WO1987002981A1 - Pharmaceutical composition containing hispiduline or a derivative thereof and utilization of such compounds in the preparation of antiasthmatic compositions - Google Patents
Pharmaceutical composition containing hispiduline or a derivative thereof and utilization of such compounds in the preparation of antiasthmatic compositions Download PDFInfo
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- WO1987002981A1 WO1987002981A1 PCT/FR1986/000379 FR8600379W WO8702981A1 WO 1987002981 A1 WO1987002981 A1 WO 1987002981A1 FR 8600379 W FR8600379 W FR 8600379W WO 8702981 A1 WO8702981 A1 WO 8702981A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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- the present invention relates to a new medicament useful more particularly as a bronchodilator, as well as the methods for its preparation.
- Theophylline is one of the most widely used bronchodilator drugs, particularly in the treatment of asthma attacks as well as in the background treatment of asthmatic disease.
- Theophylline acts by increasing the intracellular level of cyclic AMP by inhibiting its degradation by phosphodiesterase. It is this increase which is responsible for the relaxation of the smooth muscle fiber, and in particular of the bronchial fiber, base of the broncho-dilator effect of the drug.
- theophylline inhibits the degranulation of mast cells, thus preventing the release of chemical mediators from broncho-spasm.
- theophylline causes a relaxation of the smooth digestive fiber, an analeptic effect on the heart, arterial vasodilatation, a diuretic effect, a psycho-analeptic action, a stimulation of the respiratory centers.
- One of the major drawbacks of theophylline is that the margin existing between the effective therapeutic doses and the toxic doses is relatively small, this obviously hinders the prescription.
- theophylline has many undesirable effects, in particular in the event of an overdose, namely nausea, vomiting, headache, excitement, insomnia, tachycardia, which are closely related to the ancillary activities of this product.
- this product remains the treatment of choice in asthma attacks and the basic treatment of asthmatic diseases.
- the subject of the present invention is a new pharmaceutical product which can be used as a bronchodilator and in particular in the treatment of asthma, both at the level of the crisis and as a basic treatment, possibly used as a replacement for theophylline in its other applications. of the same type.
- the present invention is based on the demonstration of the bronchodilator activity of the compound called Hispidulin of formula:
- the present invention relates to the preceding compounds as medicaments, the pharmaceutical compositions containing them and their use, in particular, in the preparation of bronchodilator compositions, in particular anti-asthmatic ones.
- the compounds according to the invention have other pharmacological properties, in particular local anti-inflammatory properties. Hispidulin and its derivatives mentioned above are also useful in compositions for external topical application, whether cosmetic or dermatological.
- Hispidulin in itself is known and has already been isolated from plants, but it has not been possible to demonstrate the pharmacological activity which will be described below.
- Hispidulin can be obtained in different ways. First of all, it is possible to synthesize it by methods which have already been described or by adapting methods which are also known.
- the examples below will give an idea of the methods which can be used for the synthesis of Hispidulin.
- the product can, moreover, be prepared by extraction from different plants in which it is known that there are varying amounts of Hispidulin.
- a first extract with alcohol, ether or ketone is then treated with a chlorinated solvent.
- This chlorinated solvent extract contains Hispidulin which can be separated by chromatography.
- Hispidulin like theophylline, can be used in pharmaceutical compositions applicable by different routes, in particular Hispidulin can be administered orally, rectally or even by injection.
- Galenic shaping of Hispidulin can be carried out by known processes and with excipients adapted to each of the routes used, taking into account the solubility properties of Hispidulin.
- the dosages used will depend, of course, on the patient's condition and the type of disease to be treated and on the nature of the treatment, basic treatment, treatment of paroxysmal attacks.
- the dosage to be used will be less restrictive than with products such as theophylline.
- the extract is then treated with a mixture of water and dichloromethane (400 1 CH 2 Cl 2/200 1 H 2 O). The extraction is carried out by 6 kg.
- the CH 2 Cl 2 phase representing approximately 1.7 kg, that is to say 3.4% of the total weight of the plant, contains the active fraction Hispidulin. This CH 2 Cl 2 phase will be fractionated by chromatography in order to isolate the Hispidulin.
- the product is fixed on the silica by pulping with it.
- Hispidulin is found in the ether fraction, both by chromatography and by measurement of the inhibition of platelet aggregation on rabbit platelets (which is proportional to the Hispidulin content).
- Hispidulin a bronchodilator of choice superior to theophylline with which it will be compared in the examples which follow.
- the measurements were carried out as follows:
- the tracheae used come from male albino guinea pigs or are samples of lung tissue from surgical procedures on human patients.
- the human lung trachea or smooth muscle is prepared and cut into a spiral.
- the spirals are fixed at their ends so as to obtain a force of 8 g for the guinea pig trachea and 2 to 8 g for the human bronchus. They are each stored in an insulated organ tank filled with 10 ml of oxygenated Tyrode buffer (95% O 2 , 5% CO 2 ) and thermostatically controlled at 37 ° C. After 90 minutes of equilibration the length of the spirals is measured. Isometric sensors and physiographers are used to record changes in force.
- Relaxation of the basic tone The relaxing effect of a molecule on the basic tone has been observed by adding it to the preparation of the previously balanced muscle.
- the relaxation of an induced contraction is carried out by stimulating the preparations with the maximum dose of agonist, in this case histamine.
- the molecules to be studied are added to the bath at increasing concentrations every 5 minutes.
- the preparations are convtracted with one of the agonists chosen at the maximum or submaximal dose, then washed. They are preincubated for 30 minutes with the molecule to be studied and contracted under the same conditions as above. The heights of the agonist contacts, before and after incubation, are measured.
- the responses obtained are expressed in g / mm 2 , after dividing the response measured in g by the wet weight / length ratio.
- the contraction after incubation is expressed as a% of the first contraction which expresses a percentage of inhibition.
- Relaxation is expressed as a percentage of the maximum contraction obtained with the agonist used.
- concentration making it possible to obtain 50% of the maximum response (EC50) characterizes the sensitivity of the preparation to the molecule studied and is measured by interpolation of the curve obtained (percentage of relaxation as a function of the decimal logarithm of the concentration). The results observed were as follows: RELAXATION OF THE BASIC MUSCLE TONUS - COBAYA TRACHEA
- PRODUCTS RESPONSE (g / mm 2 ) SEM) (N) REPORT
- a virtually pure mast cell culture is constituted from bone marrow cells of mouse femurs.
- the mast cells suspended in gelatin tyrode buffer (TG), after counting, are distributed so as to have 1 ⁇ 10 6 cells / tube in 100 ⁇ l.
- the tubes are centrifuged at 300 g for 5 minutes at 4 ° C.
- the supernatants are recovered and the pellets taken up in 500 ⁇ l of TG BSA and sonicated 3 times 5 seconds. Determination of the release of ⁇ -hexosaminidase
- the supernatants are tested directly on the aggregation of the rabbit washed platelets and treated with aspirin (0.1 mM for 15 minutes).
- the measurement is carried out in an aggregameter in the presence of the CP / CPK complex.
- the amount of Paf. controls, cells stimulated in physiological saline, is maximum in these tubes and will be the 0% inhibition of the release of Paf.
- the inhibition percentage of the other tubes can thus be deducted.
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Abstract
The present invention relates to hispiduline having the formula (I), its methyl ethers and its demethylated derivative, as well as pharmaceutical compositions containing them and having a broncho-dilating and/or anti-inflammatory activity and/or for external topical application.
Description
COMPOS ITION PHARMACEUTIOUE CONTENANT DE L ' HISP IDULINE OU UN DER IVE ET UTILISATION DE CES COMPOSES DANS LA PREPARATION DE COMPOSITIONS ANTI-ASTHMATIQUES PHARMACEUTICAL COMPOSITION CONTAINING HISP IDULIN OR DERIVATIVE AND USE OF SUCH COMPOUNDS IN THE PREPARATION OF ANTI-ASTHMATIC COMPOSITIONS
La présente invention concerne un nouveau médicament utile plus particulièrement comme agent bronchodilatateur, ainsi que les procédés pour sa préparation.The present invention relates to a new medicament useful more particularly as a bronchodilator, as well as the methods for its preparation.
La théophylline est l'un des médicaments bronchodilatateurs les plus utilisés,en particulier dans le traitement des crises d'asthme ainsi que dans le traitement de fond de la maladie asthmatique.Theophylline is one of the most widely used bronchodilator drugs, particularly in the treatment of asthma attacks as well as in the background treatment of asthmatic disease.
La théophylline agit par augmentation du taux intracellulaire d'AMP cyclique en inhibant sa dégradation par la phosphodiestérase. C'est cet accroissement qui est responsable du relâchement de la fibre musculaire lisse, et notamment de la fibre bronchique, base de l'effet broncho-dilatateur du médicament.Theophylline acts by increasing the intracellular level of cyclic AMP by inhibiting its degradation by phosphodiesterase. It is this increase which is responsible for the relaxation of the smooth muscle fiber, and in particular of the bronchial fiber, base of the broncho-dilator effect of the drug.
De plus la théophylline inhibe la dégranulation des mastocytes, empêchant ainsi la libération des médiateurs chimiques du broncho-spasme.In addition, theophylline inhibits the degranulation of mast cells, thus preventing the release of chemical mediators from broncho-spasm.
Outre la broncho-dilatation, la théophylline provoque un relâchement de la fibre lisse digestive, un effet analeptique cardiaque, une vaso-dilatation artérielle, un effet diurétique, une action psycho-analeptique, une stimulation des centres respiratoires.
L'un des inconvénients majeurs de la théophylline est que la marge existant entre les doses thérapeutiques efficaces et les doses toxiques est relativement faible, ceci gêne, bien entendu, la prescription. En outre la théophylline présente de nombreux effets indésirables, notamment en cas de surdosage, à savoir nausées, vomissements, céphalées, excitation, insomnies, tachycardie, qui sont ttotamment liés aux activités annexes de ce produit.In addition to broncho-dilation, theophylline causes a relaxation of the smooth digestive fiber, an analeptic effect on the heart, arterial vasodilatation, a diuretic effect, a psycho-analeptic action, a stimulation of the respiratory centers. One of the major drawbacks of theophylline is that the margin existing between the effective therapeutic doses and the toxic doses is relatively small, this obviously hinders the prescription. In addition, theophylline has many undesirable effects, in particular in the event of an overdose, namely nausea, vomiting, headache, excitement, insomnia, tachycardia, which are closely related to the ancillary activities of this product.
Néanmoins, malgré ces inconvénients, ce produit reste le traitement de choix dans les crises d'asthme et le traitement de fond des maladies asthmatiques.However, despite these drawbacks, this product remains the treatment of choice in asthma attacks and the basic treatment of asthmatic diseases.
La présente invention a pour objet un nouveau produit pharmaceutique qui peut être utilisé comme bronchodilatateur et notamment dans le traitement de l'asthme, tant au-niveau de la crise que comme traitement de fond, éventuellement utilisé en remplacement de la théophylline dans ses autres applications de même type.The subject of the present invention is a new pharmaceutical product which can be used as a bronchodilator and in particular in the treatment of asthma, both at the level of the crisis and as a basic treatment, possibly used as a replacement for theophylline in its other applications. of the same type.
La présente invention repose sur la mise en évidence de l'activité broncho-dilatatrice du composé dénommé Hispiduline de formule :The present invention is based on the demonstration of the bronchodilator activity of the compound called Hispidulin of formula:
L'Hispiduline en elle-même est connue et a déjà été isolée à partir des plantes, mais sans qu'on ait pu mettre en évidence l'activité pharmacologique qui sera décrite ci-après.Hispidulin in itself is known and has already been isolated from plants, but it has not been possible to demonstrate the pharmacological activity which will be described below.
L'Hispiduline peut être obtenue de différentes façons. Tout d'abord, il est possible d'en effectuer la synthèse par des procédés qui ont déjà été décrits ou par l'adaptation de procédés qui sont également connus.Hispidulin can be obtained in different ways. First of all, it is possible to synthesize it by methods which have already been described or by adapting methods which are also known.
En particulier, les synthèses suivantes sont connues pour la préparation de l'Hispiduline : . la synthèse par réaction de Hoesch sur l'irétol, Phadke et al., Indian J. Chem. 5, 131 (1967) ; . la synthèse par condensation type Baker - Venkataraman, Fukui et al., J. Sci. Hiroshima Univ., Ser. A-Il, ,33, 305 (1969) ; . la synthèse de Iinuma et al., Chem. Pharm. Bull., 32, 4935 (1984) ; . la synthèse de Ahluwalia et al., Indian J. Chem., 14B, 592 (1976).In particular, the following syntheses are known for the preparation of Hispidulin:. Hoesch reaction synthesis on iretol, Phadke et al., Indian J. Chem. 5, 131 (1967); . Baker-type condensation synthesis - Venkataraman, Fukui et al., J. Sci. Hiroshima Univ., Ser. A-Il,, 33, 305 (1969); . the synthesis of Iinuma et al., Chem. Pharm. Bull., 32, 4935 (1984); . the synthesis of Ahluwalia et al., Indian J. Chem., 14B, 592 (1976).
Les rendements de ces synthèses sont faibles mais permettent la préparation de l'Hispiduline.The yields from these syntheses are low but allow the preparation of Hispidulin.
Les exemples ci-après donneront une idée des procédés qui peuvent être mis en oeuvre pour la synthèse de l'Hispiduline.
Le produit peut, en outre, être préparé par extraction à partir de différentes plantes dans lesquelles il est connu que se trouvent des quantités variables d'Hispiduline. Parmi ces plantes, il faut citer notamment plusieurs espèces appartenant aux genres botaniques :The examples below will give an idea of the methods which can be used for the synthesis of Hispidulin. The product can, moreover, be prepared by extraction from different plants in which it is known that there are varying amounts of Hispidulin. Among these plants, we should mention in particular several species belonging to the botanical genera:
AmbrosiaAmbrosia
ArnicaArnica
Baldvina CentaureaBaldvina Centaurea
DigitalisDigitalis
EupatoriumEupatorium
FlourensiaFlourensia
Helenium IvaHelenium Iva
NepetaNepeta
MillingtoniaMillingtonia
PlantagoPlantago
Scutellaria Salvia, etc.Scutellaria Salvia, etc.
Les exemples ci-après mettront en évidence des processus d'extraction permettant de préparer une Hispiduline utilisable à titre de médicament.The examples below will demonstrate extraction processes making it possible to prepare a Hispidulin which can be used as a medicament.
En particulier, à partir de la plante, un premier extrait à l'alcool, éther ou cétone est ensuite traité par un solvant chloré. Cet extrait au solvant chloré contient l'Hispiduline qui peut être séparée par chromatographie.In particular, from the plant, a first extract with alcohol, ether or ketone is then treated with a chlorinated solvent. This chlorinated solvent extract contains Hispidulin which can be separated by chromatography.
L'Hispiduline, comme la théophylline, peut être utilisée dans des compositions pharmaceutiques applicables par différentes voies, en particulier
l'Hispiduline peut être administrée par voie orale, par voie rectale ou même par injection.Hispidulin, like theophylline, can be used in pharmaceutical compositions applicable by different routes, in particular Hispidulin can be administered orally, rectally or even by injection.
Il est également possible de prévoir, notamment dans le traitement des crises asthmatiques, l'utilisation de la voie aérosol.It is also possible to provide, in particular in the treatment of asthmatic attacks, the use of the aerosol route.
La mise en forme galénique de l'Hispiduline peut être réalisée par des processus connus et avec des excipients adaptés à chacune des voies mises en oeuvre, tenant compte des propriétés de solubilité de l'Hispiduline. Les posologies mises en oeuvre dépendront, bien entendu, de l'état du patient et du type de maladie qui doit être traité et de la nature du traitement, traitement de fond, traitement des crises paroxystiques.Galenic shaping of Hispidulin can be carried out by known processes and with excipients adapted to each of the routes used, taking into account the solubility properties of Hispidulin. The dosages used will depend, of course, on the patient's condition and the type of disease to be treated and on the nature of the treatment, basic treatment, treatment of paroxysmal attacks.
Compte tenu de l'activité très importante de l'Hispiduline, la posologie à mettre en oeuvre sera moins contraignante qu'avec des produits tels que la théophylline .Given the very important activity of Hispidulin, the dosage to be used will be less restrictive than with products such as theophylline.
Les exemples ci-après permettront de mettre en évidence d'autres caractéristiques et avantages de la présente invention. EXEMPLE 1The examples below will make it possible to demonstrate other characteristics and advantages of the present invention. EXAMPLE 1
Préparation de l'Hispiduline par extraction de fleurs d'Arnica a) On fait macérer 50 kg de fleurs séchées d'Arnica montana dans de l'alcool éthylique à 60°. L'extraction est effectuée par lot de 17 kg, successivement 300, 200, 180 et 150 1 d'alcool éthylique.Preparation of Hispidulin by extracting Arnica flowers a) 50 kg of dried Arnica montana flowers are macerated in 60 ° ethyl alcohol. The extraction is carried out in batches of 17 kg, successively 300, 200, 180 and 150 l of ethyl alcohol.
L'extrait est alors traité avec un mélange d'eau et dichlorométhane (400 1 CH2Cl2/200 1 H2O) . L'extraction est effectuée par 6 kg.The extract is then treated with a mixture of water and dichloromethane (400 1 CH 2 Cl 2/200 1 H 2 O). The extraction is carried out by 6 kg.
La phase CH2Cl2 représentant environ 1,7 kg, c'est-à-dire 3,4 % du poids total de la plante, contient la fraction active Hispiduline.
Cette phase CH2Cl2 va être fractionnée par chromatographie afin d'en isoler l'Hispiduline.The CH 2 Cl 2 phase representing approximately 1.7 kg, that is to say 3.4% of the total weight of the plant, contains the active fraction Hispidulin. This CH 2 Cl 2 phase will be fractionated by chromatography in order to isolate the Hispidulin.
On utilise pour ce faire une colonne de 220 mm d'une hauteur d'environ 1 ,50 m contenant 19 kg de silice 7734 Merck 60 préparé avec un volume d'hexane d'environTo do this, a 220 mm column with a height of approximately 1.50 m containing 19 kg of silica 7734 Merck 60 prepared with a volume of hexane of approximately
100 1. Le produit est fixé sur la silice par mise en pâte avec celle-ci.100 1. The product is fixed on the silica by pulping with it.
On effectue ensuite une élution avec les solvants suivants : Solvant Volume Extrait % CH2Cl2 % d'inhibition (1) (g) hexane 200 0 toluène 200 390 23 27,5An elution is then carried out with the following solvents: Solvent Volume Extract% CH 2 Cl 2 % inhibition (1) (g) hexane 200 0 toluene 200 390 23 27.5
CH2Cl2 550 690 40,5 65CH 2 Cl 2 550 690 40.5 65
100 ) éther 500 220
19 62100) ether 500 220 19 62
54 ) AcOEt 200 70 4154) AcOEt 200 70 41
MeOH 150 207MeOH 150 207
L'Hispiduline se trouve dans la fraction éther, à la fois par chromatographie et par mesure de l'inhibition de l'agrégation plaquettaire sur plaquettes de lapins (qui est proportionnelle à la teneur en Hispiduline).Hispidulin is found in the ether fraction, both by chromatography and by measurement of the inhibition of platelet aggregation on rabbit platelets (which is proportional to the Hispidulin content).
Le produit ainsi obtenu est, après analyse et comparaison avec des échantillons préparés par la technique synthétique qui sera décrite ci-après, identifié à l'Hispiduline ayant les propriétés physico-chimiques suivantes :The product thus obtained is, after analysis and comparison with samples prepared by the synthetic technique which will be described below, identified with Hispidulin having the following physicochemical properties:
- P.M. : 300- P.M.: 300
- Point de fusion : 287-290°C - UV : 275 nm, 338 nm- Melting point: 287-290 ° C - UV: 275 nm, 338 nm
- Microanalyse : 64 % C ; 4 % H ; 32 % O- Microanalysis: 64% C; 4% H; 32% O
- Cristallise dans MeOH
- C.C.M. : dp à 366 nm, visible à 254 nm jaune à H2SO4 - Crystallizes in MeOH - CCM: dp at 366 nm, visible at 254 nm yellow at H 2 SO 4
Rf : 0,28 à CH2Cl2 95 / MeOH5,Rf: 0.28 at CH 2 Cl 2 95 / MeOH5,
EXEMPLE 2 Activité pharmacologiqueEXAMPLE 2 Pharmacological activity
Les propriétés les plus remarquables de l'Hispiduline sont, comme cela a été dit précédemment, la relaxation du tonus de base du muscle lisse et l'inhibition de la dégranulation immunologique des mastocytes. Ces propriétés faisant de l'Hispiduline un broncho-dilatateur de choix supérieur à la théophylline avec laquelle elle sera comparée dans les exemples qui suivent.The most remarkable properties of Hispidulin are, as has been said previously, the relaxation of the basic tone of smooth muscle and the inhibition of the immunological degranulation of mast cells. These properties make Hispidulin a bronchodilator of choice superior to theophylline with which it will be compared in the examples which follow.
Les mesures ont été effectuées de la façon suivante : Les trachées utilisées proviennent de cobayes mâles albinos ou bien sont des échantillons de tissus pulmonaires provenant d'interventions chirurgicales sur des patients humains.The measurements were carried out as follows: The tracheae used come from male albino guinea pigs or are samples of lung tissue from surgical procedures on human patients.
Les trachées ou les muscles lisses pulmonaires humains sont préparés et découpés en spirale. Les spirales sont fixées par leur extrémité de façon à obtenir une force de 8 g pour la trachée de cobaye et 2 à 8 g pour la bronche humaine. Elles sont stockées chacune dans une cuve à organe isolé remplie de 10 ml de tampon Tyrode oxygéné (95 % d'O2, 5 % de CO2) et thermostatées à 37°C. Après 90 minutes d'équilibration la longueur des spirales est mesurée. Des capteurs isométriques et des physiographes sont utilisés pour enregistrer les changements de force.The human lung trachea or smooth muscle is prepared and cut into a spiral. The spirals are fixed at their ends so as to obtain a force of 8 g for the guinea pig trachea and 2 to 8 g for the human bronchus. They are each stored in an insulated organ tank filled with 10 ml of oxygenated Tyrode buffer (95% O 2 , 5% CO 2 ) and thermostatically controlled at 37 ° C. After 90 minutes of equilibration the length of the spirals is measured. Isometric sensors and physiographers are used to record changes in force.
Relaxation du tonus de base L'effet relâchant d'une molécule sur le tonus de base a été observé en l'ajoutant sur la préparation du muscle préalablement équilibré.
La relaxation d'une contraction induite est effectuée en stimulant les préparations avec la dose maximale d'agoniste, en l'occurrence l'histamine. Quand le plateau est atteint, les molécules à étudier sont ajoutées dans le bain à des concentrations croissant toutes les 5 minutes.Relaxation of the basic tone The relaxing effect of a molecule on the basic tone has been observed by adding it to the preparation of the previously balanced muscle. The relaxation of an induced contraction is carried out by stimulating the preparations with the maximum dose of agonist, in this case histamine. When the plateau is reached, the molecules to be studied are added to the bath at increasing concentrations every 5 minutes.
Enfin, l'inhibition des contractions est mesurée de la façon suivante :Finally, inhibition of contractions is measured as follows:
Pour observer l'inhibition les préparations sont convtractées avec l'un des agonistes choisis à la dose maximale ou submaximale, puis lavées. Elles sont préincubées pendant 30 minutes avec la molécule à étudier et contractées dans les mêmes conditions que précédemment. Les hauteurs des contactions à l'agoniste, avant et après l'incubation, sont mesurées.To observe the inhibition, the preparations are convtracted with one of the agonists chosen at the maximum or submaximal dose, then washed. They are preincubated for 30 minutes with the molecule to be studied and contracted under the same conditions as above. The heights of the agonist contacts, before and after incubation, are measured.
Cakcul des réponsesCakcul of the answers
Les préparations n'étant pas homogènes, il était nécessaire d'uniformiser les réponses obtenues en tenant compte des variations précédentes. Les réponses obtenues sont exprimées en g/mm2, après avoir divisé la réponse mesurée en g par le rapport poids humide/longueur.As the preparations were not homogeneous, it was necessary to standardize the responses obtained taking account of the previous variations. The responses obtained are expressed in g / mm 2 , after dividing the response measured in g by the wet weight / length ratio.
Pour les protocoles d'inhibition, la contraction après incubation est exprimée en % de la première contraction qui exprime un pourcentage d'inhibition.For inhibition protocols, the contraction after incubation is expressed as a% of the first contraction which expresses a percentage of inhibition.
La relaxation est exprimée en pourcentage de la contraction maximale obtenue avec l'agoniste utilisé. La concentration permettant d'obtenir 50 % de la réponse maximale (EC50) caractérise la sensibilité de la préparation à la molécule étudiée et est mesurée par interpolation de la courbe obtenue (pourcentage de relaxation en fonction du logarithme décimal de la concentration).
Les résultats observés ont été les suivants : RELAXATION DU TONUS DE BASE DU MUSCLE - TRACHEE DE COBAYERelaxation is expressed as a percentage of the maximum contraction obtained with the agonist used. The concentration making it possible to obtain 50% of the maximum response (EC50) characterizes the sensitivity of the preparation to the molecule studied and is measured by interpolation of the curve obtained (percentage of relaxation as a function of the decimal logarithm of the concentration). The results observed were as follows: RELAXATION OF THE BASIC MUSCLE TONUS - COBAYA TRACHEA
PRODUITS REPONSE (g/mm2)
SEM) (N) RAPPORPRODUCTS RESPONSE (g / mm 2 ) SEM) (N) REPORT
Tampon - 0,05 0,02 (8) 1Buffer - 0.05 0.02 (8) 1
Théophylline 10-4 M - 0,12 0,04 (13) 2,5Theophylline 10 -4 M - 0.12 0.04 (13) 2.5
Hispiduline 10-4 M - 0,24
0,04 (8) 5Hispidulin 10 -4 M - 0.24 0.04 (8) 5
RELAXATION D'UNE CONTRACTION INDUITE PAR HISTAMINERELAXATION OF A HISTAMINE INDUCED CONTRACTION
TRACHEE DE COBAYE - COURBES DOSE-REPONSE SIGNIFICATIVEMENTCOBAYA TRACHEE - SIGNIFICANTLY DOSE-RESPONSE CURVES
SUPERPOSABLESSTACKABLE
HISPIDULINE THEOPHYLLINEHISPIDULINE THEOPHYLLINE
EC 50 8
2 x 10-5 M 5,5
1 x 10-5 MEC 50 8 2 x 10 -5 M 5.5 1 x 10 -5 M
Inhibition de la dégranulation immunologigue des mastocytes de la moëlle osseuse des sourisInhibition of immunological degranulation of mouse bone marrow mast cells
Pour la mesure de l'inhibition de cette dégranulation, on a utilisé deux marqueurs de dégranulation :For the measurement of the inhibition of this degranulation, two degranulation markers were used:
- un marqueur enzymatique : la β-hexosaminidase,- an enzymatic marker: β-hexosaminidase,
- le médiateur libéré : Paf.acéther.- the released mediator: Paf.acéther.
Les expériences sont effectuées schématiquement de la façon suivante : On constitue, à partir de cellules de moelle osseuse de fémurs de souris par cultures successives, une culture virtuellement pure en mastocytes .The experiments are carried out schematically in the following way: A virtually pure mast cell culture is constituted from bone marrow cells of mouse femurs.
L'expérience proprement dite est effectuée de la façon suivante : Protocole expérimentalThe actual experiment is carried out as follows: Experimental protocol
Les mastocytes en suspension dans du tampon tyrode gélatine (TG), après comptage, sont distribués de façon à avoir 1 x 106 cellules/tube dans 100 μl.The mast cells suspended in gelatin tyrode buffer (TG), after counting, are distributed so as to have 1 × 10 6 cells / tube in 100 μl.
100 μl IgE anti-DNP (dilué au 1 /100/aliquot) sont ajoutés de façon à sensibiliser les cellules.
Les tubes sont incubés 1 heure à 37°C dans une étuve avec CO2 et agités manuellement toutes les 15 minutes. Les cellules sont lavées 3 fois par centrifugation à 300 g pendant 5 minutes avec du TG. Lors de la dernière centrifugation, les cellules sont resuspendues dans 400 μl de TG avec l'albumine de sérum bovin (BSA).100 μl anti-DNP IgE (diluted 1/100 / aliquot) are added so as to sensitize the cells. The tubes are incubated for 1 hour at 37 ° C. in an oven with CO 2 and shaken manually every 15 minutes. The cells are washed 3 times by centrifugation at 300 g for 5 minutes with TG. During the last centrifugation, the cells are resuspended in 400 μl of TG with bovine serum albumin (BSA).
Après une incubation des cellules pendant 15 minutes au bain-marie, avec 50 μl des solutions à tester ou 50 μl des solvants utilisés (tubes témoins), 50 μl d'antigène DNP ([Ag] finale = 40 ng/ml) sont ajoutés dans les tubes dits stimulés, et 50 μl de tampon TG BSA sont ajoutés aux tubes dits non stimulés, et l'incubation est poursuivie pendant 5 minutes sous agitation. La réaction est arrêtée dans la glace par l'addition de 10 μl d'EDTA (4 mM concentration finale).After an incubation of the cells for 15 minutes in a water bath, with 50 μl of the solutions to be tested or 50 μl of the solvents used (control tubes), 50 μl of DNP antigen ([Ag] final = 40 ng / ml) are added in the so-called stimulated tubes, and 50 μl of TG BSA buffer are added to the so-called unstimulated tubes, and the incubation is continued for 5 minutes with shaking. The reaction is stopped in ice by the addition of 10 μl of EDTA (4 mM final concentration).
Les tubes sont centrifugés à 300 g pendant 5 minutes à 4°C. Les surnageants sont récupérés et les culots repris dans 500 μl de TG BSA et soniqués 3 fois 5 secondes. Dosage de la libération de la β-hexosaminidaseThe tubes are centrifuged at 300 g for 5 minutes at 4 ° C. The supernatants are recovered and the pellets taken up in 500 μl of TG BSA and sonicated 3 times 5 seconds. Determination of the release of β-hexosaminidase
50 μl de surnageant ou de solution culot sont additionnés à 450μl de substrat β-hexosaminidase et mis 1 heure à 37°C au bain-marie, sans agitation. La réaction est arrêtée avec 1,5 ml de solution d'arrêt pour chaque tube.50 μl of supernatant or pellet solution are added to 450 μl of β-hexosaminidase substrate and placed for 1 hour at 37 ° C. in a water bath, without stirring. The reaction is stopped with 1.5 ml of stop solution for each tube.
L'activité de la β-hexosaminidase dans les culots et les surnageants est mesurée au spectrophotomètre à 410 nm et le pourcentage net de libération a été calculé selon la formule :The activity of β-hexosaminidase in the pellets and the supernatants is measured with a spectrophotometer at 410 nm and the net percentage of release has been calculated according to the formula:
DO surnageant activé - DO contrôle x 100DO supernatant activated - DO control x 100
(DO surnageant activé + DO culot) - DO surnageant contrôle(DO supernatant activated + DO pellet) - DO supernatant control
(DO = densité optique).
Dosage de la libération de Paf.acéther(OD = optical density). Paf.acether release assay
Les surnageants sont testés directement sur l'agrégation des plaquettes lavées de lapin et traitées par l'aspirine (0,1 mM pendant 15 minutes).The supernatants are tested directly on the aggregation of the rabbit washed platelets and treated with aspirin (0.1 mM for 15 minutes).
La mesure est effectuée dans un agrégamètre en présence du complexe CP/CPK.The measurement is carried out in an aggregameter in the presence of the CP / CPK complex.
Une gamme étalon est effectuée avec des quantités connues de Paf.acéther synthétique.A standard range is carried out with known amounts of synthetic acetate.
Les résultats sont exprimés en équivalent ng de Paf.acéther calculés par rapport à la gamme précédente.The results are expressed in equivalent ng of Paf.acéther calculated compared to the preceding range.
La quantité de Paf. des témoins, cellules stimulées dans du sérum physiologique, est maximale dans ces tubes et sera le 0 % d'inhibition de la libération de Paf. Le pourcentage d'inhibition des autres tubes pourra être ainsi déduit.The amount of Paf. controls, cells stimulated in physiological saline, is maximum in these tubes and will be the 0% inhibition of the release of Paf. The inhibition percentage of the other tubes can thus be deducted.
Les résultats observés sont les suivants : β-HEXOSAMINIDASE Paf.ACETHERThe results observed are as follows: β-HEXOSAMINIDASE Paf.ACETHER
PRODUITS CI 50 RAPPORT CI 50 RAPPORT Théophylline 4,5±1,5 x 10-4 M 1 2,2±0,8 x 10-4M 1 (N = 4) (N = 3)PRODUCTS CI 50 REPORT CI 50 REPORT Theophylline 4.5 ± 1.5 x 10 -4 M 1 2.2 ± 0.8 x 10 -4 M 1 (N = 4) (N = 3)
Hispiduline 4,911 x 10-5M 10 4,8±1 x 10-5M 5 (N = 5) (N = 4)Hispidulin 4.911 x 10 -5 M 10 4.8 ± 1 x 10 -5 M 5 (N = 5) (N = 4)
N = nombre d'expériencesN = number of experiments
On a, en outre, vérifié qu'aux doses très fortes d'Hispiduline, c'est-à-dire 10-4 M, ou de la théophylline, 10-3 M, il n'y avait pas de lyse cellulaire.It was further verified that at very high doses of Hispidulin, that is to say 10 -4 M, or theophylline, 10 -3 M, there was no cell lysis.
Des résultats des expériences précédentes, il ressort que l'Hispiduline se présente, dans le domaine des agents broncho-dilatateurs, comme très supérieure à la théophylline.
EXEMPLE 3From the results of previous experiments, it appears that Hispidulin is, in the field of bronchodilators, very superior to theophylline. EXAMPLE 3
Des essais ayant été conduits avec la pectolinarigénineTrials having been conducted with pectolinarigenin
Claims
REVENDICATIONS
1) A titre de médicament, l'Hispiduline de formule :1) As a medicament, the Hispidulin of formula:
2) Composition pharmaceutique caractérisée en ce qu'elle comporte à titre de principe actif un médicament selon la revendication 1.2) Pharmaceutical composition characterized in that it comprises, as active principle, a medicament according to claim 1.
3) Utilisation de l'Hispiduline, de ses éthers méthyliques et/ou de son dérivé déméthylé pour la préparation de compositions pharmaceutiques broncho-dilatatrices3) Use of Hispidulin, its methyl ethers and / or its demethylated derivative for the preparation of bronchodilator pharmaceutical compositions
4) Utilisation de l'Hispiduline, de ses éthers méthyliques et/ou de son dérivé déméthylé pour la préparation de compositions pharmaceutiques anti-inflammatoires.4) Use of Hispidulin, its methyl ethers and / or its demethylated derivative for the preparation of anti-inflammatory pharmaceutical compositions.
5) Utilisation selon la revendication 3, caractérisée en ce que les compositions pharmaceutiques sont à activité anti-asthmatique.5) Use according to claim 3, characterized in that the pharmaceutical compositions are anti-asthmatic activity.
6) Composition pour application topique externe, caractérisée en ce qu'elle comporte à titre de principe actif au moins un composé de formule :6) Composition for external topical application, characterized in that it comprises, as active principle, at least one compound of formula:
7) Composition pour application topique externe selon la revendication 6, caractérisée en ce qu'il s'agit d'une composition cosmétique.
7) Composition for external topical application according to claim 6, characterized in that it is a cosmetic composition.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8516692A FR2598414B1 (en) | 1985-11-12 | 1985-11-12 | PHARMACEUTICAL COMPOSITION CONTAINING HISPIDULIN OR A DERIVATIVE AND USE OF SUCH COMPOUNDS IN THE PREPARATION OF ANTI-ASTHMATIC COMPOSITIONS |
| FR85/16692 | 1985-11-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1987002981A1 true WO1987002981A1 (en) | 1987-05-21 |
Family
ID=9324708
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/FR1986/000379 WO1987002981A1 (en) | 1985-11-12 | 1986-11-07 | Pharmaceutical composition containing hispiduline or a derivative thereof and utilization of such compounds in the preparation of antiasthmatic compositions |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0245333A1 (en) |
| FR (1) | FR2598414B1 (en) |
| WO (1) | WO1987002981A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6724188B2 (en) | 2002-03-29 | 2004-04-20 | Wavbank, Inc. | Apparatus and method for measuring molecular electromagnetic signals with a squid device and stochastic resonance to measure low-threshold signals |
| EP1556031A2 (en) * | 2002-10-22 | 2005-07-27 | Jenken Bioscience, Inc. | Chromones and chromone derivatives and uses thereof |
| US6952652B2 (en) | 2002-04-19 | 2005-10-04 | Wavbank, Inc. | System and method for sample detection based on low-frequency spectral components |
| US6995558B2 (en) | 2002-03-29 | 2006-02-07 | Wavbank, Inc. | System and method for characterizing a sample by low-frequency spectra |
| CN101891728A (en) * | 2010-05-20 | 2010-11-24 | 南京中医药大学 | Scutellarein derivative as well as preparation method and application thereof |
| US9417257B2 (en) | 2004-07-27 | 2016-08-16 | Nativis, Inc. | System and method for collecting, storing, processing, transmitting and presenting very low amplitude signals |
| US10046172B2 (en) | 2013-03-15 | 2018-08-14 | Nativis, Inc. | Controller and flexible coils for administering therapy, such as for cancer therapy |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6025923A (en) * | 1983-07-22 | 1985-02-08 | Otsuka Pharmaceut Co Ltd | 15-lipoxygenase inhibitor |
-
1985
- 1985-11-12 FR FR8516692A patent/FR2598414B1/en not_active Expired
-
1986
- 1986-11-07 WO PCT/FR1986/000379 patent/WO1987002981A1/en not_active Application Discontinuation
- 1986-11-07 EP EP19860906358 patent/EP0245333A1/en not_active Withdrawn
Non-Patent Citations (4)
| Title |
|---|
| CHEMICAL ABSTRACTS, Vol. 102, 1985, (Columbus, Ohio, US) Y. OSHIMA et al.: "Liver Protective Drugs. 13. Antihepatotoxic Principles of Salvia Plebeia Herbs", see page 62, Abstract 56094a, & Shoyakugaku Zasshi 1984, 38 (2), 201-2 * |
| CHEMICAL ABSTRACTS, Vol. 86, 1977, (Columbus, Ohio, US) F. TILLEQUIN et al.: "Isolation of 5-Hydroxy-4', 6, 7-Trimethoxyflavone from Phyllarthron Madagascariense (Bignoniaceae) Leaves", see page 366, Abstract 161175b, & Planta Med. 1977, 31(1), 76-9 * |
| CHEMICAL ABSTRACTS, Vol. 91, 1979, (Columbus, Ohio, US) L.A. GUMENYUK et al.: "Russian Houseleek-New Source of Biologically Active Compounds", see page 417, Abstract 87358y, & Farm. Zh. (Kiev) 1979, (3), 72-3 * |
| PATENTS ABSTRACTS OF JAPAN, Vol. 9, No. 138 (C-286) (1861), 13 June 1985, JP, A, 6025923 (Otsuka Seiyaku K.K.) 08-02-1985, see the Abstract "Derwent" * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6724188B2 (en) | 2002-03-29 | 2004-04-20 | Wavbank, Inc. | Apparatus and method for measuring molecular electromagnetic signals with a squid device and stochastic resonance to measure low-threshold signals |
| US6995558B2 (en) | 2002-03-29 | 2006-02-07 | Wavbank, Inc. | System and method for characterizing a sample by low-frequency spectra |
| US7081747B2 (en) | 2002-03-29 | 2006-07-25 | Nativis, Inc. | System and method for characterizing a sample by low-frequency spectra |
| US6952652B2 (en) | 2002-04-19 | 2005-10-04 | Wavbank, Inc. | System and method for sample detection based on low-frequency spectral components |
| US7412340B2 (en) | 2002-04-19 | 2008-08-12 | Nativis, Inc. | System and method for sample detection based on low-frequency spectral components |
| EP1556031A2 (en) * | 2002-10-22 | 2005-07-27 | Jenken Bioscience, Inc. | Chromones and chromone derivatives and uses thereof |
| EP1556031A4 (en) * | 2002-10-22 | 2009-03-25 | Jenken Biosciences Inc | Chromones and chromone derivatives and uses thereof |
| US9417257B2 (en) | 2004-07-27 | 2016-08-16 | Nativis, Inc. | System and method for collecting, storing, processing, transmitting and presenting very low amplitude signals |
| CN101891728A (en) * | 2010-05-20 | 2010-11-24 | 南京中医药大学 | Scutellarein derivative as well as preparation method and application thereof |
| US10046172B2 (en) | 2013-03-15 | 2018-08-14 | Nativis, Inc. | Controller and flexible coils for administering therapy, such as for cancer therapy |
| US11103721B2 (en) | 2013-03-15 | 2021-08-31 | Natives, Inc. | Controller and flexible coils for administering therapy, such as for cancer therapy |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2598414A1 (en) | 1987-11-13 |
| FR2598414B1 (en) | 1988-10-14 |
| EP0245333A1 (en) | 1987-11-19 |
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