ES2692165B2 - CONTROLLED RELEASE SYSTEM AND METHOD OF PREPARATION OF THE SAME - Google Patents

Description

DESCRIPTION

CONTROLLED RELEASE SYSTEM AND PREPARATION METHOD

OF THE SAME

Field of the invention

The present invention relates generally to the field of the administration of active principles and in particular of the controlled release thereof. More specifically, the present invention relates to the field of controlled release of active ingredients in the colon region.

Background of the invention

The continuous increase in incidence and prevalence in the world population of diseases affecting the colon, especially intestinal inflammatory diseases (ulcerative colitis and Crohn's disease, mainly), has captured the attention and effort of many researchers and the pharmaceutical industry to find solutions that are really effective. So far, the therapeutic arsenal available does not solve the growing problem.

In the last decade, different biological agents have been introduced (mainly anti-TNFa), expanding the therapeutic arsenal available for IBD. However, this is still not enough, since many patients do not respond or do not respond continuously to these treatments (they are no longer effective). In addition, its oral administration is limited by having to cross the stomach (where pH acidity degrades the compounds) and the small intestine (where a large part of the compounds can be absorbed before arrival at the site of action, decreasing the effectiveness and resulting in unwanted adverse reactions).

Most so-called "controlled release" formulations consist of systems with a matrix (in some cases) or polymer cover (in others) that responds to pH changes found throughout the gastrointestinal tract. Due to the great inter and intravariability in the subjects of the gastrointestinal pH (even higher in some cases affected by ulcerative colitis or Crohn's disease), these systems fail to be sufficiently effective.

WO2014037596 discloses a nanodevice for the controlled release of substances comprising a support coated by oligosaccharides with at least 3 units of monosaccharides, wherein at least one of the monosaccharides is galactose. These nanodevices release their charge in a specific way in senescent cells.

WO2012117140A1 discloses a thermosensitive compound for the controlled release of at least one active substance or an indicator, comprising: (a) a porous support formed of porous material, the pores of which contain the active substance or indicator; and (b) an interface layer with molecules of lipophilic nature, having a first end covalently bound to the porous support and a second end constituted by lipophilic organic chains joined by non-covalent interactions to a thermosensitive surface layer located on the surface of the porous support, covering the pores. The thermosensitive surface is constituted by organic substances of lipophilic non-polymeric nature that, above a threshold temperature, decrease their interaction with the interface layer, unblocking the entrance of the pores of the porous support and releasing the active substance or the indicator.

WO2012007623A2 discloses a controlled release system formed by a porous support capable of containing an active substance or an indicator, an oligonucleotide blocking the pores of the support and an interface layer between the porous support and the oligonucleotide which ensures the fixation between these elements. The release of the active substance is produced by hybridization of the blocking oligonucleotide of the pores with its complementary oligonucleotide.

Therefore, there is still a need in the art for a controlled release system that reliably releases an active ingredient in the colon region, avoiding as much as possible its release in other regions of the digestive system, and reducing the occurrence of Adverse effects.

Summary of the invention

To solve the problems of the prior art, the present invention provides, according to a first aspect, a controlled release system in the region of the colon for the treatment, prevention or diagnosis of diseases, preferably of diseases related to the colon. , which comprises:

- a mesoporous silica matrix;

- an active principle charged in the pores of the matrix; Y

- functionalized molecular doors on the surface that prevent the release of the active principle;

in which the molecular doors are constituted by an azoderivative compound that fragments (by reducing the azo bond) in the presence of azorreductase activity thus allowing the release of the active principle.

In the gastrointestinal tract of the human being, the azorreductase activity is specific to the local colon microbiota that produces enzymes with said activity. Therefore, the azoderivative compound of the release system according to the first aspect of the present invention is fragmentary (by reducing the azo bond) upon reaching the colon (and not before) and will allow the release of the active principle therein.

According to a second aspect, the present invention provides a method for preparing a controlled release system according to the first aspect of the invention, comprising the steps of:

a) derivatizing an azo derivative by reaction with an alkoxysilane in organic solvent;

b) encapsulate an active ingredient in a mesoporous silica matrix by reaction in organic solvent; Y

c) adding to stage b) the alkoxysilane derivatized derivative compound obtained in step a).

Brief description of the drawings

The present invention will be better understood with reference to the following drawings with illustrative and non-limiting character of the invention:

Figure 1 is a schematic representation of the encapsulation of dye (rhodamine B) or drug (hydrocortisone) in a mesoporous silica material with pores blocked by azoderivated molecules covalently anchored to the outer surface (particles S1 or S2) and their release by Enzymatic reduction of azo bonds.

Figure 2 is a schematic representation of the synthesis reaction of the molecular door that gives rise to the azoderivative 1.

Figure 3 represents the release kinetics of the encapsulated drug (hydrocortisone) from a suspension of mesoporous microparticles functionalized with the Azoderivative S2, in aqueous solution at pH = 2 (squared), pH «4.5 (circles), pH« 7.4 (triangles) and pH "7.4 in the presence of the sodium azithreducting agent sodium dithionite (SD, 2 mg / ml) (crosses).

Figure 4 depicts systemic absorption in vivo (rhodamine concentration B ^ g / ml in plasma) for subjects (Wistar rats) receiving a rhodamine B dye solution or a suspension with S1 particles.

Figure 5 depicts the specific release of the in vivo load (rhodamine concentration B ^ g / ml in the cecum, colon and stool) in subjects (Wistar rats) receiving a rhodamine B dye solution or a suspension with the particles S1.

Figure 6 shows images of rat colon of subjects sacrificed on day 10 after the induction of colitis with TNBS: A: negative control (TNBS was not administered), B: positive control (group 1, treatment: saline), C (group 2, treatment: aqueous suspension of microparticles type MCM-41 empty), D (group 3, treatment: hydrocortisone solution) and E (group 4, treatment: formulation of S2 particles in aqueous suspension).

Figure 7 shows histological images of rat colon representative of a healthy subject A and of subjects sacrificed on day 10 after the induction of colitis with TNBS: B: positive control (group 1, treatment: saline), C (group 2 , treatment: aqueous suspension of microparticles type MCM-41 empty), D (group 3, treatment: hydrocortisone solution) and E (group 4, treatment: formulation of S2 particles in aqueous suspension).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As mentioned above, in a first aspect the present invention discloses a controlled release system in the region of the colon for the treatment, prevention or diagnosis of diseases, preferably diseases related to the colon, which includes:

- a mesoporous silica matrix;

- an active principle charged in the pores of the matrix; Y

- functionalized molecular doors on the surface that prevent the release of the active principle.

The molecular doors are constituted by an azoderivative compound that fragments (by reducing the azo bond) in the presence of azorreductase activity, thus allowing the release of the active principle. In the gastrointestinal tract of the human being, the azorreductase activity is specific to the local microbiota of the colon that produces enzymes with said activity, so that the release of the active principle occurs specifically in the colon.

That is why, the system according to the present invention is suitable for various treatments in which the release of the active principle in the colon is required. It must be taken into account that some active principles, for example, antibodies, hormones and entities of protein or peptide origin, present a deficit absorption in the stomach or small intestine given the existence of a large number of proteolytic enzymes. However, when they get protected to the colon, they can be absorbed in the once released.

The system according to a preferred embodiment of the present invention is suitable for the treatment, prevention and diagnosis of colon-related diseases, such as inflammatory bowel diseases (IBD) (e.g., ulcerative colitis and Crohn's disease), so more effective (since the active ingredient is released specifically in the place of action) and with fewer adverse effects (since no active ingredient is released elsewhere in the body).

According to a preferred embodiment of the present invention, the mesoporous silica matrix is micrometric in size and has pores with a pore size of 2 to 3 nm, more preferably the mesoporous silica matrix is of the MCM-41 type.

According to an embodiment of the present invention, the active ingredient incorporated in the pores of the mesoporous silica matrix is for the treatment or prevention of an inflammatory bowel disease (such as for example ulcerative colitis or Crohn's disease), such as example an anti-inflammatory steroidal drug used orally in cases of intestinal inflammatory diseases of moderate to medium intensity, such as for example hydrocortisone.

According to another embodiment of the present invention, the molecular doors that block the pores of the mesoporous silica matrix are preferably constituted by an azoderivative compound which, in the presence of azorreductase activity, undergoes reduction of the azo bond, giving rise to a fragment (5-ASA ) that is released and that It has anti-inflammatory properties. Therefore, a double therapeutic action is produced, on the one hand, from the active principle released from the inside of the pores and, on the other hand, from the fragment released from the molecular doors.

This characteristic is shown in Figure 1, in which it can be seen that at first (left part) the dye (rhodamine B) or the active principle (hydrocortisone) is included within the pores of the matrix and blocked in them by azoderivated molecules. After undergoing azorreductase activity, both the active principle and the 5-ASA fragment (right part) are released.

According to a preferred embodiment of the invention, the aromatized compound is sodium olsalazine.

According to another embodiment of the present invention, the controlled release system further comprises magnetic nanoparticles included within the mesoporous silica matrix. In this way, the retention time of the system in the desired area (for example, in the colon) can be increased by the application of an external magnetic field.

According to a second aspect, the present invention also discloses a method of preparing a controlled release system as defined hereinbefore. The method comprises the steps of:

a) derivatizing an azo derivative by reaction with an alkoxysilane in organic solvent;

b) encapsulate an active ingredient in a mesoporous silica matrix by reaction in organic solvent; Y

c) adding to stage b) the alkoxysilane derivatized derivative compound obtained in step a).

More specifically, the method can comprise derivatizing an azo derivative with an alkoxysilane (for example, 3-aminopropyltriethoxysilane) by suspension reaction in tetrahydrofuran (THF) at room temperature (20-25 ° C) under an inert atmosphere (Ar), preferably for at least 48 hours. On the other hand, the active principle is encapsulated by vigorous agitation thereof with microparticles of mesoporous silica in tetrahydrofuran (THF) at room temperature (20-25 ° C) under an inert atmosphere of Argon (Ar), preferably for at least 8- 12 hours. Finally, the derivatized azoderivative compound obtained above is added and stirring is continued at room temperature (20-25 ° C), preferably for 4-8 hours.

In the following, the present invention will be further detailed by the following specific examples.

Synthesis of microparticles type MCM-41 (S0)

The mesoporous microparticles type MCM-41 were synthesized according to the following procedure:

First, a solution of triethanolamine (TEAH3, 25.79 g, 0.173 mol) and sodium hydroxide (NaOH, 2 ml of a 6 M solution) was heated to 120 ° C and then allowed to cool to 70 ° C. At that time, tetraethyl orthosilicate (TEOS, 11 ml, 0.045 mol) was added to the reaction and heated to 120 ° C again. The reaction was allowed to cool and when it decreased below 118 ° C n-cetyltrimethylammonium bromide (CTABr, 4.68 g, 0.013 mol) was added. When the temperature decreased to 70 ° C, 80 ml of water were added little by little distilled with constant agitation. This mixture was allowed to age in an autoclave at 100 ° C for 24 h. The resulting powder was recovered by filtration and washed with distilled water. Finally, the solid was dried in an oven at 70 ° C ("24 h). To obtain the final mesoporous SO material (type MCM-41), the solid was calcined at 550 ° C using an oxidizing atmosphere for 5 hours in order to remove the surfactant.

Synthesis of the molecular door azoderivada (1)

Sodium olsalazine (0.5 g, 1.65 mmol) was dissolved in 30 ml of an acid solution at pH 0 (28.5 ml of distilled water and 1.5 ml of 37% HCl solution). The solution was stirred for 5 minutes at room temperature (20-25 ° C) and centrifuged. The supernatant was discarded, the resulting protonated product was recovered and allowed to dry at 70 ° C for 24 h. This process was repeated 4 times to obtain 1.8 g of product 1a (see figure 2, yield:). Then, N, N'-dicyclohexylcarbodiimide (DCC, 1.042 g, 5 mmol) and N-hydroxysuccinimide (NHS, 0.59 g, 5 mmol) were dissolved in anhydrous tetrahydrofuran (THF, 25 ml). This reaction was stirred at room temperature (20-25 ° C), under inert argon (Ar) atmosphere, for 5 h. A yellowish white precipitate (DCU, dicyclohexylurea) was formed which was discarded after centrifugation. The supernatant was still stirred for 15 h (under an inert Ar atmosphere, at room temperature, 20-25 ° C). After 15 h of reaction, the centrifugation was centrifuged and the new DCU precipitate was discarded again. Then, aminopropyltriethoxysilane (APTES, 1.2 ml, 5 mmol) was slowly added and allowed to react for 24 h (under inert Ar atmosphere, at room temperature 20-25 ° C). The solvent was removed by rotaevaporation and product 1 (see Figure 2) was isolated in the form of yellow-orange oil (2.05 g, 4.05 mmol, yield: 80%).

Encapsulation of dye (rhodamine B) in juMCM-41 and functionalization with the azo derivative (synthesis of S1 particles)

Microparticles type MCM-41 (S0, 1 g) were suspended in a THF solution (40 ml) and rhodamine B (400 mg, 0.8 mmol / g S0) under inert Ar atmosphere. This solution was allowed to stir at room temperature (20-25 ° C) for 8-12 h. Then, the azo-derivative 1 (2.05 g, 4.05 mmol / g of S0) was dissolved in anhydrous THF (25 ml) and added to the microparticle / dye suspension. The reaction mixture was stirred for 6 h (under inert Ar atmosphere, at room temperature, 20-25 ° C). After washing repeatedly with ethanol and distilled water, a red solid, S1, was isolated and left to dry in a vacuum for at least 24 h.

^{Drug encapsulation (hydrocortisone) in} ju ^{MCM-41 and} functionalization with the azoderivative (synthesis of S2 particles)

Microparticles type MCM-41 (S0, 1 g) were suspended in a solution of THF (40 ml) and hydrocortisone (296 mg, 0.8 mmol / g S0) under an inert atmosphere of Ar. This solution was allowed to stir at room temperature (20-25 ° C) for 8-12 h. Then, the azo-derivative 1 (2.05 g, 4.05 mmol / g of S0) was dissolved in anhydrous THF (25 ml) and added to the microparticle / dye suspension. The reaction mixture was stirred for 6 h (under inert Ar atmosphere, at room temperature, 20-25 ° C). After repeated washing with ethanol and distilled water, a solid yellow, S2, which was allowed to dry in vacuo for at least 24 h.

Release studies of hydrocortisone and 5-ASA.

In a typical experiment, 2 mg of solid S2 was suspended in 2 ml of an aqueous solution at a selected pH (pH = 2.0, 4.5 or 7.4) and at pH = 7.4 in the presence of the dithionite-depleting agent. sodium (able to reduce azo bonds). Aliquots were collected at times t = 0.5 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h. The samples were centrifuged to guarantee the elimination of traces or traces of the solid and each sample was analyzed by HPLC, to quantify the results (with the appropriate calibration curve) of both hydrocortisone and total 5-ASA.

Results (see figure 3 and table 1): It can be seen that the release is negligible at different pH in the absence of azorreductive activity. However, the liberation of the load (hydrocortisone) in the presence of the stimulus (azorreductive activity) reaches 80% at 6 h and presents total release of 24.36 g / mg of solid at 24 h. The total release of 5-ASA at 24 h is 93 ^ g / mg of solid.

Table 1:

In vivo pharmacokinetic assays

10 Wistar rats (male, approximate weight 300 ± 30 g) were anaesthetized by in situ perfusion to implant the jugular cannula 24 h before the experiment. To do this, we used the previously described permanent jugular cannulation method (Torres-Molina et al ., 1996). The cannula thus implanted allows the collection of blood samples. The animals were randomly assigned to the following groups:

Group 1. They were administered orally 1 ml of a solution of rhodamine B (saline 750 .g ^ / ml).

Group 2. They were administered orally 1.75 ml of a suspension containing 30 mg of the S1 formulation (which should release 750 ^ g of rhodamine B, approximately).

Blood samples (0.6-0.7 ml) were collected with heparinized syringes, and replaced with an equal volume of heparinized saline (10 IU / ml) at the established sampling times (t = 15 min, 30 min , 45 min, 1 h, 2 h, 3 h and 4 h). The plasma was separated immediately by centrifugation (10000 rpm for 10 min) and frozen at -20 ° C until the samples were analyzed. At the end of 4 h, the subjects were sacrificed and the caecum, colon and faeces were collected and processed for the analysis of the presence of rhodamine B.

Results (see Figures 4 and 5): The systemic absorption for the subjects who received the rhodamine B solution reached a maximum of 0.45 ^ g / ml, while the subjects who received the S1 formulation did not show systemic absorption or were negligible. The presence of rhodamine B in the cecum, colon and stool was also evaluated. The concentrations of rhodamine B detected in the cecum and colon were markedly higher for the group that received the S1 formulation.

In vivo assays of efficacy in a model of ulcerative colitis

Induction of colon inflammation. In this particular example, the studies were carried out with male Wistar rats aged 8-12 weeks and weighing approximately 275-325 g. The animals were kept in air-conditioned rooms at 22 ± 3 ° C, humidity of 55 ± 5%, with cycles of 12 h of light / dark and free access to feed and water during the studies. To induce the model of chronic inflammation in the colon of the rat, the method described by Morris et al . (1989) with slight modifications. In summary, the rats were randomly separated in the different treatment groups, fasted for 48 h with free access to water and anesthetized with isoflurane. A rectal cannula is inserted into the colon (the tip of the cannula is approximately 8 cm from the opening of the anus). At this point, a solution of 0.6 ml of TNBS (78 mg / kg body weight) dissolved in 50% v / v ethanol (instilled total volume of 0.6 ml) is instilled. The control group received 0.6 ml of a 50% v / v ethanol solution, administered in a similar manner as above. The induction and development of the inflammation were monitored every day during the 10 days that the study lasted. On day 10 after the administration of TNBS, the rats were sacrificed with an overdose of anesthesia. The development of inflammation was evaluated with respect to the weight ratio of colon / body weight, clinical activity score and histological changes.

Design of the treatments. The rats were divided into 4 groups. Group 1 (control group, 3 rats) was given saline, group 2 (8 rats) received a suspension of empty MCM-41 microparticles, group 3 (8 rats) received a hydrocortisone solution and finally group 4 (8 rats) received a suspension of formulation S2 (microparticles type MCM-41 with encapsulated hydrocortisone and functionalized with the molecular door azoderivada 1). The administered dose of hydrocortisone was 5.58 mg / kg / day, calculated according to the dose for humans (Sandborn and Hanauer, 2003). This dose was administered orally once a day for three days in the most intensive period of inflammation of the disease (days 3, 4 and 5 after the administration of TNBS).

Results (see figures 6 and 7): Figure 6A shows the characteristic appearance of a healthy colon in a rat to which TNBS was not administered. Figure 6B shows the characteristic colon in subjects treated with saline (placebo treatment, group 1) 10 days after the colitis model was induced with TNBS. Figures 6C, 6D and 6E show the characteristic appearance of the colon in subjects of groups 2, 3 and 4, respectively. All subjects (Wistar rats) were sacrificed 10 days after rectal instillation of TNBS. The results shown in figure 6B and 6C (groups 1 and 2) are very similar, in both cases necrotic, thickened and rigid intestinal tissue is appreciated. In group 3 (figure 6D) it seems that part of the tissue begins to recover. There are still areas of necrotic and thickened tissue, although they have been reduced. However, the results for group 4 (figure 6E) show, in general, that the tissue has recovered and it seems that only small areas have not recovered completely.

Histologically, the results agree with the macroscopic results previously commented. Negative control or healthy subject shows intestinal tissue healthy: healthy mucosa, not affected, enterocytes and globular cells, and between them connective tissue (own lamina, figure 7A), muscle layer, submucosa and outer muscle layer also in perfect conditions. Groups 1 and 2 show necrosis and loss of the necrotic mucosa that is replaced by granular tissue. They also show a strong inflammatory process present in the lamina propria, submucosa and external muscular layer. In FIGS. 7B and 7C, the process of ulceration with fibrillar necrosis of the mucosal surface and granular tissue beneath the necrotic tissue can be observed. The animals of group 3 present superficial erosion, with thinning of the mucosa accompanied by the thickening of the muscular layer, and a chronic inflammatory process that affects the mucosa and submucosa with early development of lymphoid follicles. Minor areas present a normal mucosal structure but with a strong follicular hyperplasia in the outer muscular layer and areas with necrosis, mucosal loss and substitution with granular tissue and inflammation (figure 7D). Group 4 usually shows normal structure in its mucosa and a slight presence of inflammation in the muscularis propria (Figure 7E). These results suggest that the untreated group (group 1) and the group treated with the empty MCM-41 microparticles (group 2), show strong lesions and inflammation; whereas the group 4 treated with the S2 formulation shows a remarkable improvement and recovery of the lesions in the tissues, as well as a significant reduction of the inflammation, recovering almost completely the normal structure of the mucosa.

As can be seen, unlike solutions known from the state of the art, the controlled release system according to the present invention, presents a mesoporous silicon matrix that is not affected by the passage through the gastrointestinal tract, and keeps stored in its pores the active principle of choice thanks to the blockade or impediment exerted by the molecular gates covalently anchored to the surface of said matrix. These molecular doors give the system great specificity, since the release of the active principle stored in its pores will occur in the presence of a specific external stimulus, in this case azoreductase activity produced by enzymes released by certain species of the local microbiota of the colon. In the system according to the present invention, the released fragment will produce an anti-inflammatory therapeutic effect, which contributes to the repair of the damaged tissue, therefore, it will have a beneficial effect in the treatment of the target diseases. In addition, the fragment released (5-ASA), by reducing the azo bond present in the molecular gates, has anti-inflammatory pharmacological properties, so that a double therapeutic action occurs when the charge of the microparticles and said fragment is released. In this way, the developed system possesses specificity in the release of charge or controlled release of the drug in the colon, avoiding the systemic absorption of pharmacological active principles.

As will be readily appreciated by the person skilled in the art from the foregoing description, the controlled release system described in the present invention can be used to convey pharmacological active ingredients or (bio) chemical entities up to the colon by minimizing the degradation of the active compounds and undue systemic absorption, thus increasing the effectiveness of the treatment while reducing or even reducing eliminate unwanted side effects This is of interest both for the treatment or prevention of diseases affecting the colon, and for increasing the bioavailability of oral administration drugs that are preferentially absorbed in the colon and are degraded in upper tracts of the gastrointestinal tract.

Claims (15)

1. System of controlled release in the region of the colon for the treatment, prevention or diagnosis of diseases, which includes: - a mesoporous silica matrix; - an active principle charged in the pores of the matrix; Y - functionalized molecular doors on the surface that prevent the release of the active principle; wherein the molecular doors are constituted by an azoderivative compound that fragments in the presence of azorreductase activity thus allowing the release of the active principle, and wherein the molecular doors are constituted by an azoderivative compound which, in the presence of azorreductase activity, undergoes azoic bond reduction giving rise to a fragment with anti-inflammatory properties. 2. System according to claim 1, characterized in that the mesoporous silica matrix is micrometric in size and has pores with a pore size of 2 to 3 nm. System according to any one of the preceding claims, characterized in that the mesoporous silica matrix is of the MCM-41 type. 4. System according to any of the preceding claims, characterized in that the active principle is for the treatment or prevention of a disease related to the colon. System according to any one of the preceding claims, characterized in that the active principle is for the treatment or prevention of a Inflammatory bowel disease. 6. System according to any of claims 4 and 5, characterized in that the active principle is an anti-inflammatory steroidal drug. System according to claim 6, characterized in that the active principle is hydrocortisone. System according to any of claims 1 to 3, characterized in that the active principle is for the diagnosis of a disease related to the colon. 9. System according to claim 1, characterized in that the azoderivative compound is sodium olsalazine. System according to any of the preceding claims, characterized in that it also comprises magnetic nanoparticles included within the mesoporous silica matrix. 11. Method of preparing a controlled release system according to any of claims 1 to 10, comprising the steps of: a) derivatizing an azo derivative by reaction with an alkoxysilane in organic solvent; b) encapsulate an active ingredient in a mesoporous silica matrix by reaction in organic solvent; Y c) adding to stage b) the alkoxysilane derivatized derivative compound obtained in step a). 12. Method according to claim 11, characterized in that the various steps are carried out under inert atmosphere. 13. Method according to any of claims 11 and 12, characterized in that the organic solvent of The various stages is tetrahydrofuran. Method according to any of claims 11 to 13, characterized in that the various steps are carried out at room temperature. 15. Method according to any of claims 11 to 14, characterized in that the alkoxysilane used in step a) is 3-aminopropyltriethoxysilane.