WO2013050637A1

WO2013050637A1 - Oxoisoaporphine nanocapsules for treating depression

Info

Publication number: WO2013050637A1
Application number: PCT/ES2012/070678
Authority: WO
Inventors: Eduardo SOBARZO SÁNCHEZ; Miriam CEBEY LOPEZ; Jose Ángel FONTENLA; Francisco OTERO ESPINAR; Juan J TORRES LABANDEIRA; Humberto GONZÁLEZ DÁAZ; Renato GRILLO; Leonardo FERNÁNDEZ FRACETO
Original assignee: Universidade De Santiago De Compostela; Universidade Estadual Paulista "Julio De Mesquita Filho"
Priority date: 2011-10-04
Filing date: 2012-10-02
Publication date: 2013-04-11
Also published as: ES2402132A1; BR112014007922A2; ES2402132B1; BR112014007922B1

Abstract

Oxoisoaporphine nanocapsules for treating depression. Nanocapsule systems are described that incorporate compounds of formula I or II that allow controlled, timed release of said compounds. Also described are the production method, the pharmaceutical compositions and the use for preparing a drug.

Description

OXOISOAPORFIN NANOCAPULES FOR THE TREATMENT OF DEPRESSION

Technical sector

The present invention relates to nanocapsules of compounds of formula I or II and to pharmaceutical compositions comprising those nanocapsules.

Background

A group of compounds derived from oxoisoaporphins of formula I and II, were found to be selective and potent inhibitors of monoamine oxidase A (MAO-A) in ranges of IC ₅₀ values from nM to pM in in vitro tests (Eduardo Sobarzo-Sánchez , Matilde Yañez Jato, Francisco Orallo Cambeiro, Eugenio Uriarte Villares and Ernesto Cano Rubio, "Use of oxoisoaporphines and their derivatives thereof as selective inhibitors of monoamine oxidase A", 2008, WO / 2009/034216).

But it is necessary to keep in mind that the CNS is protected by the blood-brain barrier (BHE), a homeostatic defense system designed for protection against pathogens and toxins. BHE is effective in preventing the entry of potential harmful materials by regulating the entry of solutes through endothelial cells. However, this also limits the ability to access the CNS of therapeutically beneficial compounds. Although it is widely known that small molecules can pass through BHE, their access capacity is conditioned by size (<400 Da) and lipophilicity.

It is necessary to design a formulation that allows these compounds to cross the blood brain barrier so that their administration is the most appropriate.

Description of the invention

The authors of the present invention have developed stable nanoparticular systems, with high association efficiency of the compounds of formulas I and II, and which allow them to be released in a controlled manner over time. Therefore, the present invention provides a nanocapsule system suitable for incorporating the compounds of formula I and II, which has potential to cross the blood brain barrier. Thus the invention further provides selective and effective pharmaceutical compositions for the inhibition of MAO-A, and useful in the treatment of depression. Thus, in one aspect the invention is directed to a nanocapsule system characterized by an average size of less than 1 μιη comprising a polymer, a surfactant, an oil and a compound selected from the compounds of formula I and II, their salts , solvate hydrates and N-oxides,

where:

1 2 3 4 5 6 7 8 9

-R, R, RRR, R, R, R °, and R 'are each independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, cycloheteroalkyl, aryl, -OR ^b and - R ^a R ^b ;

-R ^a and R ^b are independently selected from hydrogen, alkyl or, alkenyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, or, R ^a and R ^b together form a heterocycle ring, 4 to 7 members containing 0-2 heteroatoms independently selected from oxygen, sulfur and NR ^c , where R ^c is selected from hydrogen, alkyl, and -C (0) R ^b .

In another aspect the invention is directed to a pharmaceutical composition comprising the nanocapsule systems described above.

In another aspect the invention is directed to the use of the pharmaceutical compositions described above for the preparation of a medicament. In a particular embodiment, the medicament is for the treatment of nervous disorders.

In another aspect, the invention relates to a process for the preparation of the systems of the invention as defined above, comprising:

a) preparing an organic phase comprising an organic solvent, a polymer, an oil and a compound selected from the group consisting of the compounds of formula I and II, their salts, hydrates, solvates and N-oxides, b) preparing a aqueous phase comprising at least one surfactant, c) mixing the phases prepared in steps a) and b), and

d) remove the solvent. Description of the figures

Figure 1. Scheme of polymeric nanocapsules containing OXO compounds: A: polymer; B: oil phase; C: nanocapsules; D: oxoisoporphins; E: Aqueous phase; F: Interaction of oxo compounds with the oil phase and polymer matrix.

Figure 2. OXO 1 chromatogram obtained under described chromatographic conditions.

Figure 3A Calibration curve for compound 0X01 by CLAE. Line equation: y = 0.64x + 0.176. Figure 3B: Calibration curve for the oxoisoaporphines-2 compound by CLAE. Line equation: y = 4.37x - 0.43; Figure 3C: Calibration curve for the oxoisoaporphines-3 compound by CLAE. Line equation: y = 0.98x +0.20; Figure 3D: Calibration curve for the oxoisoaporphines-4 compound by CLAE. Line equation: y = 3.99x + 2.44; Figure 3E: Calibration curve for the oxoisoaporphines-5 compound by CLAE. Line equation: y = 0.95x + 0.89; Figure 3F: Calibration curve for the oxoisoaporphines-6 compound by CLAE. Equation of the line: y = 0.87x - 3.64.

Figure 4. Scheme of the system with which the behavior of the animals was captured.

Figure 5. a) View of the vessels and mice from the position of the video camera and b) Representation of the path traveled by the animals punctured with vehicle (PCL) in the area corresponding to the vessels.

Figure 6. Immobility time of mice treated with the free molecule OXO 4 at a dose of lmg / kg. n = 8 for each group. The results are expressed in seconds as the mean ± standard error. Asterisks mark statistically significant results according to an ANO VA treatment followed by a Dunnet test. * p <0.05, ** p <0.01.

Figure 7. Immobility time of mice treated with the PCL-OXO 4 molecule (nanoencapsulated molecule) at a dose of lmg / kg. n = 8 for each group. The results are expressed in seconds as the mean ± standard error. Asterisks mark statistically significant results according to an ANO VA treatment followed by a Dunnet test. * p <0.05, ** p <0.01.

Figure 8. Immobility time of mice treated with the free molecule OXO 3 at a dose of lmg / kg. n = 8 for each group. The results are expressed in seconds as the mean ± standard error. Asterisks mark the results statistically significant according to an ANO VA treatment followed by a Dunnet test. * p <0.05, ** p <0.01.

Figure 9. Immobility time of mice treated with the PCL-OXO 3 molecule (nanoencapsulated molecule) at a dose of lmg / kg. n = 8 for each group. The results are expressed in seconds as the mean ± standard error. Asterisks mark statistically significant results according to an ANO VA treatment followed by a Dunnet test. * p <0.05, ** p <0.01.

Detailed description of the invention

The compounds of formulas I and II already showed a high selectivity in the inhibition of MAO-A against MAO-B and a higher activity compared to active ingredients such as, for example, Clorgilina and Moclobemide (WO2009034216, table 1 page 23). These advantages make them excellent candidates in the treatment of nervous disorders such as bipolar, depressive, panic, etc.

The compounds of formulas I and II may be in the form of salts, such as ammonium salt. Compounds I and II may also be in oxidized form, in which case they are N-oxides.

In a particular embodiment, in the compounds of formula I, R ¹ and R ² are each independently selected from hydrogen, halogen, cyano, alkyl, alkenyl, -OR ^b and -NR ^to R ^b ; Y

3 4 5 6 7 8 9

RRR, R, R, R ° and R 'are each independently selected from hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloheteroalkyl, aryl, - OR ^b and -NR ^a R ^b ;

where R ^a and R ^b are as defined above.

In another particular embodiment, in the compounds of formula I, R ¹ and R ² are hydrogen and

3 4 5 6 7 8 9

RRR, R, R, R ° and R 'are each independently selected from hydrogen, halogen, and -OR ^bl ; where R ^bl is selected from hydrogen and alkyl.

In another particular embodiment, in the compounds of formula I, R 1, R 2, R 3, R 6, R 7, R 8 and R ⁹ are hydrogen and R ⁴ and R ⁵ are independently selected from hydrogen, methyl or hydroxyl. In a particular embodiment, in the compounds of formula II, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are each independently selected from hydrogen, halogen, nitro, alkyl, cycloalkyl, aryl or -OR ^b ; where R ^b is as defined above.

In another particular embodiment, in the compounds of formula II, R ¹ and R ² are selected

3 4 5 6 7 8 9

between hydrogen and halogen, and R, R, R, R, R, R and R are each independently selected from hydrogen, halogen, nitro, and -OR ^bl ;

where R ^bl is selected from hydrogen and alkyl.

In a particular embodiment, the compounds of formula I and II are preferably selected from the compounds:

1. 2,3-dihydro-7H-dibenzo [de, h] quinolin-7-one

2. 7H-dibenzo [úfe, / z] quinolin-7-one

3. 5-Methoxy-2,3-dihydro-7H-dibenzo [¿faith, 2] quinolin-7-one

4. 5 -methoxy-7H-dibenzo [de, h] quinolin-7-one

5. 5-Methoxy-6-hydroxy-2,3-dihydro-7H-dibenzo [¿faith, 2] quinolin-7-one

6. 5-Methoxy-6-hydroxy-7H-dibenzo [úfe, / z] quinolin-7-one In the present invention, "alkyl" is understood to mean a linear or branched hydrocarbon chain containing no instalation, from 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, optionally substituted with one to three substituents selected from -OR ^b , - R ^to S (0) _m R ^b where m is selected from 1 to 2, -SR ^b , - S (O) _m R ^b , -S (0) _m R ^a R ^b where m is selected between 1 and 2, - R ^a R ^b , -C (0) R ^b , - C0 ₂ R ^b , -C ( 0) R ^a R ^b , - R ^a C (0) R ^b , - R ^a C (0) OR ^b , - R ^a C (0) R ^a R ^b , -CF ₃ , -OCF ₃ , cycloalkyl, cycloheteroalkyl , aryl and heteroaryl; where R ^a and R ^b are as previously defined.

"Cycloalkyl" refers to a cyclic hydrocarbon chain that does not contain any setting, from 3 to 10 carbon atoms, preferably from 5 to 6 carbon atoms. The cycloalkyl may be monocyclic, bicyclic or tricyclic and may include fused rings. Optionally the cycloalkyl may be substituted with one to three substituents selected from halogen, cyano, -OR ^b , - R ^a S (0) _m R ^b where m is selected between 1 and 2, -SR ^b , -S (O) _m R ^b , -S (0) _m R ^a R ^b where m is selected between 1 and 2, - R ^a R ^b , -C (0) R ^b , -C0 ₂ R ^b , -C (0) R ^a R ^b , - R ^a C (0) R ^b , - R ^a C (0) OR ^b , - NR ^to C (O) NR ^to R ^b , -CF ₃ , -OCF ₃ , alkyl, aryl and heteroaryl; where R ^a and R ^b are as previously defined.

"Alkenyl" refers to a linear or branched, cyclic or acyclic hydrocarbon chain, containing at least one setting, from 2 to 10 carbon atoms, preferably from 2 to 5 carbon atoms, optionally substituted with one to three substituents selected from halogen, cyano, -OR ^b , -NR ^a S (0) _m R ^b where m is selected between 1 and 2, -SR ^b , -S (O) _m R ^b , -S (O) _m NR ^a R ^b where m is selected between 1 and 2, -NR ^a R ^b , -C (O) R ^b , -C0 ₂ R ^b , -C (O) NR ^a R ^b , -NR ^a C (O) R ^b , - NR ^to C (O) OR ^b , - NR ^to C (O) NR ^to R ^b , -CF ₃ , -OCF ₃ , alkyl, cycloalkyl, cycloheteroalkyl, aryl and heteroaryl; where R ^a and R ^b are as previously defined.

"Cycloheteroalkyl" refers to a cycloalkyl containing at least one heteroatom selected from oxygen, nitrogen or sulfur, for example: pyrrolidinyl, morpholinyl, piperazinyl and piperidinyl. Optionally the cycloheteroalkyl can be substituted with one to three substituents selected from -OR ^b , -NR ^to S (O) _m R ^b where m is selected from 1 to 2, -SR ^b , -S (O) _m R ^b , - S (O) _m NR ^a R ^b where m is selected between 1 and 2, -NR ^a R ^b , -C (O) R ^b , -CO ₂ R ^b , -C (O) NR ^a R ^b , -NR ^a C (O) R ^b , -NR ^to C (O) OR ^b , - NR ^to C (O) NR ^to R ^b , -CF ₃ , -OCF ₃ , alkyl, aryl and heteroaryl; where R ^a and R ^b are as previously defined.

"Aryl" refers to an aromatic hydrocarbon of 6 to 10 carbon atoms, for example: phenyl or naphthyl; optionally the aryl may be substituted with one to three substituents selected from -OR ^b , -NR ^to S (O) _m R ^b where m is selected from 1 to 2, -SR ^b , -S (O) _m R ^b , - S (O) _m NR ^a R ^b where m is selected between 1 and 2, -NR ^a R ^b , -C (O) R ^b , - C0 ₂ R ^b , -C (O) NR ^a R ^b , -NR ^a C (O) R ^b , -NR ^to C (O) OR ^b , -NR ^to C (O) NR ^to R ^b , -CF ₃ , -OCF ₃ , alkyl, alkenyl, aryl and heteroaryl; where R ^a and R ^b are as previously defined.

"Heteroaryl" refers to an aryl containing at least one heteroatom selected from oxygen, nitrogen or sulfur, for example: pyridyl, pyrazolyl, triazolyl, pyrimidyl, isoxazolyl, indolyl and thiazolyl; optionally the heteroaryl may be substituted with one to three substituents selected from -OR ^b , -NR ^to S (O) _m R ^b where m is selected from 1 to 2, -SR ^b , -S (O) _m R ^b , - S (O) _m NR ^a R ^b where m is selected between 1 and 2, -NR ^a R ^b , - C (O) R ^b , -CO ₂ R ^b , -C (O) NR ^a R ^b , -NR ^a C (O) R ^b , -NR ^to C (O) OR ^b , -NR ^to C (O) NR ⁾ R ^b , -CF ₃ , - OCF ₃ , alkyl, alkenyl, aryl and heteroaryl; where R ^a and R ^b are as previously defined. The compounds of formula I and II are prepared according to the previously published references (Eduardo Sobarzo-Sánchez, Bruce K. Cassels, Carolina Jullian and Luis Castedo Magn. Reson. Chem. (2003) 41, 296-300; Eduardo Sobarzo-Sánchez , Julio De la Fuente and Luis Castedo Magn. Reson. Chem. (2005) 43, 1080-1083).

The nanocapsule systems of the present invention are characterized in that the average size of the particles that form it is less than 1 micrometer.

The term "average size" means the average diameter of the nanocapsule population, which comprises the system. The average size of these systems can be measured using standard procedures known to those skilled in the art, and which are described in the experimental part.

The nanocapsules of the system of the invention have an average particle size of less than 1 micrometer, that is, they have an average size of between 1 and 999 nm, preferably between 100 and 500 nm, even more preferably between 150 and 300 nm. The average particle size is mainly influenced by the composition and formation conditions thereof.

On the other hand, the nanocapsules have a negative electrical charge (measured by the potential Z). In a particular embodiment of the invention, the nanocapsules have a negative charge that can vary between -10 mV and -60 mV. In another particular embodiment, the negative charge is between -20 and -45 mV.

The zeta potential of the nanocapsules of the systems of the invention can be measured using standard procedures known to those skilled in the art, and which are described, for example, in the experimental part of the present specification. The polydispersion index of the nanocapsule system of the invention is low. In a particular embodiment, the polydispersion index of the nanocapsule system of the invention is between 0.001 and 0.7. In a particular embodiment, the polydispersion index of the systems of the invention is between 0.01 and 0.2.

In a particular embodiment, the polymer constituting the nanocapsules of the invention is selected from polystyrene, polyester, polyphosphazine, polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyacrylate, polyvinyl pyrrolidinone, polyallylamine, polyethylene, polyacrylic acid, polyethylene acrylate, polyoxyethylene, polyoxyethylene , chitosan, polyhydroxybutarate and its derivatives. In a particular embodiment, the polyester is selected from the group consisting of polycaprolactone, polyglycolic acid, polylactic acid, glycolic acid copolymer and lactic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid, polycyanoacrylate and polymethylidene malonate.

In a more particular embodiment, the polyester is selected from the group consisting of polylactic acid, polyglycolic acid, poly-epsilon-caprolactone. Preferably, the polymer is poly-epsilon-caprolactone.

In a particular embodiment, the oil is selected from a mineral, synthetic or vegetable oil. In a particular embodiment, the oil is selected from saturated and unsaturated aliphatic hydrocarbons, aromatic hydrocarbons, cyclic and acyclic hydrocarbons, saturated or unsaturated C1-C24 carbon chain triglycerides. In a more particular embodiment, C1-C6 carbon chain triglycerides. In another particular embodiment, C8-C20 carbon chain triglycerides. In a particular embodiment, the oil is selected from sunflower, olive, soybean, palm and rapeseed oil. In a particular embodiment, the oil is a triglyceride of capric and caprylic acid (Miglyol 810).

"Surfactant" is understood as any molecule composed of a hydrophobic part and a hydrophilic moiety. These molecules therefore have amphiphilic properties, which causes them to migrate to the surface between the water and the oil phase in a water / oil mixture. Thus, the hydrophilic head is maintained in the aqueous phase and the hydrophobic tail interacts with the oil by altering the surface properties of the water / oil interface and allowing the formation of an emulsion, as well as its stabilization.

In a particular embodiment, the surfactant is selected from castor oil ethoxylate, Pluronic F68 (copolymer of eulene oxide and propylene oxide), BRIJ (stearate), Tween 20 (polysorbate 20), Tween 40 (polysorbate 40), Tween 60 (polysorbate 60), Tween 80 (polysorbate 80), Sodium lauryl sulfate, Crillet 1, Crillet 4 HP, Cril let 4 NF, Cremophor RH40, Cremophor RH60, Cremophor EL, Eiocas 30, Mkkol HCO-6G, Labrasol, Acconon MC-8, Gelucire 50/13, Gelucire 44/14, MYRJ, polyoxameros, Epikuron 170, phospholipids, Span (sortean monostearate), glycerol monostearate, Capmul CM (capric / capric glyceride), Capmul MCM 8, Capmul MCM 10 , imwitor 988, Imwitor 742, immitator 308, Labrafil M 1944 CS, Labrafil M 2 125, Capryol PGMC, Capryol 90, Laurogycol, Captex 200, ethoxylated fatty acid, Oleic plurol (polyglyceryl-6-dioleate), Crill 1 (sorbitan laurate), Crill 4 (sorbitan oleate), Maisine (glycerin monolinoleate), Peceol (glycerol oleate) and Arlacel P135 (polyethylene glycol dipolhydroxystearate). In a particular embodiment, the phospholipid is lecithin.

The process described in the present invention makes it possible to obtain the systems of the invention in a simple way, under mild working conditions so that degradation of the active ingredients they comprise is avoided.

In a particular embodiment, the process for the preparation of the systems of the invention as described above, comprises:

e) preparing an organic phase comprising an organic solvent, poly-epsilon-caprolactone and a compound selected from the group consisting of the compounds of formula I and Π, their salts, hydrates, solvates and N-oxides, f) preparing a aqueous phase comprising at least one surfactant, g) mixing the phases prepared in steps a) and b), and

h) remove the solvent.

In a particular embodiment, the solvent is selected from acetone, ethanol, water, chloroform, methanol, ethyl acetate, dimethylformamide, dimethyl sulfoxide and tetrahydrofuran.

In a particular embodiment, the organic phase further comprises an oil soluble surfactant. Oil-soluble surfactants are known to those skilled in the art, and are, for example, lecithin, polysorbate 80, etc.

EXPERIMENTAL PROCEDURE

1. SYNTHESIS AND CHARACTERIZATION OF NANOPARTICLES

1.1 Material

Poly-e-caprolactone (PCL, 80,000 MW) was purchased from Sigma (Aldrich Chem. Co.), sorbitan monostearate (Span 60 ^® ) (Sigma Aldrich Chem. Co.), Polysorbate 80 (Tween 80 ^® ) (LabSynth , Brazil), triglycerides of caprylic / capric acids (Miglyol 810 ^® ) (Huís, Germany) and analytical grade acetone (LabSynth, Brazil). The solvents used in chromatography analysis were HPLC grade, acetonitrile (JT Baker ^® ) and deionized water (Milli-Q, Millipore). The solutions were filtered using Millipore (Belford, USA), 0.22 μιη nylon membranes (Belford, USA).

1.2 Methods

1.2.1 Preparation of polymeric capsules containing oxoisoaporphine derivatives

The nanocapsules of Poly-s-caprolactone (PCL-NC) empty and loaded with oxoisoaporphins (OXO) (Figure 1) were prepared by interfacial deposition (Fessi,

H.; Puiseiux, F .; Devissaguet, J. P .; 1988, Procédé de preparation de dispersible colloidaux systémes d'une substance sous form of nanocapsules. European Patent,

0274961 Al). Six types of oxoisoaporphins were associated with nanocapsules.

The method consists of mixing an oily phase in an aqueous phase. The organic phase was prepared using 100 mg of polymer (PCL), 30 ml of organic solvent (acetone), 200 mg of triglycerides of capric and caprylic acid (Miglyol 810), 40 mg of sorbitan monostearate (Span 60) and 7 mg of OXO The aqueous phase was prepared using 30 ml of aqueous solution containing 60 mg of polysorbate 80 (Tween 80). After the dissolution of the components of both phases, the organic phase was introduced little by little with the help of a funnel, in the aqueous phase with magnetic stirring. The resulting suspension was kept under stirring for 10 minutes and then the organic solvent was removed using a rotary evaporator until the final volume of 10 ml (0.70% of the Compound). All preparations were made protected from light and kept in the dark all the time.

I.2.2 Chromatographic conditions for the quantification of oxoisoaporphins High-performance liquid chromatography (FIPLC) analyzes were developed using a Varían ^® ProStar instrument, equipped with a PS 210 pump, PS 325 UV-VIS detector, Metatherm ^® oven, Phenomenex column ^® and an automatic injector. The chromatograms were processed using the Galaxy Workstation ^® software. Table 1 shows the chromatographic conditions of the compound OXOs, and the samples and the mobile phase were previously filtered through 0.22 μιη Millipore ^® nylon membranes.

1.2.3 Association efficiency measures

The total amounts (100%) of the OXO compounds present in the nanocapsule (NC) suspensions were determined by HPLC, after dilution in acetonitrile and filtration through a Millipore membrane of 0.22 μιη.

The amounts of compound associated with the NC were measured using the ultrafiltration / centrifugation method, which consists of centrifuging the NC suspension for 10 min. in ultrafiltration containers formed of regenerated cellulose of 30 kDa (Microcon, Millipore), and then analyzing the ultrafiltrates by HPLC. Only the free compound passed through the 30 kDa membrane, so that the amounts associated with the nanoparticle could be estimated by difference (Gamisans, F .;

Lacoulonche, F .; Chauvet, A .; Thorn, M .; García, M. L .; Egea, M. A. Int. J. Pharm.

1999, 179, 37-48 .; Schaffazick, S. R .; Guterres, S. S .; Freitas, L. L .; Pohlmann, A. R. Quim. Nova, 2003, 26, 726-737 .; Kilic, A. C; Capan, Y .; Vural, L; Gursoy, R. N .;

Dalkara, T .; Cuine, A .; Hincal, AAJ Microencapsulation 2005, 22, 633-641). 1.2.4 Characterization of the nanocapsules of Poly-s-caprolactone (PCL) 1.2.4.1 Measures of size and polydispersion

The light scattering technique was used to determine the average size and distribution of the nanoparticles. This analysis was carried out after dilution (1/100 v / v) of the nanoparticle suspensions, using a Malvern ^® Zetasizer HSA 3000 (United Kingdom) particle analyzer at a fixed angle of 90 ° and a temperature of 25 ° C, immediately after preparation. Each result was expressed as the measure of three trials (Govender, T .; Stolnik, S .; Garnett, M. C; Illum, L .; Davis, SSJ Control. Relay 1999, 57, 171-185.; Venkatraman, SS ; Jie, P .; Min, R; Freddy, BY C; Leong-Huat, G., Int. J. Pharm. 2005, 298, 219-232).

1.2.4.2 Zeta potential

The surface charges of the nanoparticles were assessed by zeta potential determination, using the Zetasizer HSA 3000 instrument, immediately after preparation. The analyzes were performed after dilution (as mentioned above) and the results were the averages of three measures.

EVALUATION OF COMPOUNDS

The compounds evaluated in the present invention are based on the following general formulas, corresponding to the general formula (I):

A. 2,3-DIHIDRO-7H-DIBENZO [¿/ e, / 2] QUINOLIN-7-ONA (2,3-DIHIDRO

OXOISOAPORFINA), General formula (I):

In which: a) in that if: - R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ is hydrogen, it is 2,3-dihydro-7H-dibenzo [úfe , / z] quinolin-7-one, hereinafter referred to as OXO 1; b) in that if: - R ¹ , R ² , R ³ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ is hydrogen and R ⁴ represents a methoxy, it is 5-methoxy-2,3- dihydro-7H-dibenzo [¿faith, 2] quinolin-7-one, hereinafter referred to as OXO 3; Y

1 2 3 6 7 8 9 4

c) in which if: -R, R, R, R, R, R and R is hydrogen, R represents a methoxy and R ⁵ represents a hydroxyl; it is 5-methoxy-6-hydroxy-2,3-dihydro-7H- dibenzo [de, / z] quinolin-7-one, hereinafter referred to as OXO 5. B. 7H-DIBENZO [Ufe, / 2 ] QUINOLIN-7-ONA (OXOISOAPORFINA), General Formula (II):

In which:

a) in which if: - R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ is hydrogen, it is 7H-dibenzo [de, / z] quinolin- 7-one, hereinafter called OXO 2;

b) where: - R ¹ , R ² , R ³ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ is hydrogen and R ⁴ represents a methoxy, it is 5-methoxy-7H-dibenzo [ de, / z] quinolin-7-one, hereinafter referred to as OXO 4; Y

1 2 3 6 7 8 9 4

c) in which if: -R, R, R, R, R, R and R is hydrogen, R represents a methoxy and R ⁵ represents a hydroxyl; it is 5-methoxy-6-hydroxy-7H-dibenzo [de, / z] quinolin-7-one, hereinafter referred to as OXO 6;

2. RESULTS AND DISCUSSION OF THE SYNTHESIS AND CHARACTERIZATION OF THE NANOPARTICLES

2.1 Chromatographic analysis of nanoparticles A chromatographic analysis of all the oxoisoaporphins mentioned in the present invention was performed. Figure 2 shows the OXO 1 chromatogram. This compound shows a good profile, with a symmetrical peak and with a retention time of 5.3 min.

2.2 Analytical curve determined by HPLC

The calibration curve was evaluated for all OXO compounds (Figure 3). The data of the standard curves were obtained in triplicate and showed a linear behavior with a good correlation coefficient.

2.3 Association efficiency of OXO compounds in nanoparticles

The determination of the amounts of compounds associated with the nanoparticles is especially complex due to the small size of the latter, which complicates the separation of the free and associated compound fractions (Magenheim, B .; Benita, STP Pharma Sci. 1991, 1 , 221-241 .; Soppimath, KS; Kulkarni, AR; Aminabhavi, TM J Control Relay 2001, 75, 331-345). In addition, other factors influence the association, such as the physicochemical characteristics of the compound (Guterres, SS; Fessi, H .; Barratt, G .; Devissaguet, J. -Ph .; Puisieux, F. Int. J. Pharm 1995, 113, 57-63 .; Calvo, P .; Vila-Jato, JL; Alonso, M. j. J. Pharm. Sci. 1996, 85, 530-536), the pH of the medium (Brasseur, N .; Brault, D .; Couvreur, P. Int. J. Pharm. 1991, 70, 129-135 .; Govender, T .; Stolnik, S .; Garnett, M. C; Illum, L :; Davis, SSJ Control Relay 1999, 57, 171-185), the characteristics of the particle surface or the nature of the polymer (Vila, A .; Sánchez, A .; Tobío, M .; Calvo, P .; Alonso, M. j. J Control Relay 2002, 78, 15-24), as well as the amount of drug added to the formulation (Brasseur, N .; Brault, D .; Couvreur, P. Int. J. Pharm. 1991, 70, 129 -135), the order of addition and the type of surfactant used to stabilize the polymeric surface (Schaffazick, SR; Guterres, SS; Freitas, LL; Pohlmann, AR Quim. Nova 2003, 26, 726-737).

The rate of association of OXO with PCL nanocapsules was determined by the ultrafiltration / centrifugation method using FIPLC. The degree of association of OXO was determined as the difference between its concentration in the filtrate and the total concentration (100%). Association values are shown in Table 2.

The efficiency of association of the OXO compounds in the nanocapsules formed by PCL was very high with values close to 100%, showing that there is a good affinity between the drug and the polymer (Mora-Huertas, CE; Fessi, H .; Elaissari, A. International Journal of Pharmaceutics 2010, 385, 113-142).

2.4 Characterization of the nanocapsules

The photonic correlation technique was used to determine the average particle size (such as hydrodynamic diameter) and polydispersion index. The zeta potential (mV) values were determined using a Zetasizer ZS90 (Malvern ^® ) analyzer. The values of size, polydispersion and zeta potential are shown in Table 3.

The nanoparticles presented a size distribution in the range of 244-274 nm, indicating that there were small differences in size between the formulations. The polydispersity index, which provides an indication of the particle size distribution, can also be used to assess stability. Factors that influence polydispersity include properties of the solution, the thermodynamics of the system and the method of preparation. The high polydispersity index values are indicative of the heterogeneity in the diameters of the suspended particles, while the changes over time are due to the formation of particle populations that have different diameters of those initial particles, due to aggregation processes, disintegration or degradation. A polydispersity index value smaller than 0.2 is considered ideal, as this reflects a narrow particle size range (Schaffazick, SR; Guterres, SS; Freitas, LL; Pohlmann, AR Quim. Nova, 2003, 26, 726 -737). In our case, the polydispersity values obtained are all below 0.2 (Table 3), indicating excellent particle homogeneity. Another parameter considered was the measure of zeta potential. An analysis of the zeta potential of the particles reflects their surface charges. This parameter can be influenced by the particle composition, dispersion medium, pH, and ionic strength of the medium. The nanoparticles showing values (approximately ± 30) are more stable in suspension (Mohanraj, VJ; Chen, Y. Tropical J. Pharm. Res. 2006, 5, 561-573). In all prepared formulations the potential zeta value was negative (Table 3). For the prepared formulations, the potential zeta values measured were quite high (> 35 mV), indicating that these particles probably have good stability in solution. Thus, according to the experimental synthesis and characterization data of PCL nanocapsules containing oxoisoaporphine derivatives, these supramolecular systems showed excellent association efficiency with the highest value (> 99%). The nanocapsules showed an average particle size of 260 nm, polydispersity indexes <0.2 and zeta potentials close to -40 mV. The excellent interaction obtained between the PCL nanocapsules and the active ingredients, together with good suspension stability, opens up new perspectives for the controlled release of these compounds.

3. RESULTS OF THE IN VIVO ANTIDEPRESSIVE ACTIVITY OF FREE AND ENCAPSULATED OXOISOAPORFINS General conditions

An experimental procedure used in the preclinical investigation of depression was used to perform the in vivo tests: the Forced Swim Test (FST) also known as the Porsolt test. This procedure is the most widely accepted and the most frequently used. It is a validated test to evaluate the potential in vivo antidepressant activity of new molecules in experimental animals.

The environmental conditions have remained homogeneous and animals of the same sex and with similar weights have been used.

1. The tests were carried out in an acoustically isolated room, with automatic regulation of temperature (19-21 ° C) and ambient light (on between 8:00 - 20:00, maintaining light-dark periods of 12 hours) . In addition, an environmental humidity control (45-60%) was performed.

2. Swiss male albino mice of Charles River CD1 origin have been used, weighing between 20-35 g, which were supplied by the animal husbandry of the University of Santiago de Compostela (USC).

3. The animals were arranged in polypropylene boxes (215x465x145 mm). The floor of the box was kept covered with a bed of wood chips (Lignocel®, J.

Rettenmaier &Sóhne; Rosenberg, Germany) that changed periodically. The animals were fed with commercial SAFE pellets (Scientific Animal Food and Engineering, Augy, France) and, like water, they were supplied on demand.

4. The animals were kept a minimum of 3 days under the conditions described above before carrying out the pharmacological tests. These tests were carried out during the light period between 8: 00-20: 00.

5. Both the housing and the handling and experimentation have been carried out in accordance with the standards established in the directive of the Council of the European Communities (86/609) and the Spanish legislation (RD 1201/2005 of October 10, on the protection of animals used for experimentation and other scientific purposes (BOE n ° 252, of October 21, 2005)). Methods and drugs

The in vivo method performed has as main characteristics that:

• It is a short test in time (6 minutes)

• Detects a broad spectrum of antidepressants, regardless of their mechanism.

• Automated, so it is objective.

The behavior of the animals was recorded for 6 min by an analog video camera (Sony DXC-107A, Sony Corporation, Japan), positioned on the ceiling of the room and perpendicular to the vessels with water in which the animals were introduced. The camera is connected to an adapter (Sony CMA-D2) which sends the signal to a monitor (Sony PVM-14M2E) and to two digital converters:

• A Picolo digitizer card (Euresys, Liege, Belgium), placed in one of the computer's PCI slots (Dell Dimension 8200).

• An external DVC-USB digitizer card (Dazzle), (Figure 4). The signal digitized by the Picolo card is used by the behavior analysis software (Computerized Animal Observation System -Ethovision V. 3.1.16, Noldus Information Technology, Wageningen, The Netherlands), installed on the computer that was in an adjacent room.

The EthoVision software locates the animal's center of gravity, graphically symbolized by the intersection between the coordinate axes (x, y), stores the data and allows the subsequent analysis of multiple parameters (distance traveled, speed, etc.).

All the tests were also digitized using the DVC-USB card and the Dazzle MovieStar software (V. 4.5) that recorded the video in vivo of the experiment and allows the analysis of behaviors not susceptible to automation.

The vessels with water in which the animals are introduced are illuminated by a diffused light that allowed the correct monitoring of the movements of the mice.

The parameters analyzed in the trials were the time of immobility, mobility and strong mobility (these last two not represented)

The compounds evaluated in the in vivo assays of the present invention are the most active in the in vitro human MAO-A inhibition assays reported by our research group (Eduardo Sobarzo-Sánchez, Matilde Yañez Jato, Francisco Orallo Cambeiro, Eugenio Uriarte Villares and Ernesto Cano Rubio, "Use of oxoisoaporphines and their derivatives thereof as selective inhibitors of monoamine oxidase A", 2008, WO / 2009/034216), the compounds being named OXO 3 (IC ₅₀ 13.65 nM) and OXO 4 (IC ₅₀ 0.84 nM), those used for the different tests as free and encapsulated compound and administered intraperitoneally.

Forced swimming test (FST)

The test was performed according to the method developed by Porsolt et al. (Porsolt, R. D .; Le Pichón, M .; Jalfre, M. Nature 1977, 266, 730-732) for mice. The test began with the acute administration of the molecule to be analyzed and after a set time (15, 30, 45 minutes), each mouse was introduced into a 3-liter plastic cup (19.5 cm high, 12 cm high). diameter), filled with water at a temperature of 25 ± 0.5 ° C, and with a depth of 14.5 cm. The depth of the water was chosen in such a way that the animals had to swim or float without their hind legs or their tail touching the bottom of the vessel and that they could not leave it. For the test, each mouse was introduced into the vessel for 6 minutes and, after intense initial activity, the mice acquired a characteristic immobility posture.

The parameter to be evaluated was the time that each mouse was floating (that is, the time during which the mice made only the movements necessary to keep their heads out of the water) since the shorter this time (immobility time), the longer Antidepressant activity of the molecule to be evaluated, since immobility is considered an index of despair and mood depression. In this way the mice were immobile for longer, without swimming and escape movements, in the presence of a vehicle than if an antidepressant was administered.

As Porsolt et al suggested, only the data measured during the last 4 minutes were analyzed and presented (Figures 5a and 5b).

The molecules were administered intraperitoneally (ip.) 15, 30 or 45 minutes (min.) Before the test at a dose of 1 mg / kg.

The free compounds (OXO 3 and OXO 4) were suspended in 1% carboxymethylcellulose (CMCNa) because of their hydrophobic nature. Said compound in addition to being used as a vehicle for the administration of the molecules was also used as a control substance. Experimental results-Forced swimming test (FST)

The results of the immobility time of the mice in the FST of the oxoisoaporphins OXO 4 and OXO 3, both free and nanoencapsulated molecules (PCL-OXO 4 and PCL-OXO 3) are presented.

OXO 4 immobility time

The results obtained in the test of Porsolt et al. Are shown in Figure 6. when the OXO 4 compound suspended in 1% CMCNa was evaluated at 15, 30 and 45 minutes. The CMCNa 1% is used as control. A statistically significant decrease in immobility time is observed at 15 min. (69.03 ± 8.99, p <0.01), 30 min. (100.5 ± 14.94, p <0.05) and 45min. (64, 10 ± 7.83, p <0.01) with respect to the group of animals treated only with the vehicle-control group (140.2 ± 9, 11). These data show a clear antidepressant activity of the OXO-4 molecule in the FST maintained over time. Figure 7 shows the data obtained with the encapsulated derivative of the OXO 4 molecule, the PCL-OXO 4. This figure shows a delay in the beginning of the action with a decrease in immobility time at 15min (88 , 97 ± 17.85) lower than that obtained at 30 min. (69.51 ± 12, 17, p <0.01) and 45 min (76.69 ± 13.68, p <0.05) with respect to the control (135.90 ± 13.11). OXO 3 immobility time

The results obtained with the OXO 3 molecule suspended in 1% CMCNa at 15, 30 and 45 minutes in the Porsolt et al test are shown in Figure 8. The CMCNa 1% is used as control. A reduction in immobility time to 15 min is observed. (86.88 ± 13.91, p <0.05) after administration of the new OXO 3 molecule that is higher than that observed at 30 min. (110.60 ± 10.62, n.s.). However, the greatest reduction was obtained at 45 min. (77.20 ± 13.91 p <0.01) with respect to the control (140.20 ± 9, 10). Figure 9 shows the data of the PCL-OXO 3 encapsulated derivative with reductions in immobility time at 15 (121.20 ± 17.73) and 30 min. (92.54 ± 16.44) not statistically significant with respect to the control (135.90 ± 13, 11). On the contrary, a statistically significant decrease is observed at 45 min. (73, 14 ± 9.60, p <0.05). The data presented indicate that there is a considerable delay in the appearance of antidepressant activity with respect to the free molecule (not encapsulated).

Claims

1. Nanocapsule system characterized by an average size of less than 1 μιη comprising a polymer, a surfactant, an oil and a compound selected from the compounds of formula I and II, their salts, hydrates, solvates and N-oxides ,

where:

1 2 3 4 5 6 7 8 9

2. System according to claim 1 wherein the compound is selected from the compounds:

7. 2,3-dihydro-7H-dibenzo [de, h] quinolin-7-one

8. 7H-dibenzo [úfe, / z] quinolin-7-one

9. 5 -methoxy-2,3-dihydro-7H-dibenzo [de, h] quinolin-7-one

10. 5 -methoxy-7H-dibenzo [de, h] quinolin-7-one

11. 5 -methoxy-6-hydroxy-2, 3-dihydro-7H-dibenzo [¿fe, h] quinolin-7-one

12. 5-methoxy-6-hydroxy-7H-dibenzo [úfe, / z] quinolin-7-one

3. System according to claim 1 and 2, wherein the polymer is selected from polystyrene, polyester, polyphosphazine, polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyacrylate, polyvinyl pyrrolidinone, polyallylamine, polyethylene, acid poloacyl, polymethacrylate, polysiloxane, polyoxyethylene, cellulose, chitosan, polyhydroxybutarate and its derivatives.

4. System according to claim 3, wherein the polymer is poly-epsilon-caprolactone.

5. System according to claim 1, wherein the oil is selected from saturated and unsaturated aliphatic hydrocarbons, aromatic hydrocarbons, cyclic and acyclic hydrocarbons, saturated or unsaturated C1-C24 carbon chain triglycerides.

6. System according to claim 1, wherein the oil is selected from sunflower, olive, soy, palm and rapeseed oil.

7. System according to claim 1, wherein the surfactant is selected from castor oil ethoxylate, Pluronic F68 (copolymer of ethylene oxide and propylene oxide), BRIJ (stearate), Tween 20 (polysorbate 20), Tween 40 ( polysorbate 40), Tween 60 (polysorbate 60), Tween 80 (polysorbate 80), sodium lauryl sulfate, Crillet 1, Crillet 4 HP, Crillet 4 NF, Cremophor RH40, Cremophor RH60, Cremophor EL, Etocas 30, Mkkol HCO-60, Labrasol, Acconon MC-8, Gelucire 50/13, Gelucire 44/14, MYRJ, polyoxameros, Epikuron 170, phospholi peptides, Span (sorbitan monostearate), glycerol monostearate, Capmul MCM (caprylic / capric glyceride), Capmul MCM 8, Capmul MCM 10, Iniwitor 988, Iniwitor 742, Imwitor 308, Labrafil M 1944 CS, Labrafil M 2125, Capryol PGMC, Capryol 90, LauroglycoL Captex 200, ethoxylated fatty acid, Oleic Plurol (polyglyceryl-6-dioleate), Crill 1 (laura sorbitan), Crill 4 (sorbitan oleate), Maisine (glycerin monolinoleate), Peceol (glyceril olea to) and Árlacel P135 (polyethylene glycol dipolihydroxystearate).

8. Method for the preparation of the systems, as defined in any of claims 1 to 7, comprising:

i) preparing an organic phase comprising an organic solvent, a polymer, an oil and a compound selected from the group consisting of the compounds of formula I and Π, as defined in claim 1, its salts, hydrates, solvates and N-oxides,

j) preparing an aqueous phase comprising at least one surfactant, k) mixing the phases prepared in steps a) and b), and

1) remove the solvent.

9. Pharmaceutical composition comprising nanocapsule systems as described in any of claims 1 to 7.

10. Use of nanocapsule systems as described in any of claims 1 to 7, for the preparation of a medicament.

11. Use according to claim 10, wherein the medicament is for the treatment of nervous disorders.