ES2391187B1 - NANOESTRUCTURED BIOACTIVE POLYMERIC COMPOUNDS DERIVED FROM NON-STEROID ANTI-INFLAMMATORY DRUGS (NSAIDs) and IMIDAZOL. - Google Patents

Abstract

 Polymeric compound of general Formula I # **** IMAGE-01 **** # characterized in that: A represents a residue of one or more hydrophobic acrylic or vinyl monomers carrying an NSAID derivative, where said NSAID derivative is bound to the rest of the molecule through a covalent hydrolysable bond in vivo; B represents a residue of one or more hydrophilic monomers capable of complexing with mono- or divalent cations; m and n indicate the molar fractions of monomers A and B in the polymer so that m + n is always 1, and m and n is always different from zero. # Method of obtaining the compounds of Formula I, micelles comprising said polymeric compounds of Formula I and the use of these polymeric compounds and their micelles as anti-inflammatory systems for vectorized, localized and / or controlled release of an NSAID, as directed and / or vectorized injectable systems for the release of an NSAID, to form complexes with mono- cations or divalents, such as MMP inhibitors, in bioactive coatings of medical devices, in implant coatings and fracture repair structures, as reabsorbable bioactive polymers with controlled therapeutic action, as a component in antiseptic solutions or dispersions, in dental applications, contact lenses and regeneration of vascular organs and grafts, as a non-viral vector of in teres in gene therapy.

Description

Nanostructured bioactive polymeric compounds derived from nonsteroidal anti-inflammatory drugs (NSAIDs) and imidazole.

SECTOR OF THE TECHNIQUE

The present invention is framed in the field of biomaterials of polymeric origin for the design and preparation of biocompatible formulations based on self-assembled amphiphilic macromolecular systems derived from vinylimidazole (VI) and carriers of non-steroidal anti-inflammatory drugs, hereinafter NSAIDs (hydrophobic drugs ), with release kinetics and modulated therapeutic action.

STATE OF THE TECHNIQUE

The treatment of pain associated with inflammatory processes can be approached from different perspectives. The usual treatment consists in the administration (usually orally) of NSAID drugs whose action is based on the absorption of the biologically active compound and its subsequent systemic distribution through the bloodstream. A more advanced strategy is the administration of matrix metalloproteinase inhibitor substances (MMPs) associated with inflammatory processes. In literature there are multiple references that relate MMP and inflammatory processes (Murphy, G. et al. Molecular Aspects of Medicine 2008, 29, 290–308; Parks, W. et al. Nature Reviews Immunology 2004, 4, 617-629; Matrisian , LM et al. Science 2002, 295, 2387-2392). However, to date, no treatments have been described that combine in a single formulation the two strategies just mentioned to treat inflammatory processes.

The MMPs are a family of proteases that degrade practically all the protein components of the extracellular matrix and whose basic structure consists of a series of characteristic regions among which is a carboxy-terminal catalytic domain that joins Zn and Ca so the catalytic action of MMP is inhibited by chelating agents of said metals. These MMP inhibitor chelating agents generally consist of organic molecules that have heteroatoms in their structure with free electron pairs, generally nitrogen, capable of forming metal complexes with Zn and Ca among others, necessary for the activation of said metalloproteinases. Thus, in Spanish patents No. ES2283540 and ES2172690 MMP inhibitors derived from pyridine and imidazole are described respectively, for application in the treatment of heart disease, multiple sclerosis or rheumatoid arthritis among others.

Imidazole is a compound of hydrophilic nature and ionizable capacity, whose structure is part of compounds of biological interest such as histidine or biotin. Among its chemical properties, it is worth highlighting its pH buffering capacity and complexing of metals. Thus, in the bibliography (Niu, Y. Acta Cryst. 2010, E66, m1518; Wang, ZL. Et al. Acta Cryst. 2010, C66, m311-m313; Shi, H. et al. Acta Cryst. 2010, E66 , m943-m944; Xiao,

Z. et al. Cryst Act. 2010, E66, m1040-m1041; Wang, Z.L. et al. Cryst Act. 2010, C66, m384-m386; Zheng, J.M. et al. Eur. J. Inorg. Chem. 2010, 5478-5483) Zn, Co, Cd and In metal complexes with different imidazole species are described.

The use of macromolecular systems with complexing capacity of metals involved in the activity of MMP has been described in various works. For example, in the reference (Santos, M.A. et al. Journal of Inorganic Biochemistry 2004, 98, 209-218) polymeric systems based on derivatives of amino acids capable of reducing the concentration of Cu, Ni and Zn ions and therefore susceptible to interfering with the activity of said MMPs are described.

In parallel, the use of polymeric biomaterials for the vectorization of NSAID drugs has been extensively studied. These systems are generally based on the physical encapsulation of said compounds for their release through diffusion processes. A group of NSAIDs particularly desirable for vectorization are those called Class 2, according to the Biopharmaceutical Agents Classification System proposed by Amidon et al. (Pharm. Res. 1995, 12, 413-420), including those preferably included in this invention, Ibuprofen, Ketoprofen, Naproxen and Flurbiprofen, characterized by having a reasonably good gastrointestinal permeability but low or no aqueous solubility so that its absorption is limited by its dissolution rate.

There are currently different pharmaceutical formulations intended to increase the bioavailability of these NSAIDs by oral administration such as the Self-Emulsifying Drug Administration Systems (SEDDS) (Spanish Patent No. ES2253354) based on an emulsion pre-concentrate comprising an NSAID derivative (Naproxen) ) and one or more surfactants capable of forming an emulsion in situ in contact with the gastrointestinal medium. Another strategy consists of the physical encapsulation of NSAID drugs in matrices and / or microstructured vehicles of polymeric origin for use as controlled release systems. Thus, for example, patents ES2210286 and US20100015237 respectively describe systems for the controlled release of different drugs, including NSAIDs such as Ketoprofen, Ibuprofen, Aspirin, Naproxen or Flurbiprofen.

More advanced methods are based on the incorporation of drugs and / or molecules of biological interest by chemical anchoring to polymeric complexes capable of promoting the vectorization and / or controlled release of the biologically active compound through hydrolysis processes (Duncan, R. et al. Adv Polym Sci 2006, 192, 18) for the preparation of advanced biomedical devices. An example of this strategy is the polymer derivatives described in Spanish patent # 2; ES2154242, with applicability in the field of vascular surgery and with controlled release kinetics of drugs derived from salicylic acid.

Finally, in recent years and with the aim of improving controlled drug delivery systems based on vehicles and matrices of polymeric origin, the use of amphiphilic block copolymers with the ability to self-assemble forming micellar nanostructures in various media has gained special interest. solvents or dispersants, such as the different physiological means. An example is that described in the literature by Qiao et al. (Qiao, R. et al. Colloids and Surfaces B: Biointerfaces 2009, 74, 284-292), where the synthesis of poly (-benzyl-L-aspartate) -blockpoli (vinylpyrrolidone) block copolymers capable of forming micelles in aqueous medium carrying Prednisone is detailed. The amphiphilic copolymer micelles are composed of a hydrophobic core that allows to house drugs with low solubility inside, and a hydrophilic cover responsible for its stability in physiological medium that allows its circulation and transport, favoring its dispersion and inhibiting interaction and aggregation with other compounds of a hydrophobic nature, thus considering excellent systems for the transport of this type of drugs.

However, as mentioned, to date, no invention or development is found capable of combining both strategies for the treatment of pain associated with inflammatory processes, MMP inhibition and controlled and vectorized administration of NSAIDs into a single material or formulation. An approach to this route is to use biocompatible macromolecular systems capable of presenting both functions. Macromolecular chemistry offers enormous possibilities when designing such systems, allowing the development of advanced materials with specific microstructural characteristics that allow the physical-chemical, biochemical or pharmacological properties of the prepared material to be controlled.

An unprecedented approach in the literature is that described in this invention and preferably consists of the preparation of biocompatible polymer systems derived from imidazole and NSAID carriers, such as Ibuprofen, Ketoprofen, Naproxen and Flurbiprofen among others, in which the drug It is attached to the polymer chain through covalent bonds. The different reactivity of the NSAID carrier monomer described in this invention, with respect to the imidazole derivative, in particular with respect to 1-vinylimidazole, results in the formation of polymer chains with sequences rich in the most reactive monomer followed by sequences rich in the Another monomer As a consequence of this distribution and of the hydrophobic and hydrophilic characters of the NSAID and imidazole respectively, the polymeric systems of the present invention have an amphiphilic character, which is responsible for the formation in aqueous medium of micelles by self-assembly processes. These polymeric compounds can act as vehicles for drug release in a vectorized, modulated and / or localized manner by hydrolysis of the polymer chain, while possessing the ability to reduce or even inhibit the activity of MMPs through complexation of metals such as Zn and Ca through the imidazole groups present in the hydrophilic shell of the micelles formed. Therefore, the polymer systems described in the present invention have both functionalities for the treatment of inflammatory processes; On the one hand, the transport and release of NSAIDs is achieved in a vectorized and / or controlled manner, and on the other, the inhibition of MMP by the formation of metal complexes.

DESCRIPTION OF THE INVENTION

Brief Description of the Invention

The present invention relates to a polymeric compound of general Formula I

(I)

characterized in that:

-

 A represents a residue of one or more hydrophobic acrylic or vinyl monomers bearing an NSAID derivative, wherein said NSAID derivative is linked to the rest of the molecule through a covalent hydrolysable bond in vivo;

-

B represents a residue of one or more hydrophilic monomers capable of complexing with mono or divalent cations;

-

 m and n indicate the molar fractions of monomers A and B in the polymer so that m + n is always 1, and m and n are always non-zero.

Preferably the present invention relates to a polymeric compound of Formula I wherein monomer B is an imidazole derivative, it being even more preferred that monomer B is 1-vinylimidazole.

According to a preferred embodiment, the polymeric compound of the present invention is defined by Formula Ia:

(Ia)

where: 10-R1 represents hydrogen or C1-4 alkyl;

-

R2 represents the NSAID derivative;

-

 X represents a heteroatom, preferably N and / or O;

-

 f represents an integer between 1 and 10;

-

 p represents an integer between 1 and 100;

15-m and n indicate the molar fractions of monomers A and B in the polymer so that m + n is always 1, and m and n are always non-zero.

The present invention also relates to new biocompatible and self-assembling macromolecular systems of imidazole and acrylic or vinyl monomers bearing derivatives of non-steroidal anti-inflammatory drugs (NSAIDs).

Both the compounds of Formula I described and their micelles object of the present invention can be used as anti-inflammatory systems for vectorized, localized and / or controlled drug release, as injectable systems targeted and vectorized for the release of an NSAID, to form complexes with cations

25 mono-or divalent, as MMP inhibitors, in bioactive coatings of medical devices, in implant coatings and fracture repair structures, such as reabsorbable bioactive polymers with controlled therapeutic action, as a component in antiseptic solutions or dispersions, in dental applications, contact lenses and regeneration of vascular organs and grafts, as non-viral vectors of interest in gene therapy.

Detailed description of the invention

The present invention describes a new family of polymeric compounds resulting from the radical polymerization of one or more acrylic or vinyl monomers carrying NSAID derivatives, hereinafter monomer A, and one or more monomers of hydrophilic nature, hereinafter monomer B , (Formula I) as well

as the procedure for the synthesis of said compounds, where:

(I)

-

 A represents a residue of one or more hydrophobic acrylic or vinyl monomers bearing a derivative

40 of NSAID, wherein said NSAID derivative is linked to the rest of the molecule through a covalent bond hydrolysable in vivo;

-

B represents a residue of one or more hydrophilic monomers capable of complexing with mono or divalent cations;

-

 m and n indicate the molar fractions of monomers A and B in the polymer so that m + n is always 1, and m and n are always non-zero.

As monomer B of hydrophilic nature, imidazole derivatives can be used, the use of 1-vinylimidazole (VI) being preferable.

Throughout the present invention, a residue of a polymerizable monomer, either acrylic or vinyl, is understood as the residue resulting from the polymerization of the corresponding monomer. For example, if monomer B is VI, B represents in Formula I the residue of the monomer once polymerized, as shown below:

Unless otherwise specified, the nomenclature A and B will be used throughout this description to refer interchangeably to the monomer or to the already polymerized residue. 5

Acrylic or vinyl monomers bearing NSAIDs can be prepared by forming the covalent bond between a reactive NSAID derivative and the reactive group of the chosen spacer compound. The spacer group that binds the drug to the rest of the polymer chain must contain hydrolysable groups that allow the subsequent release of the drug in the physiological medium. The most used functional groups for their activity

Hydrolytics are anhydrides, carbonates, esters, urethanes and amides. A preferred group of compounds of the present invention are those polymeric compounds of Formula I wherein the hydrolysable covalent bond is of the carboxylic ester and / or amide type.

A particular object of the present invention is a polymer compound derived from imidazole belonging to the general Formula Ia

(Ia) 20 where:

-

R1 represents hydrogen or C1-4 alkyl;

-

R2 represents an NSAID derivative;

-

 X represents a heteroatom, preferably N and / or O;

-

 f represents an integer between 1 and 10; 25-p represents an integer between 0 and 100;

-

 m and n indicate the molar fractions of monomers A and B in the polymer so that m + n is always 1 and m and n are always non-zero. .

Thus, an object of this invention is the process of preparing a polymeric compound of

Formula Ia which is obtained by radical polymerization of monomer A (acrylic derivative of NSAID) with monomer VI, in the molar proportions m and n respectively (m + n always being equal to 1, and m and always greater than 0), in the presence of a thermal radical initiator in a suitable solvent.

Although the present invention covers all NSAIDs, a preferred group of compounds of Formula Ia are those where R2 represents Ibuprofen, Ketoprofen, Naproxen or Flurbiprofen.

Another preferred group of compounds are those of Formula Ia where p represents an integer between 0 and 50, preferably 1, and f represents an integer between 1 and 10, preferably 2; especially preferred are those compounds where R2 represents Ibuprofen, Ketoprofen, Naproxen or

40 Flurbiprofen.

A preferred group of compounds are those in which R 1 represents a (C 1-4) alkyl, preferably methyl.

A preferred group of compounds of Formula Ia are those in which m is between 0.01 and 0.35 and n between 0.65 and 0.99. Another preferred group of compounds are those in which m is between 0.35 and 0.65 and n between 0.35 and 0.65. Another group of compounds are those in which m is between 0.65 and 0.99 and n between 0.01 and 0.35.

Preferably, the polymeric compounds of Formula Ia according to the present invention are characterized by having an average molecular weight between 3,000 and 100,000 Daltons. Preferably, the polymeric compounds of Formula Ia of the present invention have an average molecular weight between 10,000 and 60,000 Daltons.

The polymeric compounds of Formula I can be prepared through known methods of radical polymerization. For example, they can be prepared by dissolving polymerization of the desired monomers within a suitable solvent in the presence of a radical polymerization initiator, at a temperature and for a certain time.

As suitable solvents, dioxane, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), isopropanol or chloroform may be used, the use of polar solvents such as DMF or DMSO being preferred.

As initiator any of those described in the literature can be used to carry out radical polymerizations, for example, benzoyl peroxide or 2,2'-azobisisobutyronitrile (AIBN), the use of the latter being more preferable. Preferred initiator amounts are 0.001 to 0.1 M.

The reaction temperature depends on the initiator used. In general, a temperature range between 45 and 75 ° C will be preferred.

As for the reaction time, it will preferably be from 6 to 48 h, with a reaction time between 18 and 30 h being especially preferred.

Conventional methods are used for the isolation of the polymeric compounds of Formula I, for example, by precipitation in a suitable solvent such as water. Another method of isolation and preferred for the case of these systems, is the dialysis of the polymeric compound in a suitable solvent, preferably water, using dialysis membranes with a pore size of 3,500 Dalton or greater.

The different reactivity of the NSAID-bearing monomer A described in this invention with respect to the VI, results in the formation of polymer chains with sequences rich in the most reactive monomer followed by sequences rich in the other monomer. As a consequence of this distribution and of the hydrophobic and hydrophilic characters of monomers A and VI respectively, the polymeric systems of the present invention have an amphiphilic character, which is responsible for the formation in aqueous medium of micelles by self-assembly processes. Therefore, a preferred object of this invention is the formation of polymers carrying hydrophobic drugs (according to Formula Ia) with compositional gradient, and therefore with amphiphilic character, obtained by conventional radical polymerization techniques.

The polymeric compounds of Formula Ia of this invention in aqueous medium tend to reorganize and self assemble forming micellar structures, in which the hydrophilic part (chains rich in 1-vinylimidazole) is oriented outwardly forming a cover that protects the hydrophobic interior (chains rich in NSAIDs). These particles have spherical morphology and an average particle size between 5 and 500 nm (depending on the composition of the polymeric compound). Therefore, another object of this invention is the preparation of nanoparticles formed from polymers of Formula Ia by self-assembly processes, as well as their use in injectable systems for the release of the drug in situ.

The polymers of Formula I of the present invention are biocompatible so they can be used in the coating of biomedical devices since they do not exhibit cytotoxicity.

As mentioned above, the mechanism of controlled release of the drug is through hydrolysis of the covalent bond that binds it to the polymer chain. Therefore, it is the object of this invention to use polymeric compounds of Formula I as controlled NSAID release systems, which show great potential for the preparation of therapeutic formulations with modulated and / or vectorized anti-inflammatory activity depending on physical conditions. -chemistry of the environment.

Since monomer B, preferably an imidazole derivative, and more preferably 1-vinylimidazole, is capable of forming complexes with different metals, another object of the present invention is the use of the polymeric compounds of Formula I for the formation of metal complexes. , with special interest in those metals relevant for the reduction or even inhibition of MMPs such as Zn and Ca. Additionally, since MMPs require metals to carry out their function, and that monomer B has the ability to complex metals, The object of the present invention is the application of the polymeric compounds of Formula I for use in biomedical applications as MMP inhibitors.

Another object of the invention is the use of polymeric compounds of Formula I as non-viral vectors of interest in gene therapy.

Another object of the invention is the use of polymeric compounds of Formula I as components of antiseptic solutions or dispersions.

Another object of the present invention is the use of polymeric compounds of Formula I as components in the implant coating.

Another object of the present invention is the use of polymeric compounds of Formula I as reabsorbable bioactive polymers with controlled therapeutic action.

A further object of the present invention is the use of micelles comprising a polymeric compound of Formula Ia as anti-inflammatory systems for vectorized, localized and / or controlled release of an NSAID, as injectable systems directed and / or vectorized for the release of an NSAID. , to form complexes with mono- or divalent cations, such as MMP inhibitors, in bioactive coatings of medical devices, in implant coatings and fracture repair structures, as reabsorbable bioactive polymers with controlled therapeutic action, as a component in antiseptic solutions or dispersions , in dental applications, contact lenses and regeneration of organs and vascular grafts, as a non-viral vector of interest in gene therapy.

Brief Description of the Figures

-

 Figure 1: Scheme of the synthesis of methacrylic monomers carrying Ibuprofen (Example 1) and Ketoprofen (Example 2).

-

 Figure 2: Structure of the copolymers obtained from the monomers derived from NSAIDs and VI described in Examples 4 and 5.

-

 Figure 3: 1 H-NMR spectrum (300 MHz, DMSO-d6) of copolymer p (HEI-co-VI) 20:80 of Example 4.

-

 Figure 4: 1H-NMR spectrum (300 MHz, DMSO-d6) of the 50:50 p (HEI-co-VI) copolymer of Example 4.

-

 Figure 5: 1 H-NMR spectrum (300 MHz, DMSO-d6) of copolymer p (HEI-co-VI) 80:20 of Example 4.

-

 Figure 6: 1H-NMR spectrum (300 MHz, DMSO-d6) of copolymer p (HKT-co-VI) 20:80 of Example 5.

-

 Figure 7: 1 H-NMR spectrum (300 MHz, DMSO-d6) of copolymer p (HKT-co-VI) 50:50 of Example 5.

-

 Figure 8: 1H-NMR spectrum (300 MHz, DMSO-d6) of copolymer p (HKT-co-VI) 80:20 of Example 5.

-

 Figure 9: Image obtained by scanning electron microscopy of the nanoparticles of copolymer p (HKT-co-VI) 20:80 obtained according to Example 6.

-

 Figure 10a: Ibuprofen release profile of the copolymers of Example 4 (pH 7) following the method described in Example 7.

-

 Figure 10b: Ibuprofen release profile of the copolymers of Example 4 (pH 10) following the method described in Example 7

-

 Figure 11a: Ketoprofen release profile of the copolymers of Example 5 (pH 7) following the method described in Example 8.

-

 Figure 11b: Ketoprofen release profile of the copolymers of Example 5 (pH 10) following the method described in Example 8.

-

 Figure 12: Results of the in vitro biocompatibility MTT test of the p (HEI-co-VI) 20:80, 50:50 and 80:20 systems, according to the method described in Example 9.

EXAMPLES

The following Examples are included by way of illustration of the preparation and applications of the compounds of the present invention and in no way be construed as limiting the scope of application.

Example 1.

Synthesis of Ibuprofen-derived methacrylic monomer (HEI).

The synthesis scheme is represented in Figure 1 where R is Ibuprofen. A solution of Ibuprofen (100 mmol) in thionyl chloride (70 ml) was refluxed for 4 h. The excess thionyl chloride was removed by distillation, thus obtaining the corresponding acid chloride. Then, it was esterified with 2-hydroxyethyl methacrylate (HEMA). On a solution of HEMA (25 mmol) and triethylamine (25 mmol) in diethyl ether (100 ml) another of the above acid chloride (25 mmol) dissolved in diethyl ether (50 ml) was added dropwise at room temperature and in inert atmosphere At 24 h, triethylamine hydrochloride was filtered and the filtrate was washed with a 5% solution of sodium bicarbonate (2x100 ml). The organic phase was dried over anhydrous magnesium sulfate and finally the solvent was removed under reduced pressure. The overall reaction yield was 90%.

1 H-NMR (CDCl 3, 300 MHz, 25 ° C): 7.23 (d, 2H, J = 8.1 Hz), 7.11 (d, 2H, J = 8.1 Hz), 6.07 (s , 1H), 5.56 (t, 1H, J = 1.5 Hz), 4.31-4.36 (m, 4H), 3.75 (c, 1H, J = 7.2 Hz), 2 , 47 (d, 2H, J = 7.2 Hz), 1.92 (s, 3H), 1.81-1.92 (m, 1H), 1.52 (d, 3H, J = 7.2 Hz), 0.93 (d, 6H, J = 6.6 Hz).

Example 2

Synthesis of Ketoprofen-derived methacrylic monomer (HKT).

The synthesis scheme is depicted in Figure 1 where R is Ketoprofen. A solution of Ketoprofen (100 mmol) in thionyl chloride (70 ml) was refluxed for 4 h. The excess thionyl chloride was removed by distillation, thus obtaining the corresponding acid chloride. Then, it was esterified with 2-hydroxyethyl methacrylate (HEMA). On a solution of HEMA (25 mmol) and triethylamine (25 mmol) in diethyl ether (100 ml) another of the above acid chloride (25 mmol) dissolved in diethyl ether (50 ml) was added dropwise at room temperature and in inert atmosphere At 24 h, triethylamine hydrochloride was filtered and the filtrate was washed with a 5% solution of sodium bicarbonate (2x100 ml). The organic phase was dried over anhydrous magnesium sulfate and finally the solvent was removed under reduced pressure. The overall reaction yield was 87%.

1 H-NMR (CDCl 3, 300 MHz, 25 ° C): 7.78-7.84 (m, 3H), 7.44-7.73 (m, 6H), 6.05 (s, 1H), 5, 56 (d, 1H, J = 1.5 Hz), 4,324.39 (m, 4H), 3.87 (c, 1H, J = 7.2 Hz), 1.91 (s, 3H), 1, 58 (d, 3H, J = 7.2 Hz).

Example 3

Study of the reactivity relationships of monomeric pairs of VI with acrylic derivatives of NSAIDs (HEI derived from Ibuprofen or HKT-derived from Ketoprofen).

This example describes the calculation of the reactivity ratios using 1 H-NMR in situ to determine the microstructural organization of the polymer chains. The method described in the following reference was used to calculate these values: Macromolecules 2002, 35 (6), 2036-2041.

Copolymerizations of the various products were carried out in DMSO-d6. Several concentrations of comonomers A: B (in order to cover the entire concentration range) of 2 pairs of monomers (HEI: VI and HKT: VI) were studied. The following table shows the results obtained.

rIBU = 5.16 rVI = 0.01 rHKT = 8.06 rVI = 0.04

Table I. Reactivity ratios of the monomer pairs HEI: VI, HKT: VI.

In all cases we obtain that NSAID acrylic derivatives are much more reactive than VI. From these values, the microstructural distribution of the monomeric sequences can be deduced: initially rich chains will be formed in the NSAID-derived monomer (hydrophobic in nature) ending in VI-rich domains (hydrophilic in nature). This compositional gradient along the macromolecular chain gives the described systems the amphiphilic character responsible for the organization and self-assembled in an accusative medium for the formation of micelles.

Example 4

Synthesis of copolymers p (HEI-co-VI).

Copolymers were prepared by reacting the HEI monomer obtained as described in Example 1 and VI as hydrophilic monomer from compositions in the HEI feed: VI (% -molar) of 20:80, 50:50 and

80:20 The structure of these copolymers is shown in Figure 2 where R is Ibuprofen.

The copolymerization reaction was carried out by dissolving the monomers (0.25 M) in DMSO and AIBN (1.5x10-2 M). This solution was deoxygenated by a stream of nitrogen for 15 minutes at room temperature. The reaction mixture was maintained at 60 ° C for 24 h. Then, the reaction mixture was purified by dialysis in water for 4-5 days. Finally the product was frozen and lyophilized until constant heavy.

5 The 1 H-NMR spectra of the synthesized copolymers are shown in Figures 3, 4 and 5.

The composition of the copolymers prepared was calculated from the corresponding 1 H-NMR spectra. Table I shows the composition values obtained in the copolymers. 10

p (HEI-co-VI)

Feeding

20:80 50:50 80:20

Copolymer

25:75 56:44 91: 9

Mn (Da)

13,413 17,660 16,580

Mw (Da)

21,811 32,406 32,163

Mw / Mn

1.62 1.83 1.94

Tg (ºC)

48 46 27

Table I. Molar composition in the feed and in the copolymer. Average molecular weights in number (Mn) and in weight (Mw), polydispersity Mw / Mn and glass transition temperatures (Tg) of the prepared copolymers.

15 By size exclusion chromatography (SEC) the average number (Mn) and weight average (Mw) molecular weights of the copolymers were determined using a Perkin-Elmer chromatograph equipped with a 250 Series isocratic pump and an index detector. Series 200 refraction. Samples were eluted through three series connected columns of PL-gel polystyrene-divinylbenzene of 500, 104 and 105 nm pore size (Polymer Laboratories) at 70 ° C. DMF with 0.1% LiBr and a flow of 0.3 ml / min was used as the mobile phase. For him

20 calibrated methyl polymethacrylate standards of molecular weight between 10,300 and 480,000 D. were used. The results are summarized in Table I.

The glass transition temperature (Tg) of the copolymers was measured with a differential scanning calorimeter (Perkin Elmer DSC8500). For this, the dried sample was deposited in an aluminum capsule

25 sealed and heated at a constant rate of 10 ° C / min with an N2 flow of 20 ml / min. The sweeps were performed in the temperature range of -30 to 180 ° C. The Tg was determined as the midpoint of the transition of the heat capacity observed in the thermogram corresponding to the second scan. The Tg values obtained are shown in Table I.

30 Example 5.

Synthesis of the copolymers p (HKT-co-VI).

Copolymers were prepared by reaction of the HKT monomer obtained as described in Example 2 and VI from compositions in the HKT: VI (% -molar) feed of 20:80, 50:50 and 80:20. The structure of these copolymers is shown in Figure 2 where R is Ketoprofen.

The synthesis and characterization of this family of copolymers was carried out following the same procedure explained in Example 3, but using as a NSAID derivative the methacrylic derivative of Ketoprofen (HKT).

The 1 H-NMR spectra of the synthesized copolymers are shown in Figures 6, 7 and 8.

Table II shows the results obtained from the characterization of said copolymers p (HKT-co-VI). Four. Five

p (HKT-co-VI)

Feeding

20:80 50:50 80:20

Copolymer

23:77 51:49 97: 3

Mn (Da)

44,406 33,382 27,141

Mw (Da)

54,083 56,963 32,290

Mw / Mn

1.21 1.71 1.19

Tg (ºC)

61 59 46

Table II Molar composition in the feed and in the copolymer. Average molecular weights in number (Mn) and in weight (Mw), polydispersity Mw / Mn and glass transition temperatures (Tg) of the prepared copolymers.

Example 6

Preparation of nanoparticles from copolymers of Formula I.

Nanoparticles were prepared from the copolymers p (HEI-co-VI) and p (HKT-co-VI) synthesized according to Examples 4 and 5 respectively. For this, the copolymer (10 mg / ml) was dissolved in a water-miscible organic solvent (acetone, ethanol). Then 1 ml of this solution was added dropwise and with vigorous stirring, over 25 ml of water. It was stirred an additional 10 minutes and sonicated for 5 minutes to favor the dispersion of the nanoparticles.

The size of the polymer particles was determined by dynamic light scattering and polarized light scattering measurements. The morphology and their size were analyzed by scanning electron microscopy (SEM). The results obtained for all the compositions studied showed that the nanoparticles have a spherical morphology (Figure 9) and an average size, depending on the conditions used, between 30 and 170 nm.

Example 7

Ibuprofen release test contained in copolymers p (HEI-co-VI).

The release of Ibuprofen contained in the copolymers obtained according to Example 4 was evaluated by an in vitro assay at 37 ° C and in different buffered solutions (pH 7 and 10). For this, disks of approximately 100 mg of copolymer (compositions 20:80, 50:50 and 80:20) were immersed in 10 ml of buffered solution. Aliquots were taken periodically for analysis that was replenished with fresh medium. The amount of Ibuprofen released at different times was determined by UV spectroscopy (Perking Elmer Lambda 16) (= 263 nm) and is represented in Figure 10.

Example 8

Ketoprofen release test contained in copolymers p (HKT-co-VI).

The release of Ketoprofen contained in the copolymers obtained according to Example 5 was evaluated by an in vitro assay at 37 ° C and in different buffered solutions (pH 7 and 10). For this, disks of approximately 100 mg of copolymer (compositions 20:80, 50:50 and 80:20) were immersed in 10 ml of buffered solution. Aliquots were taken periodically for analysis that was replenished with fresh medium. The amount of Ketoprofen released at different times was determined by UV spectroscopy (Perking Elmer Lambda 16) (= 255 nm) and is represented in Figure 11.

Example 9

In vitro biocompatibility test of copolymers of Formula I p (HEI-co-VI).

The biocompatibility test was performed using a primary culture of human endothelial cells obtained from umbilical vein (Human Umbilical Vein Endothelial cells, HUVEC; Pharmakine DPK-HUVEC). The cultures were maintained and multiplied at 37 ° C in an atmosphere with 5% CO2, using MEP-modified culture medium HEPES (Sigma), supplemented with 10% fetal bovine serum (SBF; Gibco), 1% of a solution of penicillin-streptomycin (10,000 U / ml penicillin and 10 mg / ml streptomycin; Sigma), 0.1% of endothelial growth factor (EGF; Sigma) and 0.1% of sodium heparin (Sigma) . Thermanox® discs were used as a non-cytotoxic negative control and a 1% solution of Triton-X100 (Merck) in culture medium as a positive control.

Cytotoxicity of leachates. MTT test.

The purpose of the MTT test is the quantification of the cytotoxicity of the products released over a period of 7 days by the p (HEI-co-VI) 20:80, 50:50 and 80:20 systems.

In order to obtain the leachates necessary to perform this test, 5 ml of complete culture medium without fetal bovine serum were kept at 37 ° C, 7 days and with continuous stirring the TMX control and experimental group samples. After 1, 2 and 7 days, 5 ml of the medium was collected, replacing it with another 5 ml of fresh medium.

After obtaining all of the leachates, cell seeding was carried out at a concentration of 105 cells / ml in complete culture medium (100 μl of cell concentrate / well), after which the samples were incubated 24 h at 37 ° C in an atmosphere with 5% CO2. After this time, the culture medium was exchanged for the leachates previously obtained (n = 8), keeping them in contact with the cells for another 24 h at 37 ° C. Upon completion of this second incubation, the contents of the wells were removed, adding instead a solution of the MTT reagent (0.5 mg / ml). After 4 h at 37 ° C in an atmosphere with 5% CO2, the contents of the wells were extracted to add 100 µl of DMSO on each of them. Next, the optical density reading at 570 nm was performed.

The absorbance values obtained are subtracted from the values measured for the target, and are relativized with respect to the values read for the negative control (leachates of the TMX control) obtaining the relative cell viability with respect to the control (% VR), a from the following Formula:

TWO DO

% VR

B DOC DOUB

where, DOS, DOB and DOC are the measurements of optical density of the sample, the blank (dissolution of the MTT reagent in culture medium introduced in wells without cells) and the negative control, respectively.

As shown in Figure 12, the results obtained in the MTT test indicate that leachates from

the products studied do not present any toxicity, but a high cell viability (similar to that obtained

for the TMX control material), ensuring the biocompatibility of the systems described in this patent.

Claims (23)

1. Polymeric compound of general Formula I 5 (I) characterized in that:

-

 A represents a residue of one or more hydrophobic acrylic or vinyl monomers bearing an NSAID derivative, wherein said NSAID derivative is linked to the rest of the molecule through a covalent hydrolysable bond in vivo;

10-B represents a residue of one or more hydrophilic monomers capable of complexing with mono or divalent cations;

-

 m and n indicate the molar fractions of monomers A and B in the polymer so that m + n is always 1, and m and n are always non-zero.

A polymeric compound according to claim 1, characterized in that monomer B is an imidazole derivative.

3. A polymeric compound according to claim 2, characterized in that monomer B is 1-vinylimidazole.

A polymeric compound according to any one of the preceding claims, characterized in that the in vivo hydrolysable covalent bond is of the carboxylic ester or amide type.

5. A polymeric compound according to any one of the preceding claims, characterized in that the polymeric compound is defined by Formula Ia:  (Ia) where:

-

R1 represents hydrogen or C1-4 alkyl; 30-R2 represents the NSAID derivative;

-

 X represents a heteroatom, preferably N and / or O;

-

 f represents an integer between 1 and 10;

-

 p represents an integer between 1 and 100;

-

 m and n indicate the molar fractions of monomers A and B in the polymer so that m + n is always 1, and m and n are always non-zero.

6. A compound according to claim 5, characterized in that p represents an integer between 1 and 50. A compound according to any one of claims 5 or 6, characterized in that R1 represents a methyl group, X represents oxygen, f = 2 and p = 1. 8. A polymeric compound according to any one of the preceding claims, characterized in that the NSAID it is Ibuprofen, Ketoprofen, Naproxen or Flurbiprofen. Four. Five 9. A polymeric compound according to any one of claims 5 to 8 characterized in that m is between 0.01 and 0.35 and n between 0.65 and 0.99. 10. A polymeric compound according to any one of claims 5 to 8 characterized in that m is between 0.35 and 0.65 and n between 0.35 and 0.65.

eleven.

A polymeric compound according to any one of claims 5 to 8 characterized in that m is

between 0.65 and 0.99 and n between 0.01 and 0.35. 12

12.

A polymeric compound according to any one of claims 5 to 11 characterized by having an average molecular weight between 3,000 and 100,000 Daltons.

13.

A polymeric compound according to claim 12, characterized by having an average molecular weight between 10,000 and 60,000 Daltons.

14.

A micelle comprising a polymeric compound of Formula Ia as defined in any one of claims 5 to 13.

fifteen.

A process for preparing a polymer compound of Formula I, as defined in any one of claims 1 to 13, characterized in that it comprises the radical polymerization of monomers A and B in the myn molar proportions, respectively, within a suitable solvent and in the presence of a polymerization reaction initiator.

16.

A process for preparing nanometric polymer micelles with a size between 5 and 500 nm by self-assembly processes comprising the use of a polymeric compound of Formula Ia as defined in any one of claims 5 to 13.

17.

Use of a polymeric compound of Formula I as defined in any one of claims 1 to 13 or a micelle as defined in claim 14 as an NSAID release system in a vectorized, localized and / or controlled manner.

18.

Use of a polymeric compound of Formula I as defined in any one of claims 1 to 13 or a micelle as defined in claim 14 as a component in the bioactive coating of biomedical devices.

19.

Use of a polymeric compound of Formula I as defined in any one of claims 1 to 13 or a micelle as defined in claim 14 for complex formation with mono or divalent cations.

twenty.

Use of a polymeric compound of Formula I as defined in any one of claims 1 to 13 or a micelle as defined in claim 14 for application as an MMP inhibitor.

twenty-one.

Use of a polymeric compound of Formula I as defined in any one of claims 1 to 13 or a micelle as defined in claim 14 as a component in the implant coating.

22

Use of a polymeric compound of Formula I as defined in any one of claims 1 to 13 or a micelle as defined in claim 14 as a component in the preparation of antiseptic solutions or dispersions.

2. 3.

Use of a polymeric compound of Formula I as defined in any one of claims 1 to 13 or a micelle as defined in claim 14 as a targeted and / or vectorized injectable system for the release of NSAIDs.

24.

Use of a polymeric compound of Formula I as defined in any one of claims 1 to 13 or a micelle as defined in claim 14 as a non-viral vector of interest in gene therapy.

25.

Use of a polymeric compound of Formula I as defined in any one of claims 1 to 13 or a micelle as defined in claim 14 as a reabsorbable bioactive polymer with controlled therapeutic action.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201130651 SPAIN Date of submission of the application: 04.24.2011 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

X

S. DAVARAN et al., "Hydrophilic copolymers prepared from acrylic type derivatives of Ibuprofen containing hydrolizable thioester bond", European Polymer Journal, 1998, vol. 34, nº 2, pages 187-192, see Scheme 2, Table 2, example 5. 1-13,15.17

TO

M. BABAZADEH, "Synthesis and study of controlled release of ibuprofen form the new acrylic type polymers", International Journal of Pharmaceutics, 2006, vol. 316, No. 1-2, pages 68-73, see Scheme 2. 1-25

TO

C. PAREJO et al., "Controlled release of NSAIDs bound to polyacrylic carrier systems", Journal of Materials Science: Materials in Medicine, 1998, vol. 9, pages 803-809, see Schemes 1 and 2. 1-25

TO

A. GALLARDO et al., "NSAIDs bound to methacrylic carriers: Microstructural characterization and in vitro release analysis", Journal of Controlled Release, 2001, vol. 71, No. 1, pages 127-140, see Figure 1. 1-25

TO

US 20100247654 A1 (G-H. HSIUE et al.) 30.09.2012, figure 6; sections [0005] - [0026]. 1-25

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 19.09.2012

Examiner E. Dávila Muro Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201130651 CLASSIFICATION OBJECT OF THE APPLICATION C08F220 / 06 (2006.01) C08F226 / 06 (2006.01) A61K31 / 787 (2006.01) Minimum documentation sought (classification system followed by classification symbols) C08F, A61K Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, XPESP, CAPLUS, REGISTRY, BIOSIS, MEDLINE State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201130651 Date of Written Opinion: 19.09.2012 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 14,16,18-25 Claims 1-13,15,17 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims 14,16,18-25 Claims 1-13,15,17 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201130651  1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

S. DAVARAN et al., European Polymer Journal, 1998, vol. 34, no. 2, pages 187-192

 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The invention relates to a polymeric compound of the general formula I - [A] m- [B] n-where fragment A represents one or several hydrophobic acrylic or vinyl monomers bearing an NSAID derivative bound to the rest of the molecule by a covalent hydrolysable bond in vivo, and fragment B represents one or more hydrophilic monomers capable of complexing with mono / divalent cations, in particular a 1-vinylimidazole derivative. The invention also relates to the preparation of compound I, to the nanometric polymeric micelles comprising the compound I as well as its preparation, and to the use of compound I or the micelles obtained therefrom to obtain controlled release systems of NSAIDs and in various pharmacological and biomedical applications. Document D01 discloses hydrophilic copolymers prepared from methacrylic derivatives of ibuprofen, in particular the compound S-methacryloxyethyl-�-methyl-4 (2-methylpropyl) benzene thioacetate (MOETE), in which the drug is separated from the methacrylic fragment by means of an oxyethylene spacer and a hydrolysable thioester bond. This monomer is obtained by reacting ibuprofen, activated with 1,1-carbonylimidazole, with 2-mercaptoethanol in anhydrous THF in the presence of sodium hydride, followed by reaction with methacryloyl chloride in anhydrous THF in the presence of pyridine (see page 188, Scheme1 ). The methacrylic derivative of ibuprofen MOETE is copolymerized with hydrophilic monomers such as methacrylic acid, methacrylamide and vinylimidazole as the third component, as well as with polyethylene glycol methacrylate, in a suitable solvent (DMF, MeOH) at 60-70 ° C and in the presence of AIBN as the initiator of the polymerization (see pages 188189, Scheme 2, Table 2, in particular example 5). Polymeric compounds are obtained with hydrophilic and hydrophobic monomers carrying derivatives of an NSAID compound. The copolymers of the methacrylic derivative of ibuprofen MOETE with the vinyl monomers of Scheme 2 disclosed in D01 correspond exactly to the polymeric compounds of formulas I and Ia of the invention, R1 = methyl, X = O, S and R2 = ibuprofen, thus as the process of obtaining them from the radical copolymerization of vinyl hydrophilic monomers MA, MAAm, VIm and PEGM with the hydrophobic acrylic monomer carrying ibuprofen in the presence of a polymerization initiator. Also in D01 the use of said polymeric compounds as systems for the localized and controlled release of NSAIDs is disclosed. Consequently, the characteristics of claims 1-13,15,17 are already known from what is disclosed in document D01, so they are not considered new or inventive in view of the state of the known art (articles 6.1 and 8.1 LP 11/1986). No documents have been found in the state of the art that disclose polymeric micelles comprising the copolymers of formula I, or their preparation from the self-assembly of said copolymers. Nor have documents been found that collect the use of polymeric compounds I or micelles derived therefrom in coatings of biomedical devices or implants, in complexing systems of mono / divalent cations, in MMP inhibitor systems, antiseptic solutions, bioactive polymers. absorbable, injectable systems or non-viral vectors for gene therapy. Therefore, the characteristics of claims 14,16,18-25 are considered new and with inventive activity and industrial application according to articles 6.1 and 8.1 LP 11/1986. State of the Art Report Page 4/4