ES2402132A1 - Oxoisoaporphine nanocapsules for treating depression - Google Patents

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

Field of the art The present invention relates to nanocapsules of compounds of formula I or 11 and to pharmaceutical compositions comprising those nanocapsules.

Background A group of compounds derived from oxoisoaporphins of formula I and 11, were found to be selective and potent inhibitors of monoamine oxidase A (MAO-A) in ranges of ICso 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 ofmonoamino oxidase A", 2008, W0I2009 / 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 the size "400 Da) and by 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 nanoparticular systems

stable, with high association efficiency of the compounds of formulas I and 11, 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 11, which has the 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.

1 llIi

Thus, in one aspect the invention is directed to a nanocapsule system characterized by an average size of less than 1 J.I1ll comprising a polymer, a surfactant, an oil and a compound selected from the compounds of formula 1 and 11, its salts, hydrates, solvates and N-oxides,

where: _R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each selected in a way independent of hydrogen, alkyl, cycloalkyl, alkenyl, cycloheteroalkyl, aryl, _ORb and_NR3Rb;

10Ra and Rb are independently selected from hydrogen, alkyl or, alkenyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, or, Ra and Rb together form a heterocycle ring, 4 to 7 members containing 0-2 heteroatoms independently selected from oxygen, sulfur and N-Re, where Re is selected from hydrogen, alkyl, and -C (O) Rb •

In another aspect the invention is directed to a pharmaceutical composition comprising the nano capsule systems described above. In another aspect the invention is directed to the use of the pharmaceutical compounds 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 1 and 11, their salts, hydrates, solvates and N-oxides,

B) preparing an aqueous phase comprising at least one surfactant, c) mixing the phases prepared in steps a) and b), and d) removing 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 the OXOl compound by CLAE. Equation of the line: y = 0.64x + 0.176. Figure 38: 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 lmglkg. n = 8 for each group. The results are expressed in seconds as the mean ± standard error. Asterisks mark the statistically significant results according to an ANOV A treatment followed by a Dunnet test. * p <0.05, ** p <O, Ol. Figure 7. Immobility time of mice treated with the PCL-OXO 4 molecule (nanoencapsulated molecule) at a dose of lmglkg. n = 8 for each group. The results are expressed in seconds as the mean ± standard error. Asterisks mark the statistically significant results according to an ANOVA treatment followed by a Dunnet test. * p <0.05, ** p <O, OI. Figure 8. Immobility time of mice treated with the free molecule OXO 3 at a dose of 1 mglkg. n = 8 for each group. The results are expressed in seconds as the mean ± standard error. Asterisks mark the statistically significant results according to an ANOV A treatment followed by a Dunnet test. * p <0.05, ** p <O, OI. 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 the statistically significant results according to an ANOVA treatment followed by a Dunnet test. * p <0.05, ** p <O, OI.

Detailed description of the invention

The compounds of formulas I and 11 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 Moc1obemide (W02009034216, 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 11 may be in the form of salts, such as ammonium salt. Compounds I and 11 may also be in oxidized form, in which case they are N-oxides.

In a particular embodiment, in the compounds of formula 1, R1 and R2 are each independently selected from hydrogen, halogen, cyano, alkyl, alkenyl, _ORb and_NRaRb; and R3, R5, R6, R7, R8 and R9 are each independently selected from hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloheteroalkyl, aryl, -ORb Y-NRaRb; where Ra and Rb are as defined above.

In another particular embodiment, in the compounds of formula 1, R1 YR2 are hydrogen and R3, R4, R5, R6, R7, R8 and R9 are each independently selected from hydrogen, halogen, and _ORb1; where Rbl is selected from hydrogen and alkyl.

In another particular embodiment, in the compounds of formula 1, R1, R2, R3, R6, R7, R8 and R9 are hydrogen and R4 and R5 are independently selected from hydrogen, methyl

or hydroxyl.

In a particular embodiment, in the compounds of formula 1I, R1, R2, R3, R \ R5, R6, R7 R8 and R9

, are each independently selected from hydrogen, halogen, nitro, alkyl, cycloalkyl, aryl or _ORb; where Rb is as defined above.

In another particular embodiment, R8 is selected in compounds of formula 1I, R1 and R2

between hydrogen and halogen, and R3, R5, R6, R7, and R9 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 1 and II are preferably selected from the compounds:

one.

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

2.

7 H-dibenzo [de, h] quinolin-7 -one

3.

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

Four.

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

5.

5-methoxy-6-hydroxy-2,3-dihydro-7H-dibenzo [de, h] quinolin-7 -one

6.

5-methoxy-6-hydroxy-7H-dibenzo [de, h] quinolin-7-one

In the present invention, "alkyl" is understood to mean a linear or branched hydrocarbon chain that does not contain any setting, of 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, optionally substituted with one to three substituents selected from _ORb, -NRaS (O) rnRb where m is selected between 1 and 2, _SRb, -S (O) rnRb, -S (O) rnNRaRb where m is selected between 1 and 2, _NRaRb, -C (O) Rb, C02Rb, _C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, -NRaC (O) NRaRb, -CF3, -OCF3,

Ra Rb

cycloalkyl, cycloheteroalkyl, aryl and heteroaryl; where and 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, -ORb, -NRaS (O) rnRb where m is selected from 1 and 2, _SRb, -S (O) rnRb, -S (O) rnNRaRb where m is selected between 1 and 2, _NRaRb, -C (O) Rb, -C02Rb, _C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, NRaC (O) NRaRb, -CF3, -OCF3, alkyl, aryl and heteroaryl; where Ra and Rb 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 selected substituents between halogen, cyano, _ORb, -NRaS (O) rnRb where m is selected between 1 and 2, _SRb, -SeO) rnRb, -S (O) rnNRaRb where m is selected between 1 and 2, _NRaRb, -C (O ) Rb, -C02Rb, _C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb,

NRaC (O) NRaRb, -CF3, -OCF3, alkyl, cycloalkyl, cycloheteroalkyl, aryl and

heteroaryl; where Ra and Rb 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 may be substituted with one to three substituents selected from _ORb, -NRaS (O) rnRb where m is selected from 1 to 2, _SRb, -SeO) rnRb, -S (O) rnNRaRb where m is selected from 1 to 2, _NRaRb, -C (O) Rb, -C02Rb, -C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, NRaC (O) NRaRb, -CF3, -OCF3, alkyl, aryl and heteroaryl; where Ra and Rb are as previously defined. "Aryl" refers to an aromatic hydrocarbon of 6 to 1O carbon atoms, for example: phenyl or naphthyl; optionally the aryl may be substituted with one to three substituents selected from _ORb, -NRaS (O) rnRb where m is selected from 1 to 2, -SRb, -SeO) rnRb, -S (O) rnNRaRb where m is selected from 1 and 2, _NRaRb, -C (O) Rb, C02Rb, -C (O) NRaRb, _NRaC (O) Rb, -NRaC (O) ORb, -NRaC (O) NRaRb, -CF3, -OCF3,

Ra

alkyl, alkenyl, aryl and heteroaryl; where 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 _ORb, -NRaS (O) rnRb where m is selected from 1 to 2, _SRb, -SeO) rnRb, -S (O) rnNRaRb where m is selected from 1 to 2, _NRaRb, C (O) Rb, -C02Rb, -C (O) NRaRb, _NRaC (O) Rb, -NRaC (O) ORb, _NRaC (O) NRaRb, -CF3,

OCF3, alkyl, alkenyl, aryl and heteroaryl; where Ra and Rb are as previously defined.

The compounds of formula 1 and 11 are prepared according to the references published previously (Eduardo Sobarzo-Sánchez, Bruce K. Cassels, Carolina Jullian and Luis Castedo Magn. Resan. Chem. (2003) 41, 296-300; Eduardo Sobarzo-Sánchez , Julio De la Fuente and Luis Castedo Magn. Resan. 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 I 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 I micrometer, that is, they have an average size between I and 999 nm, preferably between 100 and 500 nm, even more preferably between 150 and 300 nm. The average size of the particles is mainly influenced by the composition and the conditions of their formation.

On the other hand, nanocapsules have a negative electrical charge (measured by the Z potential). 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 CI-C24 carbon chain triglycerides. In a more particular embodiment, C 1 -C 6 carbon chain triglycerides. In another particular embodiment, C8-C20 carbon chain triglycerides. In a particular embodiment, the oil is selected from sunflower, olive, soy, 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 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, polyoxamers, Epikuron 170, phospholipids, Span (sorbitan monostearate), glycerol monostearate, Capmul MCM (caprylic / capric glyceride), Capmul MCM 8, Capmul MCM 10, Imwitor 988, Imwitor 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 (sorbitan laurate), CriIl 4 (sorbitan oleate), Maisine (glycerin monolinoleate), Peceol (glyceryl oleate) and Arlacel P l35 (polyethylene glycol dipolihydroxystearate). 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-epsiloncaprolactone and a compound selected from the group consisting of the compounds of formula 1 and 11, their salts, hydrates, solvates and N-oxides,

f) preparing an aqueous phase comprising at least one surfactant, g) mix 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 a surfactant. oil soluble Oil-soluble surfactants are known by the skilled in the art, and are for example, lecithin, polysorbate 80, etc.

EXPERIMENTAL PROCEDURE

1. SYNTHESIS AND CHARACTERIZATION OF NANOPARTICLES 1.1 Material

Poly-s-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®) (Hüls, 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 11m nylon membranes (Belford, USA).

1.2 Methods

1.2.1 Preparation of polymeric capsules containing oxoisoapoñmas derivatives The nano-capsules of Poly-E-caprolactone (PCL-NC) empty and loaded with oxoisoaporphins (OXO) (Figure 1) were prepared by interfacial deposition (Fessi, H .; Puiseiux, F .; Devissaguet, JP; 1988, Procédé de preparation de colloidaux dispersible systems 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.

1.2.2 Chromatographic conditions for the quantification of oxoisoapoñmas High-performance liquid chromatography (HPLC) analyzes were developed using a Varian® 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 Millipore® nylon membranes of

0.22

11 m.

1.2.3

Measures of association efficiency The total amounts (100%) of the compound OXO present in the suspensions

Table 1. Chromatographic conditions of compound OXO.

OXO 1,2,3,4

OX05 OX06

Mobile phase

Acetonitrile / water (50:50, v / v) Acetonitrile / water (50:50, v / v) Methanol / water (60:40, v / v)

Injection volume

20 / lL 20 / lL 20 / lL

Flow rate Mobile phase

2mLlmin 2 mLlmin 1 mLlmin

Wavelength (A.)

370 nm, 410 nm, 370 nm 385nm 241 nm

Chromatographic column

Phenomenex Gemini 5 / l Cl8 110A, 150 mm x 4.60 mm Phenomenex Gemini 5 / l Cl8 110A, 250 mm x 4.60 mm Phenomenex Gemini 5 / l Cl8 110A, 250 mm x 4.60 mm

Column temperature

Ambient Ambient Ambient

5 nanocapsule (NC) were determined by HPLC, after dilution in acetonitrile and filtration through a Millipore membrane of 0.22 / lm. 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 30 kDa regenerated cellulose

10 (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 .; Espina, M .; Garcia, ML ; Egea, MA Int. J. Pharm. 1999, 179, 37-48 .; Schaffazick, SR; Guterres, SS; Freitas, LL; Pohlmann, AR

15 Chem. Nova, 2003, 26, 726-737 .; Kilic, A. C .; Capan, Y .; Vural, l .; Gursoy, R. N .; Dalkara, T .; Cuine, A .; Hincal, A. A. J. Microencapsulation 2005, 22, 633-641).

1.2.4 Characterization of the nanocapsules of Poly-E-caprolactone (PCL)

1.2.4.1 Size and polydispersion measures

The light scattering technique was used to determine the average size and distribution of the nanoparticles. This analysis was carried out after the dilution (11100 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, MC; Illum, L .; Davis, SSJ Control. Release 1999, 57, 171-185.; Venkatraman, SS; Jie ,

10 P .; Min, F .; Freddy, B. Y. 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 the zoning potential, 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 20 general formulas, corresponding to the general formula (1):

A. 2,3-DIHIDRO-7H-DIBENZO [de, h] QUINOLIN-7-0NA (2,3-DIHIDRO

OXOISOAPORFINA), General formula (1): R3 R2

(one)

In that: a) in which if: -R1, R2, R3, R4, R5, R6, R7, R8 and R9 is hydrogen, it is 2,3-dihydro-7H-dibenzo [de, h] quinolin- 7-one, hereinafter referred to as OXO 1;

a methoxy, it is 5-methoxy-2,3-dihydro-7 H-dibenzo [de, h] quinolin-7 -one, called

5 hereafter OXO 3; and e) in which if: -R1, R2, R3, R6, R7, R8 and R9 is hydrogen, R4 represents a methoxy and R5 represents a hydroxyl; it is 5-methoxy-6-hydroxy-2,3-dihydro-7 Hdibenzo [de, h] quinolin-7-one, hereinafter referred to as OXO 5.

10 B. 7H-DIBENZO [de, h] QUINOLIN-7-0NA (OXOISOAPORFINE), General Formula (11):

In which:

15 7H-dibenzo [de, h] quinolin-7-one, hereinafter referred to as OXO 2;

a methoxy, it is 5-methoxy-7H-dibenzo [de, h] quinolin-7-one, hereinafter referred to as OXO 4; Y

e) in which if: -R1, R2, R3, R6, R7, R8 and R9 is hydrogen, R4 represents an R5

20 methoxyl and represents a hydroxyl; it is 5-methoxy-6-hydroxy-7Hdibenzo [de, h] quinolin-7 -one, hereinafter referred to as OXO 6;

and and

2. DISCUSSION RESULTS OF THE SYNTHESIS

CHARACTERIZATION OF THE NANOPARTICLES

25 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

5 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 Efficiency of association of OXO compounds in nanoparticles The determination of the amounts of compounds associated with nanoparticles is

10 especially complex due to the small size of the latter, 10 which complicates the separation of free and associated compound fractions (Magenheim, B .; Benita, S.

T. P. Pharma Sci. 1991,1,221-241 .; Soppimath, K. S .; Kulkami, A. R .; Aminabhavi,

T. M. J Control Release 2001, 75, 331-345). In addition, other factors influence the association, such as the physicochemical characteristics of the compound (Guterres, S. 15 S .; Fessi, H .; Barratt, G .; Devissaguet, J. -Ph .; Puisieux, F. Int J Pharm. 1995, 113, 57-63; Calvo, P .; Vil a-Jato, JL; Alonso, MJ 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 .; Gamett, MC; Illum, L :; Davis, SS J Control. Release 1999,57, 171-185), the characteristics of the surface of the particle or the nature of the

20 polymer (Vil a, A .; Sánchez, A .; Tobío, M .; Calvo, P .; Alonso, MJ J Control Release 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 method centrifugation using HPLC. 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.

Table 2. Association efficiency values of compound OXOs with PCL-NC.

Samples

Association Efficiency (%)

NC + OXO 1

99.60 ± 0.07

NC + OX02

95.10 ± 0.39

NC + OX03

99.51 ± 0.22

NC + OX04

96.26 ± 0.11

NC + OX05

99.59 ± 0.09

NC + OX06

99.50 ± 0.12

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, TO.

5 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 values

10 zeta potential (mV) were determined using a Zetasizer ZS90 (Malvem®) analyzer. The values of size, polydispersion and zeta potential are shown in Table 3.

Table 3. NC + OXO size, polydispersion and zeta potential values

Samples

Particle Size (nm) Polydispersion Zeta potential (mV)

NC

267.8 ± 0.40 0.126 ± 0.026 -37.4 ± 0.55

NC + OXOl

244.2 ± 4.31 0.106 ± 0.008 -36.6 ± 0.51

NC + OX02

271.9 ± 4.79 0.130 ± 0.017 -39.2 ± 0.40

NC + OX03

247.2 ± 1.63 0.132 ± 0.019 -39.9 ± 0.25

NC + OX04

245.5 ± 1.30 0.104 ± 0.014 -39.3 ± 1.16

NC + OX05

273.9 ± 1.76 0.140 ± 0.015 -37.4 ± 1.05

NC + OX06

249.8 ± 6.89 0.160 ± 0.043 -35.0 ± 0.80

15 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. The factors that influence polydispersity include properties of the solution, the system dynamics 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, since 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, V. J .; Chen, Y. Tropical J. Pharm. Res. 2006, 5, 561-573). In all prepared fonnulations the potential zeta value was negative (Table 3). For prepared fonnulations, 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

THE FREE AND ENCAPSULATED OXOISOAPORFINS

General conditions

To perform the tests in vivo, an experimental procedure used in the preclinical investigation of depression was used: 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.

one.

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

2.

Swiss male albino mice of Charles River CD 1 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 &S6hne; Rosenberg, Germany) that were 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.

Four.

The animals were kept for 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 housing and 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 Spanish legislation (RD 1201/2005 of October 10, on the protection of animals used for experimentation and other scientific purposes (BOE No. 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 (Dazzle) digitizer card, (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 Etho Vision 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 immobility, mobility and strong mobility time (these last two not represented) The compounds evaluated in the in vivo tests of the present invention are the most active in the in vitro MAO inhibition tests -A human 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 ofmonoamine oxidase A", 2008, WO / 2009/034216), the compounds called OXO 3 (lCso 13.65 nM) and OXO 4 (lCso 0.84 nM), those used for the various 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 Pichon, 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 <O, Ol), 30 min (100.5 ± 14.94, p <0.05) and 45min. (64.10 ± 7.83, p <O, Ol) 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, 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 <O, Ol) and 45 min (76.69 ± 13.68, p <0.05) with respect to of 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 CMCN at 1% is used as control. A reduction in immobility time is observed at 15 min (86.88 ±

13.91, p <0.05) after administration of the new OXO 3 molecule that is greater 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 <O, Ol) 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 to 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 (7)

1. System of nanocapsules characterized by an average size of less than 1 Jlm that it comprises a polymer, a surfactant, an oil and a compound selected from the compounds of formula 1 and n, their salts, hydrates, solvates and N-oxides, R3 R2 R3 R2 R.t R1 R4 R1 R5 R5 Rg Rg Rs Rs R7 R7 (1) (11) 5 where: _R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each selected in a way independent of hydrogen, alkyl, cycloalkyl, alkenyl, cycloheteroalkyl, aryl, _ORb and_NRaRb; 10 _Ra and Rb are independently selected from hydrogen, alkyl or, alkenyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, or, Ra and R b together form a heterocycle ring, 4 to 7 members containing 0-2 heteroatoms independently selected from oxygen , sulfur and N_Rc, where RC is selected from hydrogen, alkyl, and -C (O) Rb. 2. System according to claim 1 wherein the compound is selected from among compounds: 2,3-dihydro-7 H-dibenzo [de, h] quinolin-7 -one 7H-dibenzo [de, h] quinolin-7-one 5-methoxy-2,3-dihydro-7H-dibenzo [de, h] quinolin-7-one 5-Methoxy-7 H-dibenzo [de, h] quinolin-7-one 5-methoxy-6-hydroxy-2,3-dihydro-7 H-dibenzo [de, h] quinolin-7-one 5-methoxy -6-hydroxy-7 H-dibenzo [de, h] quinolin-7 -one 3. System according to claim 1 and 2, wherein the polymer is selected from among 25 between polystyrene, polyester, polyphosphazine, polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyacrylate, polyvinyl pyrrolidinone, polyallylamine, polyethylene, polyacrylic acid, polymethacrylate, polysiloxane, polyoxyethylene, cellulose, chitosan, and polyhydroxyarate derivatives.

Four.

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 C 1-C24 carbon chain triglycerides

or unsaturated

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 ethoxyate, 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, polyoxameres, Epikuron 170, phospholipids, Span (sorbitan monostearate), glycerol monostearate, Capmul MCM (caprylic / capric glyceride), Capmul MCM 8, Capmul MCM 10 , Imwitor 988, Imwitor 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 (sorbitan laurate ), Crill 4 (sorbitan oleate), Maisine (glycerin monolinoleate), Peceol (glyceryl oleate) and Arlacel P135 (polyethylene glycol dipolihydroxystearate).

8.

Process 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 1 and n, as defined in claim 1, their salts, hydrates, solvates and N -oxides, j) preparing an aqueous phase comprising at least one surfactant, k) mix the phases prepared in steps a) and b), and 1) remove the solvent. nanocapsules nanocapsules

TO \ ~ .B and • D o 0.5 1.5 2 25 3 ~ 5 4 4.5 5 5.5 6 6.5 7 Time (min) FIGURE 2

«

20 15 10

OR

10 twenty 30 40 50 60 Concentration (~ g / mL) 70 80 90

FIGURE 3A

45 40 35

• •

-; - 30 :: :) ~ 25 ro ~ «20

fifteen

10

2

3 4 5 6 7 8 Concentration (llg / mL) 9 10 eleven

FIGURE 3B

2 3 4 5 6 7 8 9 10 11 Concentration (llg / mL) FIGURE 3C 45 40 35

-

30

=> oc :( - ro ~ and:( 20 15 10 • I 2 3 4 5 6 7 8 9 10 11 Concentration (llg / mL) 3D FIGURE

Concentration (¡.tg / m L) Concenlration (llg / mL) FIGURE 3F .... ~ -. "..".

-

FIGURE 4 FIGURE 5a FIGURE Sb ... ~ 240 or 220 «200 ~ 180> 160 O 140 ~ 120 z 100 w 80 O 60 ~ 40 ~ 20 ~ O - ~ 240 or 220 «200 OR :: J > Or 140 ~ 120 z 100w 80 OR OR or... ~ 20 w O .... CMCNa + OX04 (1 mg / kg) * ** • ** FIGURE 6 PCL-OX04 (1 mg / kg) * ** ........... ........... ........... ........... ........... ........... ........... •··········one ........... ........... ........... ........... FIGURE 7 CMCNa + OX03 (1mg / kg) ro - 240 O 220 ~ 200 ~ 180> 160 O 140 :: iE 120 z 100 w 80 O 60 ~ 40 :: iE 20 ~ O FIGURES PCL-OX03 (1 mg / kg)

-

! e 240 Or 220 ~ 200

-

l 180

> Or 140 ~ 120 100 w 80 OR • * OR or .. 40 z w O T [1 ~ 20 ~ ú '"~~ ~~ ~~ that ~ ";) CS ~ FIGURE 9