylazine causes depression of the heart rate due to first and second degree atrioventricular block.
  • Xylazine should be used with caution or avoided in animals with: gastrointestinal problems, liver disease, respiratory depression or pharyngeal or laryngeal dysfunction, patients with heart disease or diseases in the urinary tract.
  • Accepromazine is probably the most common tranquilizer used in veterinary medicine and corresponds to 2-acetyl-10 - (- 3-dimethylaminopropyl). Its effect is based on the central blockade of excitatory dopaminergic receptors, which results in tranquilizing, antihemetic and hypothermic effects. In addition to causing a decrease in motor activity in all animals, at high doses, they cause extrapyramidal or cataleptic effects.
  • the central catecolamine blockade produces a peripheral ⁇ -adrenergic block that causes peripheral vasodilation and hypotension, so its use in hypovolemic patients with cardiac alterations should be avoided. It causes a mild anticholinergic effect, which explains that at the digestive level there is a depression of gastrointestinal motility. It also produces a decrease in body temperature in all animal species and a significant decrease in respiratory rate.
  • Accepromazine can be administered intravenously, intramuscularly, subcutaneously or orally in dogs, cats and horses. Being a liver metabolizing drug should be avoided in patients with problems in this organ and, particularly, should be avoided in breeds of brachycephalic dogs such as boxer, Pekingese, buldog since they are especially sensitive to it.
  • Neuroactive steroids according to the present invention are suitable for the treatment of those states controlled by the action of the neurotransmitter (gamma) -aminobutyric acid (GABA), as substances that exhibit activity against pain, anxiety and insomnia and also as anesthetics.
  • GABA neurotransmitter
  • the difference with our technology is that it is not a new molecule that we want to safeguard, and its structure differs with our initiative.
  • Some pharmacological antecedents of steroidal derivatives that act on the central nervous system show that they are capable of interacting with membrane receptors, mainly in neurons and produce a rapid change in the excitability of the central nervous system, causing a depressing effect on it.
  • the present technology comprises a veterinary tranquilizer / sedative formulation comprising at least one neurosteroid with tranquilizing activity, preferably but not exclusively 1,4-androstadien-3,17-dione (ADD), a tranquilizer of the phenothiazine derivative type, preferably but not exclusively acepromazine maleate; and pharmaceutically acceptable excipients.
  • ADD 1,4-androstadien-3,17-dione
  • a tranquilizer of the phenothiazine derivative type preferably but not exclusively acepromazine maleate
  • pharmaceutically acceptable excipients pharmaceutically acceptable excipients.
  • acepromazine in a concentration range between 1-100 mg / Kg, approximately
  • 1,4-androstadien-3,17-dione in a concentration range between 50-100 mg / Kg, approximately
  • the combined use of these two molecules manages to significantly reduce the motor activity of the individual, unlike the effect caused by the individual use of said molecules.
  • ADD 1,4-androstadiene-3,17-dione
  • the anxiolytic capacity of ADD is evaluated in male rats.
  • the light / dark test is carried out, which consists in measuring the residence time of the individuals both in the dark compartment and in the light compartment of a box enabled for it, for 15 min for each treatment group.
  • Figure 1 shows the effects of ADD (50-100 mg / kg) compared to the anxiolytic effects of diazepam (0.5 mg / kg).
  • the control group (animals without treatment), has a homogeneous behavior that is characterized by the permanence of the animals in the field of darkness almost 100% of the observation time (15 min), recording a time of exploration of the light field of 10 ⁇ 3.5 seconds, which is equivalent to less than 1.2% of the total duration of the experiment. Animals treated with diazepam, two minutes after the drug is administered, begin to perform intermittent scans to the light field, registering a significantly longer time in the light field at the end of the experiment (p ⁇ 0.001) than the control group (close to 2000%).
  • Animals treated with ADD do not modify the behavior of permanence in the dark light areas of the box. As the dose increases, no behavioral changes are observed, however unlike the control group, animals treated with ADD have visible degrees of immobility in the dark sector of the experimental box. Unlike diazepam, ADD shows no anxiolytic effect.
  • n number of rats per group.
  • the intramuscular route is used to administer steroids, in order to simulate a real clinical situation, but it can also be administered intravenously.
  • the effect of ADD starts on average at 5 minutes post administration in all animals, therefore the measurements start from that moment to not interfere with the results. Preliminary tests show that the duration of the ADD effect is approximately 30 minutes and the maximum effect is reached 10 minutes after administration.
  • the rats Five minutes after the administration of the respective treatments, the rats are placed in a transparent acrylic box (47 x 35 x 30 cm). Spontaneous mobility is measured by direct observation, recording the number of lifts and grooming time for 10 min.
  • This group has an average of 23.5 ⁇ 3.9 quadrant changes during the measurement period, which is combined with some vertical movements where the rats stand on their hind limbs and rest their hands on the vertical part of the box ( raised), sniffing and exploring its perimeter.
  • the average number of lifts observed is 18.8 ⁇ 2.7. All this activity is associated with grooming intervals, which consist of stereotyped cleaning movements performed by individuals. This activity is observed in a total time of 112.5 ⁇ 8 seconds.
  • Accepromazine produces bradycardia and direct myocardial depression, which can aggravate hypotension and even decompensate those patients with congestive heart failure.
  • acepromazine and 1,4-androstadien-3,17-dione, allows to reduce the dose of acepromazine by 50% and achieve considerable calming effects, as observed in rats, which has a direct impact on the reduction of adverse effects presented by acepromazine and that restrict its clinical use.
  • the patch-clamp technique is performed in full cell mode.
  • ADD registration by this technique shows potentiating effects of GABA evoked currents (2.5 ⁇ ) in hippocampal neurons of rat embryos, as shown in Figure No. 5A.
  • ADD modulation was rapidly and completely reversible.
  • ADD induces a potentiation of GABAergic currents of approximately 30%, which translates into a significant reduction in GABA EC 50 from 8.9 to 6.2 ⁇ .
  • the steroids were obtained by biotransformation processes, they were grown in fungal strains in liquid medium for 120 h, with agitation of 120 rpm. After 96 h of culture, the steroidal substrate of plant origin was added at a concentration that ranged between 500-2000 mg / l. Samples of 20 ml were taken daily, in order to assess the biotransformer capacity of the strains. The hydroxylation effect of steroids was evaluated by parameters; pH of initial medium, temperature, etc.
  • the culture is filtered by separating the mycelium from the liquid medium.
  • the liquid solution was extracted with ethyl acetate subsequently evaporated, finally obtaining a total extract with the biotransformation products.
  • the spheroid for experimentation was dissolved in a solution of 10% polyethoxylated castor oil and 90% physiological serum, and then microagitated at a temperature of 45 ° C for 10 minutes.
  • Accepromazine was used as a reference drug to compare tranquilizer and sedative activity and Diazepam as a control drug to assess the anxiolytic effect.
  • the steroid administration route was intramuscular (i.m) and the doses used were determined by preliminary tests. Doses were administered in a range between 30 to 150 mg / kg, without exceeding a final injection volume of 1 ml.
  • Spontaneous motor activity Five minutes after administration of the steroid the rats were placed in a transparent acrylic box of dimensions 47 x 35 x 30 cm. Spontaneous mobility was measured by direct observation, recording the number of lifts and grooming time for 10 min.
  • Light / Dark Scan This test has been widely used to measure anxiety in rodents. It consists of using an acrylic box of dimensions 60 x 50 x 30cm, which is divided into two zones, one of darkness and another of light, communicated by a 10 x 7cm door, which allows easy movement in both fields. The residence time in each field was recorded, before and after administration of the steroid, and of the drugs, for 15 minutes. After each test, the box was carefully cleaned with 70% alcohol.
  • Locomotive activity This test was performed to evaluate the locomotive activity of individuals. A 47 x 25 cm box was used, which was divided into four quadrants of equal dimensions. Five minutes after administration of the steroid, the total number of quadrant changes was recorded by direct observation for a time of 10 min.
  • Hippocampal neurons from rat embryos (C57BL / j6) of 18 days gestation were cultured. Neurons were used, in electrophysiological tests, at 12 days of culture. Culture medium with 80% MEM was used; 10% fetal bovine serum, 10% horse serum, 1 ml of supplementary nutrients.
  • Electrophysiological tests The full-cell patch-clamp technique was used to obtain records of the effects of all steroids, with an Axon 200-B amplifier.
  • the micropipettes were filled with: 140 mM KCI, 10 mM BAPTA, 10 mM HEPES (pH 7.4), 4 mM MgCI 2 , 0.3 mM GTP and 2 mM ATP-Na 2 , 300 mOSM.
  • the external solution contained: 150 mM NaCI, 5.4 mM KCI, 2.0 mM CaCI 2 , 1.0 mM MgCI 2 , 10 mM HEPES (pH 7.4) and 10 mM glucose.
  • the fixing potential was -60 mV.
  • the records were made with a 5 kHz low pass bessel filter and a gain of 5 mV / pA.
  • the currents were measured in the presence of 2.5 ⁇ of GABA alone and co-applied with 10 ⁇ of different ADDs. Variations were represented as a percentage of the control ( * p ⁇ 0.05; * * p ⁇ 0.01)

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  • Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
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Abstract

The invention relates to technology for the veterinary domain, corresponding to a tranquilising/sedating pharmaceutical formulation, for reducing motor activity, comprising at least one neurosteroid with tranquilising activity and a tranquiliser of the phenothiazine derivative type, used as a pre-anaesthetic in mammals, preferably in small animals and in high-risk patients.

Description

FORMULACIÓN FARMACEUTICA TRANQUILIZANTE/SEDANTE A BASE DE NEUROESTEROIDES Y UN DERIVADO FENOTIAZÍNICO PARA USO EN TRANQUILIZING / SEDANT PHARMACEUTICAL FORMULATION BASED ON NEUROSTEROIDS AND A PHENOTIAZINIC DERIVATIVE FOR USE IN
MAMIFEROS. Mammals
Sector técnico Technical sector
La presente tecnología esta orientada al sector veterinario, destinada al manejo de pacientes, especialmente de alto riesgo, que deben someterse al efecto de tranquilizantes. La invención corresponde a una formulación farmacéutica, tranquilizante/sedante, que comprende al menos un neuroesteroide y un tranquilizante del tipo derivado fenotiazínico, que se utiliza como pre-anestésico en mamíferos. This technology is aimed at the veterinary sector, aimed at the management of patients, especially high-risk patients, who must undergo the effect of tranquilizers. The invention corresponds to a pharmaceutical formulation, tranquilizer / sedative, comprising at least one neurosteroid and a tranquilizer of the phenothiazine derivative type, which is used as a pre-anesthetic in mammals.
Técnica Anterior Previous Technique
En medicina veterinaria, se utilizan fármacos con efecto tranquilizante y sedante para facilitar el manejo del paciente en procedimientos como: colocación de sondas, toma de radiografías, traslados de animales, captura de animales silvestres, entre otros. Frecuentemente, el término tranquilizante y sedante se utilizan como sinónimos, sin embargo, el primero no produce una depresión del grado de conciencia al aumentar la dosis, en cambio los sedantes, al incrementar la dosis, deprimen de forma significativa el sistema nerviosos central. In veterinary medicine, drugs with a calming and sedative effect are used to facilitate patient management in procedures such as: placement of probes, radiography, animal transfers, capture of wild animals, among others. Frequently, the term tranquilizer and sedative are used as synonyms, however, the former does not produce a depression of the degree of consciousness by increasing the dose, instead the sedatives, by increasing the dose, significantly depress the central nervous system.
Los investigadores definen el término sedación/tranquilización, como la disminución del grado de conciencia caracterizada por una disminución en la actividad motora y por un tiempo de respuesta más lento frente a un estímulo. Los sedantes generan depresión del sistema nervioso central a dosis altas, en cambio dosis altas de tranquilizantes, dan lugar a signos extrapiramidales como temblores musculares.  The researchers define the term sedation / reassurance, as the decrease in the degree of consciousness characterized by a decrease in motor activity and a slower response time to a stimulus. Sedatives generate depression of the central nervous system at high doses, instead high doses of tranquilizers, give rise to extrapyramidal signs such as muscle tremors.
A veces el término sedación se utiliza para indicar un estado hipnótico incompleto, estudios indican que las acciones sedantes e hipnóticas de los anestésicos generales actúan por distintos mecanismos. Por ejemplo, dosis sedantes de propofol reducen principalmente la actividad de neuronas de la corteza cerebral, mientras que dosis hipnóticas, disminuyen en forma significativa el flujo sanguíneo y el metabolismo de estructuras subcorticales como tálamo, cerebro medio, formación reticular.  Sometimes the term sedation is used to indicate an incomplete hypnotic state, studies indicate that the sedative and hypnotic actions of general anesthetics act by different mechanisms. For example, sedative doses of propofol mainly reduce the activity of neurons in the cerebral cortex, while hypnotic doses significantly decrease blood flow and metabolism of subcortical structures such as thalamus, midbrain, reticular formation.
Un número importante de fármacos, que incluyen tranquilizantes, sedantes y anestésicos generales, modulan receptores ionotrópicos GABAA) prolongando la apertura de canales de cloruro que median la inhibición sináptica rápida, generando corrientes postsinápticas inhibitorias. También se ha relacionado a procesos como la regulación de los estados de vigilia, ansiedad, tensión muscular, memoria y convulsiones epileptiformes. A significant number of drugs, including tranquilizers, sedatives and general anesthetics, modulate GABA ionotropic receptors A) prolonging the opening of chloride channels that mediate rapid synaptic inhibition, generating inhibitory postsynaptic currents. It has also been related to processes such as regulation of wakefulness, anxiety, muscle tension, memory and epileptiform seizures.
GABA es uno de los principales neurotransmisores inhibitorio del sistema nervioso central. En mamíferos, tiene una acción hiperpolarizante al aumentar el influjo de CI" en la neurona postsináptica; aproximadamente entre el 70% y el 90% del neoestriado utiliza GABA como neurotransmisor inhibidor y existe una compleja y estrecha relación entre las neuronas GABAérgicas y dopaminérgicas, donde GABA actúa deprimiendo la vía dopaminérgica. GABA is one of the main inhibitory neurotransmitters of the central nervous system. In mammals, it has a hyperpolarizing action by increasing the influence of IC " on the postsynaptic neuron; approximately 70% to 90% of the neostriate uses GABA as an inhibitory neurotransmitter and there is a complex and close relationship between GABAergic and dopaminergic neurons, where GABA acts by depressing the dopaminergic pathway.
Dentro de los tranquilizantes más utilizados en medicina veterinaria, en Chile están los derivados fenotiazínicos (acepromazina, clorpromazina, etc.) y entre los sedantes destacan las benzodiazepinas (diazepam) y los agonistas a2 adrenérgicos (xilacina). De estas drogas, si bien es cierto cumplen el objetivo, provocan efectos secundarios no deseados que ponen en riesgo la vida del paciente. El grado de profundidad en la sedación, en animales pequeños, se cuantifica mediante la observación de distintas conductas Among the most commonly used tranquilizers in veterinary medicine, in Chile are the phenothiazine derivatives (acepromazine, chlorpromazine, etc.) and among the sedatives are benzodiazepines (diazepam) and 2 adrenergic agonists (xylazine). Of these drugs, while it is true that they meet the objective, they cause unwanted side effects that put the patient's life at risk. The degree of sedation depth in small animals is quantified by observing different behaviors.
El Diazepam es el fármaco ampliamente utilizado por sus efectos sedantes, ansiolíticos, e hipnóticos., Sin embargo, se ha observado que la acción sedante de esta benzodiazepina cuyo blanco molecular es el receptor GABAA; es escasa en animales pequeños y su uso en forma independiente no resulta útil en la realización de procedimientos diagnósticos o terapéuticos; por otra parte, la relajación muscular que produce, lo hace ser el agente de elección en anestesia disociativa.  Diazepam is the drug widely used for its sedative, anxiolytic, and hypnotic effects. However, it has been observed that the sedative action of this benzodiazepine whose molecular target is the GABAA receptor; It is scarce in small animals and its use independently is not useful in performing diagnostic or therapeutic procedures; On the other hand, the muscle relaxation that it produces, makes it the agent of choice in dissociative anesthesia.
La Xilacina: es el clorhidrato de 2 (2,6-dimetifenilamino)-4H-5,6-dihidro-1 ,3- tiazina y, farmacológicamente, se clasifica como analgésico y sedante. La xilacina es un agonista a2 adrenérgico, aunque también se ha observado que actúa sobre receptores colinérgicos, serotorinérgicos, histamínicos H2 y opiáceos. La administración de xilacina produce depresión de la frecuencia cardiaca por bloqueo auriculoventricular de primer y segundo grado. A nivel respiratorio, genera una disminución del número de respiraciones por minuto al deprimir los centros respiratorios del sistema nervioso central, además de provocar emesis (vómitos) a los pocos minutos de administrado el fármaco. La xilacina debe emplearse con precaución o evitarse en animales con: problemas gastrointestinales, enfermedad hepática, con depresión respiratoria o disfunción faríngea o laríngea, pacientes con cardiopatías o con enfermedades en las vías urinarias. La acepromazina es, probablemente, el tranquilizante de uso más común en medicina veterinaria y corresponde a 2-acetil-10-(-3-dimetilaminopropil). Su efecto se basa en el bloqueo central de los receptores dopaminérgicos excitatorios, lo que deriva en efectos tranquilizantes, antiheméticos e hipotérmicos. Además de provocar una disminución de la actividad motora en todos los animales, a dosis altas, originan efectos extrapiramidales o catalépticos. Xylazine: is 2 (2,6-dimethiphenylamino) -4H-5,6-dihydro-1, 3-thiazine hydrochloride and, pharmacologically, is classified as an analgesic and sedative. Xylazine is an adrenergic agonist at 2 , although it has also been observed that it acts on cholinergic, serotorinergic, H2 histamine and opioid receptors. The administration of xylazine causes depression of the heart rate due to first and second degree atrioventricular block. At the respiratory level, it generates a decrease in the number of breaths per minute by depressing the central nervous system respiratory centers, in addition to causing emesis (vomiting) within a few minutes of administering the drug. Xylazine should be used with caution or avoided in animals with: gastrointestinal problems, liver disease, respiratory depression or pharyngeal or laryngeal dysfunction, patients with heart disease or diseases in the urinary tract. Accepromazine is probably the most common tranquilizer used in veterinary medicine and corresponds to 2-acetyl-10 - (- 3-dimethylaminopropyl). Its effect is based on the central blockade of excitatory dopaminergic receptors, which results in tranquilizing, antihemetic and hypothermic effects. In addition to causing a decrease in motor activity in all animals, at high doses, they cause extrapyramidal or cataleptic effects.
El bloqueo catecolamínico central produce un bloqueo α-adrenérgico periférico que ocasiona vasodilatación periférica e hipotensión, por lo que se debe evitar su uso en pacientes hipovolémicoso con alteraciones cardiacas. Provoca un leve efecto anticolinérgico lo que explica que a nivel digestivo se produzca una depresión de la motilidad gastrointestinal. Además produce una baja de la temperatura corporal en todas las especies animales y una disminución significativa en la frecuencia respiratoria.  The central catecolamine blockade produces a peripheral α-adrenergic block that causes peripheral vasodilation and hypotension, so its use in hypovolemic patients with cardiac alterations should be avoided. It causes a mild anticholinergic effect, which explains that at the digestive level there is a depression of gastrointestinal motility. It also produces a decrease in body temperature in all animal species and a significant decrease in respiratory rate.
La acepromazina se puede administrar por vía endovenosa, intramuscular, subcutánea u oral en perros, gatos y equinos. Por ser una droga de metabolización hepática debe evitarse su uso en pacientes con problemas en este órgano y, particularmente, se debe evitar en razas de perros braquicéfalos como bóxer, pequinés, buldog ya que son especialmente sensibles a ella.  Accepromazine can be administered intravenously, intramuscularly, subcutaneously or orally in dogs, cats and horses. Being a liver metabolizing drug should be avoided in patients with problems in this organ and, particularly, should be avoided in breeds of brachycephalic dogs such as boxer, Pekingese, buldog since they are especially sensitive to it.
En general, el uso de estas drogas en animales resulta ser un desafío debido a que se deben utilizar con máxima precaución, sobretodo en aquellos pacientes de alto riesgo (geriátricos, politraumatizados, shock, hipovolémicos, etc.) para evitar una excesiva depresión del sistema nervioso central o agravar los estados de hipotensión. Es por esta razón que se necesitan nuevas formulaciones que reduzcan el riesgo y que las dosis utilizadas sean mínimas, para evitar muertes accidentales. Algunos documentos encontrados que guardan relación con la presente invención se detallan a continuación:  In general, the use of these drugs in animals turns out to be a challenge because they should be used with maximum caution, especially in those high-risk patients (geriatric, polytrauma, shock, hypovolemic, etc.) to avoid excessive system depression. central nervous or aggravate hypotension states. It is for this reason that new formulations are needed that reduce the risk and that the doses used are minimal, to avoid accidental deaths. Some documents found that relate to the present invention are detailed below:
En la patente de invención de la oficina de patentes de invención estadounidense US2005176976 (2005), GABA a modulating neurosteroids, resguarda una familia de moléculas neuroesteroidales, que al ser administradas activan los receptores GABAA induciendo anestesia, permitiendo el tratamiento del stress, anormalidades del sistema nervioso central, etc. La diferencia con nuestra tecnología radica en que las moléculas que se utilizan son estructuralmente distintas y el efecto que ejercen por si solas es diferente a lo que se desea proteger. La patente de invención de la oficina de patentes de la República Checa CZ9904214 (2001), Novel neuroactive steroids, processes of their preparation and use, describe esteroides con actividad neuronal, y un proceso para la preparación de éstos, caracterizado por la introducción de un átomo de flúor en la posición 3 (alfa). Los esteroides neuroactivos según la presente invención son adecuados para el tratamiento de aquellos estados controlados por la acción del neurotransmisor (gamma)- aminobutírico (GABA), como sustancias que presentan actividad contra el dolor, la ansiedad y el insomnio y además como anestésicos. Al igual que la solicitud de patente anterior, la diferencia con nuestra tecnología radica en que no es una molécula nueva lo que se quiere salvaguardar, además su estructura difiere con nuestra iniciativa. In the invention patent of the US Patent Office US2005176976 (2005), GABA a modulating neurosteroids, protects a family of neurosteroidal molecules, which when administered activate GABAA receptors inducing anesthesia, allowing the treatment of stress, system abnormalities central nervous, etc. The difference with our technology is that the molecules used are structurally different and the effect they exert on their own is different from what you want to protect. The patent of invention of the patent office of the Czech Republic CZ9904214 (2001), Novel neuroactive steroids, processes of their preparation and use, describes steroids with neuronal activity, and a process for the preparation of these, characterized by the introduction of a fluorine atom in position 3 (alpha). Neuroactive steroids according to the present invention are suitable for the treatment of those states controlled by the action of the neurotransmitter (gamma) -aminobutyric acid (GABA), as substances that exhibit activity against pain, anxiety and insomnia and also as anesthetics. Like the previous patent application, the difference with our technology is that it is not a new molecule that we want to safeguard, and its structure differs with our initiative.
La solicitud de patente de invención de la oficina de patentes de invención de Estados Unidos US4213981 (1980), Injectable anesthesic, la tecnología protege una composición de un nuevo anestésico que comprende una mezcla determinada de acepromazina o sus sales farmacéuticamente aceptables y midaflur. Esta composición produce un plano quirúrgico de anestesia útil en mamíferos, reduciendo sustancialmente los efectos secundarios que plantea la utilización de midaflur por sí solo. Se establece una gran diferencia, respecto a lo que se desea proteger, ya que en esta innovación se produce un efecto sinérgico entre estos dos compuestos lo que significa una gran diferencia entre estas dos tecnologías. The invention patent application of the United States Patent Office US4213981 (1980), Injectable anesthesic, the technology protects a composition of a new anesthetic comprising a certain mixture of acepromazine or its pharmaceutically acceptable salts and midaflur. This composition produces a surgical plane of anesthesia useful in mammals, substantially reducing the side effects posed by the use of midaflur alone. A great difference is established, with respect to what you want to protect, since this innovation produces a synergistic effect between these two compounds, which means a great difference between these two technologies.
Divulgación de la invención Disclosure of the invention
Algunos antecedentes farmacológicos de derivados esteroidales que actúan sobre el sistema nervioso central, demuestran que éstos son capaces de interactuar con receptores de membrana, principalmente en neuronas y producen un cambio rápido en la excitabilidad del sistema nervioso central, causando un efecto depresor sobre éste.  Some pharmacological antecedents of steroidal derivatives that act on the central nervous system, show that they are capable of interacting with membrane receptors, mainly in neurons and produce a rapid change in the excitability of the central nervous system, causing a depressing effect on it.
La presente tecnología comprende una formulación farmacéutica tranquilizante/sedante, veterinaria que comprende al menos un neuroesteroide con actividad tranquilizante, preferentemente pero no exclusivamente 1 ,4-androstadien- 3,17-diona (ADD), un tranquilizante del tipo derivado fenotiazínico, preferentemente pero no exclusivamente Acepromazina maleato; y excipientes farmacéuticamente aceptables. La gran ventaja comparativa de esta formulación con lo existente, es que el neuroesteroide potencia el efecto del derivado fenotiazínico y a su vez ésta asociación reduce el efecto adverso que provoca el uso independiente de acepromazina, principalmente en casos de pacientes críticos, ya que produce bradicardia y depresión miocárdica directa por lo que puede agravar e incluso descompensar a aquellos pacientes con insuficiencia cardiaca. Esta asociación es una interesante herramienta farmacológica, con la cual, se obtiene un manejo más seguro de los pacientes de alto riesgo o con problemas cardiovasculares, problema que hasta el momento no ha sido solucionado con las formulaciones existentes  The present technology comprises a veterinary tranquilizer / sedative formulation comprising at least one neurosteroid with tranquilizing activity, preferably but not exclusively 1,4-androstadien-3,17-dione (ADD), a tranquilizer of the phenothiazine derivative type, preferably but not exclusively acepromazine maleate; and pharmaceutically acceptable excipients. The great comparative advantage of this formulation with what exists is that the neurosteroid enhances the effect of the phenothiazine derivative and in turn this association reduces the adverse effect caused by the independent use of acepromazine, mainly in cases of critical patients, since it produces bradycardia and Direct myocardial depression so it can aggravate and even decompensate those patients with heart failure. This association is an interesting pharmacological tool, with which, a safer management of patients at high risk or with cardiovascular problems is obtained, a problem that so far has not been solved with existing formulations
La asociación de acepromazina (en un rango de concentración entre 1-100 mg/Kg, aproximadamente) y 1 ,4-androstadien-3,17-diona (en un rango de concentración entre 50-100 mg/Kg, aproximadamente), permite disminuir la dosis de acepromazina en un 50% y alcanza mejores efectos tranquilizantes, lo que se demuestra experimentalmente. El uso en conjunto de estas dos moléculas logra disminuir, de forma considerable, la actividad motora del individuo, a diferencia del efecto que provoca el uso individual de dichas moléculas.  The association of acepromazine (in a concentration range between 1-100 mg / Kg, approximately) and 1,4-androstadien-3,17-dione (in a concentration range between 50-100 mg / Kg, approximately), allows Decrease the dose of acepromazine by 50% and achieve better calming effects, which is demonstrated experimentally. The combined use of these two molecules manages to significantly reduce the motor activity of the individual, unlike the effect caused by the individual use of said molecules.
Para demostrar la efectividad de la formulación descrita, se realizan diversos ensayos en animales los cuales abarcan ensayos in vivo, que incluye evaluación de la actividad motora (actividad locomotora, número de levantadas, tiempo de acicalamiento) y ansiedad; y ensayos in vitro que incluye pruebas electrofisiológicas con la técnica de patch-clamp.  To demonstrate the effectiveness of the described formulation, various tests are carried out on animals which include in vivo tests, which includes evaluation of motor activity (locomotive activity, number of lifts, grooming time) and anxiety; and in vitro assays that include electrophysiological tests with the patch-clamp technique.
En principio, en un ensayo ciego, se evalúa la capacidad neuroactiva de una serie de 15 compuestos esteroidales (neuroesteroídes), obtenidos por procesos de hemisíntesis y biotransformación. Se realiza una determinación previa in vitro a los 15 neuroesteroides y, de forma paralela, se realizan pruebas in vivo. De la pesquisa preliminar se selecciona 1,4-androstadiene-3,17-diona (ADD), como un esteroide con un perfil interesante, ya que provoca una depresión en la conducta motora de los animales la que se manifiesta en la disminución de la capacidad exploratoria e inmovilidad durante un período de tiempo superior a los 30 min, ésta observación se correlaciona con los registros in vitro en donde dicho neuroesteroide potencia corrientes GABAérgicas. Además, presenta una solubilidad mayor en comparación a otras moléculas de similares características farmacológicas. En todos los casos experimentales, el efecto de ADD se compara con los vehículos y los patrones de referencia para cada estado funcional evaluado (sedación, anestesia, ansiedad) que son acepromazina como tranquilizante, alfaxolona como sedante (datos no mostrados) y diazepam como ansiolítico. In principle, in a blind trial, the neuroactive capacity of a series of 15 steroidal compounds (neurosteroids), obtained by processes of hemisynthesis and biotransformation. A previous determination is made in vitro at 15 neurosteroids and, in parallel, tests are performed in vivo. From the preliminary investigation, 1,4-androstadiene-3,17-dione (ADD) is selected as a steroid with an interesting profile, since it causes a depression in the motor behavior of animals which is manifested in the decrease in exploratory capacity and immobility for a period of time greater than 30 min, this observation correlates with in vitro records where said neurosteroid potentiates GABAergic currents. In addition, it has a higher solubility compared to other molecules of similar pharmacological characteristics. In all experimental cases, the effect of ADD is compared with the vehicles and reference patterns for each functional status evaluated (sedation, anesthesia, anxiety) that are acepromazine as a tranquilizer, alphaxolone as a sedative (data not shown) and diazepam as an anxiolytic .
Se evalúa la capacidad ansiolítica de ADD, en ratas macho. Para esto se realiza la prueba de luz/oscuridad, que consiste en medir el tiempo de permanencia de los individuos tanto en el compartimiento oscuro como en el compartimiento claro de una caja habilitada para ello, durante 15 min para cada grupo de tratamiento.  The anxiolytic capacity of ADD is evaluated in male rats. For this, the light / dark test is carried out, which consists in measuring the residence time of the individuals both in the dark compartment and in the light compartment of a box enabled for it, for 15 min for each treatment group.
En la Figura 1 se observan los efectos de ADD (50-100 mg/Kg) en comparación con los efectos ansiolíticos del diazepam (0,5 mg/Kg). El grupo control (animales sin tratamiento), tiene un comportamiento homogéneo que se caracteriza por la permanencia de los animales en el campo de oscuridad casi el 100% del tiempo de observación (15 min), registrando un tiempo de exploración del campo de luz de 10±3,5 segundos, lo que equivale a menos de un 1 ,2% del tiempo total de duración del experimento. Los animales tratados con diazepam, a los dos minutos después que se administra el fármaco, comienzan a realizar exploraciones intermitentes al campo de luz, registrándose al final del experimento un tiempo de permanencia en el campo de luz significativamente mayor (p<0,001 ) que el grupo control (cercano a un 2000%). Los animales tratados con ADD no modifican la conducta de permanencia en las zonas de luz oscuridad de la caja. Al aumentar la dosis, no se observan cambios en la conducta, sin embargo a diferencia del grupo control, los animales tratados con ADD presentan grados visibles de inmovilidad en el sector oscuro de la caja de experimentación. A diferencia de diazepam, ADD no muestra efecto ansiolítico.  Figure 1 shows the effects of ADD (50-100 mg / kg) compared to the anxiolytic effects of diazepam (0.5 mg / kg). The control group (animals without treatment), has a homogeneous behavior that is characterized by the permanence of the animals in the field of darkness almost 100% of the observation time (15 min), recording a time of exploration of the light field of 10 ± 3.5 seconds, which is equivalent to less than 1.2% of the total duration of the experiment. Animals treated with diazepam, two minutes after the drug is administered, begin to perform intermittent scans to the light field, registering a significantly longer time in the light field at the end of the experiment (p <0.001) than the control group (close to 2000%). Animals treated with ADD do not modify the behavior of permanence in the dark light areas of the box. As the dose increases, no behavioral changes are observed, however unlike the control group, animals treated with ADD have visible degrees of immobility in the dark sector of the experimental box. Unlike diazepam, ADD shows no anxiolytic effect.
En relación a los ensayos para evaluar la actividad motora espontánea, los resultados para los distintos tratamientos son resumidos en la tabla N°1. La actividad motora fue evaluada por pruebas de actividad locomotora y actividad espontánea como número de levantadas y tiempo de acicalamiento. In relation to the trials to evaluate the spontaneous motor activity, the results for the different treatments are summarized in table N ° 1. Activity motor was evaluated by tests of locomotive activity and spontaneous activity such as number of lifts and grooming time.
Tabla 1. Efectos de ADD sobre la actividad motora espontánea en ratas*. Table 1. Effects of ADD on spontaneous motor activity in rats * .
Figure imgf000008_0001
Figure imgf000008_0001
' Los datos son promedios n = número de ratas por cada grupo. El tiempo total de medición 10 minutos iniciándose el experimento a los 5 minutos de administrados los tratamientos a los animales.  'The data are averages n = number of rats per group. The total measurement time 10 minutes starting the experiment 5 minutes after the treatments were administered to the animals.
De preferencia, se utiliza la vía intramuscular para administrar los esteroides, con el objetivo de simular una situación clínica real, pero también se puede administrar por vía endovenosa. El efecto de ADD se inicia en promedio a los 5 minutos post administración en todos los animales, por lo tanto las mediciones se inician a partir de ese momento para no interferir con los resultados. En las pruebas preliminares se observa que la duración del efecto de ADD es de aproximadamente 30 minutos y el efecto máximo se alcanza a los 10 min después de su administración. Preferably, the intramuscular route is used to administer steroids, in order to simulate a real clinical situation, but it can also be administered intravenously. The effect of ADD starts on average at 5 minutes post administration in all animals, therefore the measurements start from that moment to not interfere with the results. Preliminary tests show that the duration of the ADD effect is approximately 30 minutes and the maximum effect is reached 10 minutes after administration.
De forma previa se observa que ADD provoca un efecto depresor rápido en los individuos, sin una profundización tardía de sus efectos; esto es una ventaja clínica o quirúrgica ya que, en el caso de asociar ADD a anestesia general, se puede evitar una profundización excesiva y tardía de la anestesia por el efecto sedante o, en su defecto, una recuperación prolongada que pondría en riesgo el proceso quirúrgico o la vida del paciente. Por lo tanto, esta característica que presenta ADD, desde un punto de vista práctico es útil en clínica para su uso en animales pequeños. Previously it is observed that ADD causes a rapid depressing effect in individuals, without a delayed deepening of its effects; This is a clinical or surgical advantage since, in the case of associating ADD with general anesthesia, excessive and late deepening of anesthesia can be avoided due to the sedative effect or, failing that, a prolonged recovery that would put the process at risk surgical or life of patient. Therefore, this feature presented by ADD, from a practical point of view, is useful clinically for use in small animals.
Cinco minutos después de la administración de los respectivos tratamientos, las ratas se colocan en una caja de acrílico transparente (47 x 35 x 30 cm). La movilidad espontánea se mide por observación directa, registrándose el número de levantadas y el tiempo de acicalamiento por 10 min  Five minutes after the administration of the respective treatments, the rats are placed in a transparent acrylic box (47 x 35 x 30 cm). Spontaneous mobility is measured by direct observation, recording the number of lifts and grooming time for 10 min.
La actividad motora exhibida por el grupo control (n=7) muestra que los individuos, una vez colocados en la caja de experimentación, realizan desplazamientos amplios, siempre cerca de los bordes laterales de la caja moviéndose por los distintos cuadrantes. Este grupo tiene en promedio 23,5 ± 3,9 cambios de cuadrantes durante el período de medición, lo cual se combina con algunos movimientos verticales donde las ratas se levantan en sus miembros posteriores y apoyan las manos en la parte vertical de la caja (levantadas), olfateando y explorando el perímetro de ésta. El número de levantadas promedio que se observa es de 18,8 ± 2,7. Toda esta actividad se asocia a intervalos de acicalamiento, que consisten en movimientos estereotipados de limpieza que realizan los individuos. Esta actividad se observa en un tiempo total de 112,5 ± 8 segundos.  The motor activity exhibited by the control group (n = 7) shows that the individuals, once placed in the experimentation box, make wide displacements, always close to the lateral edges of the box moving through the different quadrants. This group has an average of 23.5 ± 3.9 quadrant changes during the measurement period, which is combined with some vertical movements where the rats stand on their hind limbs and rest their hands on the vertical part of the box ( raised), sniffing and exploring its perimeter. The average number of lifts observed is 18.8 ± 2.7. All this activity is associated with grooming intervals, which consist of stereotyped cleaning movements performed by individuals. This activity is observed in a total time of 112.5 ± 8 seconds.
El grupo de ratas tratadas con ADD 50mg/kg (n=7) presenta una disminución significativa de la actividad locomotora ya que reduce el número de cambio de cuadrantes en un 82,1 ± 3,3%, en relación a la actividad mostrada por el grupo control, luego al aumentar la dosis a 100mg/kg esta depresión de la actividad se potencia a un 88,6 ± 2,2% (Fig. 2). Además reduce significativamente (*p<0,05) el número de levantadas, disminuyendo en un 64, 4±6, 4%, respecto al grupo control Al aumentar la dosis a 100 mg/kg de ADD, se observa una reducción similar de un 66,4±6,6% (Fig. 3), sin existir un incremento en el efecto en función de las dosis ensayadas (50-100 mg/kg). Cada condición experimental fue expresada como porcentaje del control (n=7); *p< 0,05; **p<0,01. The group of rats treated with ADD 50mg / kg (n = 7) shows a significant decrease in locomotive activity since it reduces the number of quadrant changes by 82.1 ± 3.3%, in relation to the activity shown by the control group, then increasing the dose to 100mg / kg this depression of activity is enhanced to 88.6 ± 2.2% (Fig. 2). It also significantly reduces (* p <0.05) the number of lifts, decreasing by 64, 4 ± 6, 4%, compared to the control group. When increasing the dose to 100 mg / kg of ADD, a similar reduction of 66.4 ± 6.6% (Fig. 3), without an increase in effect depending on the doses tested (50-100 mg / kg). Each experimental condition was expressed as a percentage of the control (n = 7); * p <0.05; * * p <0.01.
Los resultados que se obtienen en las pruebas de acicalamiento, se representan en la figura N°4 La administración de ADD reduce significativamente el tiempo de acicalamiento (p<0,001 ), se observa que los animales tratados con 50mg/kg de ADD registran una disminución del 88,5 ± 4,8% del comportamiento respecto del grupo control. Al aumentar la dosis de ADD a 100 mg/kg, no se observa una depresión mayor en el tiempo de acicalamiento (88,3 ± 4,7). El tiempo total de acicalamiento se mide por 10 min. Los resultados se expresan como porcentaje del control. La conducta de acicalamiento se elimina completamente para los últimos dos tratamientos (n=7); **p<0,01 ; ***p<0,001. En todos los experimentos no hay diferencias significativas en la depresión de la actividad motora entre 50 y 100 mg/kg de ADD. The results obtained in the grooming tests are represented in Figure No. 4 The administration of ADD significantly reduces the grooming time (p <0.001), it is observed that animals treated with 50mg / kg of ADD record a decrease 88.5 ± 4.8% of the behavior with respect to the control group. By increasing the dose of ADD to 100 mg / kg, no major depression is observed at the time of grooming (88.3 ± 4.7). The total grooming time is measured for 10 min. The results are expressed as a percentage of the control. The conduct grooming is completely eliminated for the last two treatments (n = 7); * * p <0.01; *** p <0.001. In all experiments there are no significant differences in depression of motor activity between 50 and 100 mg / kg of ADD.
Los resultados obtenidos se resumen en la tabla 2 y muestran que ADD provoca una disminución significativa en la actividad locomotora, tiempo de acicalamiento y en el número de levantadas; efecto similar al observado con acepromazina (Figuras 2-4), tranquilizante ampliamente utilizado en animales pequeños, cuyos resultados son predecibles y confiables. Por lo tanto, se comparan los efectos de ADD con acepromazina.  The results obtained are summarized in Table 2 and show that ADD causes a significant decrease in locomotive activity, grooming time and in the number of lifts; effect similar to that observed with acepromazine (Figures 2-4), tranquilizer widely used in small animals, whose results are predictable and reliable. Therefore, the effects of ADD are compared with acepromazine.
Al realizar estas mismas pruebas motoras con acepromazina se observa que el grupo de ratas tratadas con 2,5 mg/kg de acepromazina, disminuye significativamente su actividad locomotora (**p<0,01 , n=7), reduciéndose en un 28,4 ± 4,5%, respecto al grupo control durante los diez minutos de observación. El efecto depresor de la actividad locomotora se repite en forma significativa en el número de levantadas (*p< 0,05) en relación al control, disminuyendo la conducta en un 33,6 ± 7,6%. También se observa que el acicalamiento disminuye significativamente, un 8,9 ± 2,3% (**p<0,01 ) respecto al tiempo registrado por el grupo control para la misma conducta. When performing these same motor tests with acepromazine, it is observed that the group of rats treated with 2.5 mg / kg of acepromazine significantly decreases their locomotor activity (* * p <0.01, n = 7), reducing by 28, 4 ± 4.5%, with respect to the control group during the ten minutes of observation. The depressing effect of the locomotive activity is repeated significantly in the number of lifts ( * p <0.05) in relation to the control, reducing the behavior by 33.6 ± 7.6%. It is also observed that grooming decreases significantly, 8.9 ± 2.3% (** p <0.01) with respect to the time recorded by the control group for the same behavior.
Los resultados observados en la disminución de la actividad motora con ADD (50-100 mg/kg) son similares a los que se obtienen con acepromazina en las tres pruebas mencionadas (Figs. 2-4).  The results observed in the decrease in motor activity with ADD (50-100 mg / kg) are similar to those obtained with acepromazine in the three tests mentioned (Figs. 2-4).
Al combinar ADD 100mg/kg y Acepromazina 2,5 mg/kg, se observa que la actividad locomotora disminuye en un 94,5 ± 1 ,9% (**p<0,01 , n=7) en relación al grupo control (Fig.1). La disminución del número de cambio de cuadrantes es considerable respecto a los grupos de animales tratados con una sola droga. En cuanto al número de levantadas, a los 10 min de observación, disminuye en un 84,7 ± 7,4%(*p< 0,05) respecto al grupo control, y al igual que la conducta anterior, las ratas tratadas con la asociación de estas dos moléculas logran una mayor depresión de la actividad motora que los animales tratados con una sola droga. En relación a la observación de acicalamiento, el grupo de animales tratados con la asociación de compuestos, no presenta conducta de acicalamiento, se suprime en un 100%. Este resultado no se observa en ninguno de los tratamientos anteriores. When combining ADD 100mg / kg andcepromazine 2.5 mg / kg, it is observed that the locomotive activity decreases by 94.5 ± 1.9% (* * p <0.01, n = 7) in relation to the control group (Fig. 1). The decrease in the number of change of quadrants is considerable with respect to the groups of animals treated with a single drug. As for the number of lifts, at 10 min of observation, it decreases by 84.7 ± 7.4% (* p <0.05) with respect to the control group, and like the previous behavior, the rats treated with the association of these two molecules achieve a greater depression of motor activity than animals treated with a single drug. In relation to the observation of grooming, the group of animals treated with the association of compounds, does not exhibit grooming behavior, is suppressed in 100%. This result is not observed in any of the previous treatments.
Al reducir la concentración de ambos compuestos a la mitad, (acepromazina 1 mg/kg y ADD 50mg/kg); la asociación de estas moléculas mantienen una disminución significativa de la actividad locomotora en un 93,4 ± 2,1% (*p< 0,05), conservando un efecto depresor mayor que aquellos en los cuales se administra una sola droga. Aún cuando se disminuye en un 50% la dosis usual, la depresión del número de levantadas también es considerable, 92 ± 8% respecto al grupo sin tratamiento, manteniendo su potencia depresora sobre la actividad motora al igual que las pruebas anteriores. Y, respecto al acicalamiento, los animales que se tratan con esta asociación manifiestan un 100% de depresión de la conducta de acicalamiento durante el periodo de experimentación. By reducing the concentration of both compounds in half, (acepromazine 1 mg / kg and ADD 50mg / kg); the association of these molecules maintains a significant decrease in locomotive activity by 93.4 ± 2.1% (* p <0.05), while retaining a depressing effect greater than those in which a single drug is administered. Even when the usual dose is reduced by 50%, the depression of the number of lifts is also considerable, 92 ± 8% compared to the group without treatment, maintaining its depressing power on motor activity as well as the previous tests. And, regarding grooming, animals treated with this association show 100% depression of grooming behavior during the period of experimentation.
La mayor depresión en la actividad motora para las tres pruebas aplicadas (Figs.2-4) se registra al momento de asociar ambas moléculas ADD y acepromazina. Este efecto sinérgico se mantiene a pesar de disminuir las dosis previas en un 50%.  The greatest depression in motor activity for the three tests applied (Figs. 2-4) is recorded at the moment of associating both ADD and acepromazine molecules. This synergistic effect is maintained despite decreasing previous doses by 50%.
El uso asociado de estas dos moléculas (ADD y acepromazina) logra disminuir considerablemente la actividad motora en comparación a los efectos que provocan al usarlas de forma independiente; además de las pruebas motoras realizadas, es importante destacar que los animales que reciben este tratamiento, tienen un comportamiento distinto y se observan evidentemente más relajados, que los animales controles y la mayor parte del tiempo, permanecen en posición decúbito esternal, efecto que no se observa con las drogas utilizadas en forma separada. En estudios animales, la pérdida del tono postural es usado para reconocer el estado hipnótico, sin embargo, las ratas tratadas no pierden el reflejo de retirada por lo cual no se puede hablar de un efecto hipnótico, lo que da certeza del efecto tranquilizante/sedante como mecanismos de acción.  The associated use of these two molecules (ADD and acepromazine) significantly decreases motor activity compared to the effects they cause when used independently; In addition to the motor tests performed, it is important to highlight that the animals that receive this treatment, have a different behavior and are evidently more relaxed, than the control animals and most of the time, remain in a sternal position, an effect that is not Observe with drugs used separately. In animal studies, the loss of the postural tone is used to recognize the hypnotic state, however, the treated rats do not lose the withdrawal reflex, so we cannot speak of a hypnotic effect, which gives certainty of the calming / sedative effect as mechanisms of action.
Este efecto se puede explicar por la potenciación de la depresión obtenida por ADD y el bloqueo de la vía dopaminérgica por acepromazina. Esto, desde el punto de vista clínico es una interesante herramienta terapéutica para su uso como terapia pre- anestésica en animales pequeños y de razas altamente sensibles a la acción de acepromazina.  This effect can be explained by the potentiation of depression obtained by ADD and the blockage of the dopaminergic pathway by acepromazine. This, from the clinical point of view, is an interesting therapeutic tool for its use as a pre-anesthetic therapy in small animals and of breeds highly sensitive to the action of acepromazine.
También es importante pensar en pacientes de alto riesgo tales como geriátricos, politraumatizados, hipovolémicos. La acepromazina produce bradicardia y depresión miocárdica directa por lo que puede agravar la hipotensión e incluso descompensar a aquellos pacientes con insuficiencia cardiaca congestiva.  It is also important to think of high-risk patients such as geriatric, polytrauma, hypovolemic. Accepromazine produces bradycardia and direct myocardial depression, which can aggravate hypotension and even decompensate those patients with congestive heart failure.
La asociación de acepromazina y 1 ,4-androstadien-3,17-dione, permite disminuir la dosis de acepromazina en un 50% y alcanzar efectos tranquilizantes considerables, según lo observado en ratas, lo que tiene una directa repercusión en la reducción de los efectos adversos presentados por la acepromazina y que restringen su uso clínico. ADD disminuye el tiempo de acicalamiento y reduce la actividad locomotora, de forma significativa, en aproximadamente un 90% de depresión de la actividad a una dosis de 100 mg/kg de ADD (Figura 9, n=7); Esto sugiere un mecanismo de acción similar entre ambas moléculas. Sin embargo, la asociación entre estas moléculas demuestra una significativa potenciación de los efectos obtenidos en forma independiente; más interesante aún, resulta el hecho de que los efectos se mantienen aun cuando se disminuye la dosis usual de acepromazina de 2,5 a 1 mg/Kg, respecto a los obtenidos en forma individual tanto por ADD (50 y 100 mg/Kg) como para acepromazina (2,5 mg/Kg) (Fig 9-1 1 ). The association of acepromazine and 1,4-androstadien-3,17-dione, allows to reduce the dose of acepromazine by 50% and achieve considerable calming effects, as observed in rats, which has a direct impact on the reduction of adverse effects presented by acepromazine and that restrict its clinical use. ADD decreases grooming time and significantly reduces locomotive activity by approximately 90% of activity depression at a dose of 100 mg / kg of ADD (Figure 9, n = 7); This suggests a similar mechanism of action between both molecules. However, the association between these molecules demonstrates a significant potentiation of the effects obtained independently; Even more interesting is the fact that the effects are maintained even when the usual dose of acepromazine is reduced from 2.5 to 1 mg / kg, compared to those obtained individually by both ADD (50 and 100 mg / kg) as for acepromazine (2.5 mg / kg) (Fig 9-1 1).
En relación a las pruebas in vitro, se realiza la técnica de patch-clamp en modalidad de célula completa. El registro de ADD por esta técnica muestra efectos potenciadores de las corrientes evocadas GABA (2,5 μΜ) en neuronas hipocampales de embriones de rata, tal como se observa en la figura N° 5A., la modulación de ADD fue rápida y completamente reversible. ADD induce una potenciación de las corrientes GABAérgicas de aproximadamente un 30%, lo que se traduce en una reducción significativa de la EC50 de GABA de 8,9 a 6,2 μΜ. La curva concentración-respuesta para GABA 2,5 μΜ (cuadrado negro) y GABA 2,5 μΜ co-aplicado con ADD 10 μΜ (cuadro blanco) muestra que EC50 se reduce significativamente, alrededor de un 31%. (n=6, *p<0,05). (Fig.5B, n=6). Esto permite sugerir un mecanismo de acción sinérgico de potenciación de la depresión motora, entre una activación dopaminérgica inducida por acepromazina y una GABAérgica por ADD, a su vez nos permite descartar un efecto sumatorio sobre una misma diana farmacológica. Paralelamente, el mecanismo de acción para ADD se sugiere de acuerdo a los resultados obtenidos in vitro donde 1 ,4-androstadiene-3,17-diona potencia corrientes GABAérgicas, al igual que lo hacen drogas con reconocida actividad anestésico/sedantes como barbitúricos, alfaxolona, diazepam, etc. Ejemplos de aplicación In relation to in vitro tests, the patch-clamp technique is performed in full cell mode. ADD registration by this technique shows potentiating effects of GABA evoked currents (2.5 μΜ) in hippocampal neurons of rat embryos, as shown in Figure No. 5A., ADD modulation was rapidly and completely reversible. . ADD induces a potentiation of GABAergic currents of approximately 30%, which translates into a significant reduction in GABA EC 50 from 8.9 to 6.2 μΜ. The concentration-response curve for GABA 2.5 μΜ (black square) and GABA 2.5 μΜ co-applied with 10 μΜ ADD (white box) shows that EC50 is significantly reduced, around 31%. (n = 6, * p <0.05). (Fig. 5B, n = 6). This allows us to suggest a synergistic mechanism of potentiation of motor depression, between a dopaminergic activation induced by acepromazine and a GABAergic by ADD, in turn allows us to rule out a summation effect on the same pharmacological target. At the same time, the mechanism of action for ADD is suggested according to the results obtained in vitro where 1,4-androstadiene-3,17-dione potentiates GABAergic currents, as do drugs with recognized anesthetic / sedative activity such as barbiturates, alphaxolone , diazepam, etc. Application examples
El estudio se llevó a cabo en 2 etapas: primero se identificó la actividad de la molécula y luego se caracterizó y cuantificó su efecto. The study was carried out in 2 stages: first the activity of the molecule was identified and then its effect was characterized and quantified.
En primera instancia, se realizó un ensayo ciego, utilizando moléculas que fueron identificadas con un código, el que fue asignado en forma arbitraria, simbolizado por las letras ADD que representan la palabra neuroesteroide, y un número consecutivo del 1 al 15. Posteriormente, se identifica la molécula.  In the first instance, a blind test was carried out, using molecules that were identified with a code, which was assigned arbitrarily, symbolized by the letters ADD that represent the word neurosteroid, and a consecutive number from 1 to 15. Subsequently, it Identify the molecule.
Obtención de derivados esteroidales Obtaining steroidal derivatives
Los esteroides fueron obtenidos por procesos de biotransformación, se cultivaron en cepas de hongos en medio líquido por 120 h, con agitación de 120 rpm. Transcurridas 96 h de cultivo, se adicionó el sustrato esteroidal de origen vegetal a una concentración que osciló entre los 500-2000 mg/l. Diariamente se tomaron muestras de 20 mi, con el fin de evaluar la capacidad biotransformadora de las cepas. Se evaluó el efecto de hidroxilación de los esteroides por parámetros; pH de medio inicial, temperatura, etc. The steroids were obtained by biotransformation processes, they were grown in fungal strains in liquid medium for 120 h, with agitation of 120 rpm. After 96 h of culture, the steroidal substrate of plant origin was added at a concentration that ranged between 500-2000 mg / l. Samples of 20 ml were taken daily, in order to assess the biotransformer capacity of the strains. The hydroxylation effect of steroids was evaluated by parameters; pH of initial medium, temperature, etc.
El cultivo se filtra separando el micelio del medio líquido. La solución líquida fue extraída con acetato de etilo posteriormente evaporado, obteniéndose finalmente un extracto total con los productos de biotransformación.  The culture is filtered by separating the mycelium from the liquid medium. The liquid solution was extracted with ethyl acetate subsequently evaporated, finally obtaining a total extract with the biotransformation products.
Luego cada compuesto fue purificado y caracterizado en un cromatógrafo gas- masa y resonancia magnética nuclear.  Each compound was then purified and characterized in a gas chromatograph and nuclear magnetic resonance.
El esferoide para la experimentación fue disuelto en una solución de 10% aceite de ricino polietoxilado y 90% de suero fisiológico, para luego ser microagitado a una temperatura de 45°C, por 10 minutos.  The spheroid for experimentation was dissolved in a solution of 10% polyethoxylated castor oil and 90% physiological serum, and then microagitated at a temperature of 45 ° C for 10 minutes.
Animales Animals
Se ocuparon ratas macho Wistar, con un rango promedio de peso durante el estudio de 160 a 350 gr. Estos animales se colocaron en jaulas individuales, con alimentación y agua a libre demanda. La temperatura de la sala fue de 20 ± 1°C y se manejaron en un ciclo luz oscuridad de 12/12 h. Los animales se utilizaron una sola vez. Drogas utilizadas Male Wistar rats were occupied, with an average weight range during the study of 160 to 350 gr. These animals were placed in individual cages, with food and water on demand. The room temperature was 20 ± 1 ° C and they were handled in a 12/12 h dark light cycle. The animals were used only once. Drugs used
Se utilizó Acepromazina, como droga de referencia para comparar la actividad tranquilizante y sedante y Diazepam, como droga control para evaluar el efecto ansiolítico. Accepromazine was used as a reference drug to compare tranquilizer and sedative activity and Diazepam as a control drug to assess the anxiolytic effect.
Tratamiento Treatment
Se utilizaron tres ratas, para cada condición experimental. La vía de administración del esteroide fue intramuscular (i.m) y las dosis utilizadas fueron determinadas por pruebas preliminares. Se administraron dosis en un rango comprendido entre 30 a 150 mg/kg, sin sobrepasar un volumen final de inyección de 1 mi. Three rats were used, for each experimental condition. The steroid administration route was intramuscular (i.m) and the doses used were determined by preliminary tests. Doses were administered in a range between 30 to 150 mg / kg, without exceeding a final injection volume of 1 ml.
Pruebas in vivo Evaluación de la conducta In vivo tests Behavior assessment
Actividad motora espontánea. Cinco minutos después de la administración del esteroide las ratas fueron colocadas en una caja de acrílico transparente de dimensiones 47 x 35 x 30 cm. La movilidad espontánea se midió por observación directa, registrándose el número de levantadas y el tiempo de acicalamiento durante 10 min. Spontaneous motor activity Five minutes after administration of the steroid the rats were placed in a transparent acrylic box of dimensions 47 x 35 x 30 cm. Spontaneous mobility was measured by direct observation, recording the number of lifts and grooming time for 10 min.
Exploración luz/oscuridad. Este test ha sido ampliamente utilizado para medir ansiedad en roedores. Consiste en utilizar una caja de acrílico de dimensiones 60 x 50 x 30cm, la que se encuentra dividida en dos zonas, una de oscuridad y otra de luz, comunicadas por una puerta de 10 x 7cm, que permite un fácil desplazamiento en ambos campos. Se registró el tiempo de permanencia en cada campo, antes y después de la administración del esteroide, y de los fármacos, por 15 minutos. Después de cada test la caja se limpió, cuidadosamente, con alcohol al 70%. Light / Dark Scan This test has been widely used to measure anxiety in rodents. It consists of using an acrylic box of dimensions 60 x 50 x 30cm, which is divided into two zones, one of darkness and another of light, communicated by a 10 x 7cm door, which allows easy movement in both fields. The residence time in each field was recorded, before and after administration of the steroid, and of the drugs, for 15 minutes. After each test, the box was carefully cleaned with 70% alcohol.
Actividad locomotora. Se realizó esta prueba para evaluar la actividad locomotora de los individuos. Se utilizó una caja de 47 x 25 cm, la cual se divididió en cuatro cuadrantes de iguales dimensiones. A los cinco minutos después de la administración del esteroide, se registró por observación directa el número total de cambios de cuadrantes por un tiempo de 10 min. Locomotive activity This test was performed to evaluate the locomotive activity of individuals. A 47 x 25 cm box was used, which was divided into four quadrants of equal dimensions. Five minutes after administration of the steroid, the total number of quadrant changes was recorded by direct observation for a time of 10 min.
Pruebas ¡n vitro In vitro tests
Cultivos celulares. Se cultivaron neuronas hipocampales de embriones de ratas (C57BL/j6) de 18 días de gestación. Las neuronas fueron utilizadas, en las pruebas electrofisiológicas, a los 12 días de cultivo. Se utilizó medio de cultivo con 80% MEM; 10% suero bovino fetal, 10% suero de caballo, 1 mi de nutrientes suplementarios. Cell cultures. Hippocampal neurons from rat embryos (C57BL / j6) of 18 days gestation were cultured. Neurons were used, in electrophysiological tests, at 12 days of culture. Culture medium with 80% MEM was used; 10% fetal bovine serum, 10% horse serum, 1 ml of supplementary nutrients.
Pruebas electrofisiológicas. Se utilizó la técnica de patch-clamp en modalidad de célula completa para obtener registros de los efectos de todos los esteroides, con un amplificador Axon 200-B. Las micropipetas fueron llenadas con: 140 mM KCI, 10 mM BAPTA, 10 mM HEPES (pH 7.4), 4 mM MgCI2, 0.3 mM GTP y 2 mM ATP-Na2, 300 mOSM. La solución externa contenía: 150 mM NaCI, 5.4 mM KCI, 2.0 mM CaCI2, 1.0 mM MgCI2, 10 mM HEPES (pH 7.4) y 10 mM glucosa. El potencial de fijación fue de -60 mV. Los registros se realizaron con un filtro bessel de pasa baja de 5 kHz y una ganancia de 5 mV/pA. Las corrientes fueron medidas en presencia de 2.5 μΜ de GABA solo y co-aplicado con 10 μΜ de diferentes ADD. Las variaciones fueron representadas como porcentaje sobre el control (*p< 0.05; ** p<0.01) Electrophysiological tests The full-cell patch-clamp technique was used to obtain records of the effects of all steroids, with an Axon 200-B amplifier. The micropipettes were filled with: 140 mM KCI, 10 mM BAPTA, 10 mM HEPES (pH 7.4), 4 mM MgCI 2 , 0.3 mM GTP and 2 mM ATP-Na 2 , 300 mOSM. The external solution contained: 150 mM NaCI, 5.4 mM KCI, 2.0 mM CaCI 2 , 1.0 mM MgCI 2 , 10 mM HEPES (pH 7.4) and 10 mM glucose. The fixing potential was -60 mV. The records were made with a 5 kHz low pass bessel filter and a gain of 5 mV / pA. The currents were measured in the presence of 2.5 μΜ of GABA alone and co-applied with 10 μΜ of different ADDs. Variations were represented as a percentage of the control ( * p <0.05; * * p <0.01)
Análisis de datos. Se utilizó para el análisis la prueba t-Student y ANOVA. Los datos se consideraran significativos con un 95% de confianza (*p< 0.05). Analysis of data. The t-Student and ANOVA test was used for the analysis. The data will be considered significant with 95% confidence (* p <0.05).

Claims

Reivindicaciones Claims
1. Una formulación farmacéutica, tranquilizante/sedante, para disminuir la actividad motora de un individuo CARACTERIZADO porque comprende al menos un neuroesteroide con actividad tranquilizante, preferentemente pero no exclusivamente, 1 ,4-androstadien-3,17-diona; un tranquilizante del tipo derivado fenotiazínico, y excipientes farmacéuticamente aceptables. 1. A pharmaceutical formulation, tranquilizer / sedative, to decrease the motor activity of a CHARACTERIZED individual because it comprises at least one neurosteroid with tranquilizing activity, preferably but not exclusively, 1,4-androstadien-3,17-dione; a tranquilizer of the phenothiazine derivative type, and pharmaceutically acceptable excipients.
2. Una formulación farmacéutica, según reivindicación N° 1 , CARACTERIZADO porque el tranquilizante del tipo derivado fenotiazínico comprende, preferentemente pero no exclusivamente, Acepromazina maleato 2. A pharmaceutical formulation, according to claim No. 1, CHARACTERIZED in that the tranquilizer of the phenothiazine derivative type comprises, preferably but not exclusively, acepromazine maleate
3. Una formulación farmacéutica, según reivindicación N° 1 , CARACTERIZADO porque la concentración del derivado fenotiazínico comprende un rango de concentración entre 1-100 mg/Kg. 3. A pharmaceutical formulation according to claim 1, CHARACTERIZED in that the concentration of the phenothiazine derivative comprises a concentration range between 1-100 mg / Kg.
4. Una formulación farmacéutica, según reivindicación N° 1 , CARACTERIZADO porque la concentración de 1,4-androstadien-3,17-diona comprende un rango entre 50-100 mg/Kg. 4. A pharmaceutical formulation according to claim 1, CHARACTERIZED in that the concentration of 1,4-androstadien-3,17-dione comprises a range between 50-100 mg / Kg.
5. Una formulación farmacéutica, según reivindicación N° 1 , CARACTERIZADO porque la asociación de acepromazina y 1 ,4-androstadien-3,17-diona reduce la dosis de acepromazina en un 50%. 5. A pharmaceutical formulation according to claim 1, CHARACTERIZED in that the association of acepromazine and 1,4-androstadien-3,17-dione reduces the dose of acepromazine by 50%.
6. Una formulación farmacéutica, según reivindicación N° 1 , CARACTERIZADO porque la asociación de acepromazina y 1 ,4-androstadien-3,17-diona reduce la actividad locomotora del individuo en al menos un 90%. 6. A pharmaceutical formulation according to claim No. 1, CHARACTERIZED in that the association of acepromazine and 1,4-androstadien-3,17-dione reduces the locomotor activity of the individual by at least 90%.
7. Una formulación farmacéutica, según reivindicación N° 1 , CARACTERIZADO porque 1 ,4-androstadien-3,17-diona induce una potenciación de las corrientes GABAérgicas de aproximadamente un 30%. 7. A pharmaceutical formulation according to claim No. 1, CHARACTERIZED in that 1,4-androstadien-3,17-dione induces a potentiation of GABAergic currents of approximately 30%.
8. Una formulación farmacéutica, según reivindicación N° 1 , CARACTERIZADO porque se administra por vía intramuscular y endovenosa 8. A pharmaceutical formulation according to claim 1, CHARACTERIZED because it is administered intramuscularly and intravenously
9. Un uso de una formulación farmacéutica, tranquilizante/sedante, CARACTERIZADO porque se utiliza como pre-anestésico en mamíferos preferentemente, en animales pequeños y de razas altamente sensibles a la acción de acepromazina. 9. A use of a pharmaceutical formulation, tranquilizer / sedative, CHARACTERIZED because it is used as a pre-anesthetic in mammals, preferably in small animals and of breeds highly sensitive to the action of acepromazine.
10. Un uso de una formulación farmacéutica, tranquilizante/sedante, según reivindicación N° 8, CARACTERIZADO porque se utiliza en pacientes de alto riesgo, preferentemente en pacientes hipotenso e hipovolémicos, y/o con problemas cardiovasculares 10. A use of a pharmaceutical formulation, tranquilizer / sedative, according to claim 8, CHARACTERIZED because it is used in high-risk patients, preferably in hypotensive and hypovolemic patients, and / or with cardiovascular problems
PCT/CL2011/000040 2010-07-19 2011-07-15 Tranquilising/sedating pharmaceutical formulation based on neurosteroids and a phenothiazine derivative for use in mammals WO2012009823A1 (en)

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WO2009082039A1 (en) * 2007-12-26 2009-07-02 Eisai R & D Management Co., Ltd. Ampa receptor antagonists for epilepsy, mental disorders or deficits in sensory organ

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