WO2020194034A1

WO2020194034A1 - Nanoparticle of chitosan and cyclodextrin containing encapsulated interferon and pharmaceutical compositions that contain it

Info

Publication number: WO2020194034A1
Application number: PCT/IB2019/052510
Authority: WO
Inventors: Rodrigo Antonio NAVES PICHUANTE; Luis Fernando GONZÁLEZ LÓPEZ; Felipe Andrés OYARZÚN AMPUERO
Original assignee: Universidad De Chile
Priority date: 2019-03-27
Filing date: 2019-03-27
Publication date: 2020-10-01
Also published as: AR118494A1

Abstract

The present invention refers to nanoparticles containing interferon encapsulated with chitosan and cyclodextrin and a pharmaceutical composition containing them, specially designed to be used by a non-invasive alternative administration route to a patient, in order to improve the therapeutic efficiency of treatments with interferons, particularly those needing that said molecule reaches the target organ of interest directly.

Description

NANOPARTICLE OF CHITOSAN AND CYCLODEXTRIN CONTAINING ENCAPSULATED INTERFERON AND PHARMACEUTICAL COMPOSITIONS

THAT CONTAIN IT

Technical Field

The present invention is related to the technical field of nanomedicine particularly with nanoparticles (NPs) of an active compound encapsulated in a matrix of biocompatible polysaccharides. Specifically, the present invention refers to a NP that contains interferon associated with a matrix of chitosan and cyclodextrin and pharmaceutical compositions comprising said NP, a pharmaceutically acceptable excipient and optionally, a further active therapeutic agent.

Background

The development of nanotechnology in the pharmaceutical and biotechnological industry has contributed to a significant improvement in the administration of active compounds in the organism. With the use of nanotechnology is possible to improve the administration of drugs that are poorly soluble in water, to direct a drug to a specific tissue or cell group, or even to achieve that a drug goes through endothelial membranes whose cells are closely connected (Farokhzad OC., Langer R. Impact of Nanotechnology on Drug Delivery. ACS Nano. 2009. 27;3(1 ):16-20). In addition, it allows the generation of new pharmaceutical compositions to change the routes of administration of a drug to less invasive routes, in order to improve the effectiveness of a treatment, to decrease the dose and improve the patient’s adherence to said treatment.

Although the advantages of this kind of technologies are known, its use is still not usual, and the pharmaceutical compositions remain conventional for the administration by invasive routes. This is the case, for example, of all interferons currently approved by the Food and Drug Administration of the United States (FDA) which are useful for the treatment of a series of diseases whose registered administration route is by injection (subcutaneous, intramuscular, intravenous). Interferons (IFN) are proteins produced and secreted by cells of the immune system in response to the presence of pathogens and even to tumor cells. These proteins have an immunomodulating, antiviral and antiproliferative biological activity and are used for the treatment of multiple diseases. For example, Interferon gamma 1 b (IFN-g 1 b, ACTIMMUNE^®) is indicated for the treatment of infections associated with chronic granulomatous disease or to delay the progression of patients with malignant and severe osteoporosis; both Interferon beta 1 a (IFN-b 1 a, AVONEX^®, REBIF^®, PLEGRIDY^®), and Interferon beta 1 b (IFN-b 1 b, BETASERON^®, EXTAVIA^®) are indicated for the treatment of multiple sclerosis; Interferon alfa 2b (IFN-a 2a, INTRON A^®) is indicated for the treatment of hairy cell leukemia, malignant melanoma, follicular lymphoma, condyloma acuminatum, Kaposi Sarcoma related to AIDS, chronic hepatitis C, chronic hepatitis B; PEGylated interferon alfa 2a (PEGASYS^®) is indicated for the treatment of chronic hepatitis C; and PEGylated interferon alfa 2b is also indicated for the treatment of chronic hepatitis C.

In the particular case of treatments with IFN-b whose therapeutic target is located in the central nervous system, it is known that current drugs have a low effectiveness, largely due to the administration is by systemic route. As a consequence, treatments must be administrated in high dose and high frequencies, causing various undesirable adverse effects in patients.

In the state of the art, some researchers have proposed methods of nano and microencapsulation of interferon and pharmaceutical compositions containing it. For example, the patent document US 2016/0067338 A1 discloses compositions containing NPs with polymeric coatings for transferring therapeutically active substances. It describes a list of polymers for possible use but indicate that are particularly preferred the polymers or copolymers based on a-hidroxy-carboxylic acids such as polylactic acid, polylactides, polyglycolic acid, polyglycolides and copolymers thereof, more preferably polyols (for example, polyethylene glycol) and polyacids such as polyacrylic acids and carbohydrates and sugar, particularly dextrans. It also mentions that NPs are suitable for a series of therapeutically active substances such as anti-cancer drugs, antibiotics, hormones, immunomodulators, among others.

On the other hand, the patent document US 2008/0292712 A1 refers to formulations for controlled and prolonged release of bioactive molecules such as therapeutic proteins, peptides and oligonucleotides. These formulations are based on micro and nanoparticles formed by the combination of biodegradable synthetic polymers such as PLA, PGA and copolymers thereof. The bioactive molecules are coupled to hydrophilic polymers such as polyethylene glycol or polypropylene glycol and then are formulated as micro or nanoparticles to achieve controlled drug release. In a preferred execution of the invention described in US 2008/0292712 A1 , the bioactive molecule is selected from the group consisting of IFN-a, IFN-b, I FN-y, erythropoietins, interleukins, among others.

The US 6,465,425 B1 patent document refers to sustained-release compositions of proteins containing free sulfhydryl, particularly refer to IFN-b. The composition comprises biocompatible polymers such as PLA, PGA, PLGA, polycarbonates, polyesteramides, polyanhydrides, polyaminoacids, polyorthoesters, polydioxanone, polyalkylene alkylates, copolymers or polyethylene glycol and polyorthoester, biodegradable polyurethane, polyacrylates, ethylene-vinyl acetate polymers and other cellulose acetates replaced with acyls, non-degradable polyurethanes, polystyrenes, vinyl polychloride, vinyl polyfluoride, polyvinylimidazole, chlorosulfonate polyolefins, polyethylene oxide, mixtures and copolymers thereof; and at least one surfactant. Preferably, the invention comprises NPs of IFN-b of sizes between 1 and 1000 pm.

As can be seen from the previous background, all described formulations involve synthesis processes that use organic solvents which has certain technical disadvantages, for example, it makes the synthesis process more complex and expensive, it puts at risk the conformational stability of proteins contained in them and last but not least, leaves organic residues which are slowly biodegraded and undesirable from the environmental point of view. On the other hand, there are few technologies aimed to the development of nanostructures containing interferon that are truly effective. In fact, to date there is no commercial product containing nanoencapsulated interferon. For this reason, new alternatives are required for the administration of interferon in a patient, in order to improve the treatments currently available through a nanostructure which optimizes the delivery of the active.

Summary of the invention

The present invention refers to a nanoparticle containing interferon and that comprises said interferon encapsulated in a nanovehicle that contains chitosan (CS) and cyclodextrin (CD).

In a preferred execution of the invention, the interferon is selected from the group that consists of type I interferon, type II interferon and type III interferon, more preferably, the interferon is IFN-b.

In another execution of the invention, the NP has a proportion CS/CD which is determined according to the ratio of electrical charges between them, which is between 0.25 and 1.40, more preferably between 0.70 and 1.30, and more preferably yet the electrical charges ratio CS /CD is between 0.75 and 1.25.

In another particular execution, the CD is sulfobutylether-p-cyclodextrin (SBE-p-CD). Preferably, the NP described in the present invention has a size range between 150 to 300 nm.

The present invention also refers to a pharmaceutical composition of interferon which comprises NPs containing interferon associated with CS and CD, and a pharmaceutically acceptable excipient. Brief description of the figures

FIG. 1 shows spherical NPs containing IFN-b (NP/IFN-b) and of size concordant with the measurements in DLS (approximately 200 nm). (A) Blank NPs (without IFN- b). (B) NPs loaded with 50,000 IU of IFN-b. FIG. 2 shows the functional activity of IFN-b released from NPs of CS/SBE^-CD with charge ratio of 0.75 in a range of 0 to 120 hours.

FIG. 3 shows the cell viability of murine cultures of fibroblasts L(tk-) (A) or splenocytes (B) after incubation with different amounts of IFN-b encapsulated in NP (NP/IFN-b). The magnitude of each column represents, at least, the average of three independent measurements with their respective standard deviation.

FIG. 4 shows a diagram of experimental design for the therapeutic evaluation by nasal or systemic route of naked IFN-b or included in NPs of CS/SBE^-CD.

FIG. 5 shows the clinical progression of mice induced with EAE daily treated and during two weeks by intranasal route (in) with a nanoformulation consisting of IFN- b encapsulated in NP (inNP/IFN-b), naked IFN-b (inlFN-b), blank NPs (inNP), or systematically by intraperitoneal route (ip) with IFN-b (iplFN-b) or PBS (ipPBS).

FIG. 6 (A-E) shows the therapeutic effect of nasal administration of NP/IFN-b regarding the other groups of treatment administered at different dose and frequency. FIG. 7 (A-B) shows the analysis of the clinical score variation of EAE in response to the treatment with NP/IFN-b administered by nasal route in different dose and frequency.

Detailed description of the invention

The present invention refers to NPs of an immunomodulating compound encapsulated to a nanostructure of CS and CD and a pharmaceutical composition that contains them. More specifically, the present invention refers to a NP specially designed for the encapsulation of interferon and a pharmaceutical composition that contains it, to be administrated by a non-invasive alternative route to a patient, with the aim of improving the therapeutic efficiency of treatments with immunomodulators, particularly in those treatments where is required that the immunomodulating molecule directly reaches the target organ of interest.

All the technical and scientific terms used to describe the present invention have the same meaning understood for a person with basic knowledge in the technical field in question. Nevertheless, in order to define more clearly the scope of the invention, hereunder is included a list of the terminology used in this description.

It must be understood by“nanoparticle”, any particle with a size between 1 and 1000 nm, according to the size definitions of NPs with pharmaceutical purposes (DeMarino C, Schwab A, Pleet M, Mathiesen A, Friedman J, El-Hage N, Kashanchi F. Biodegradable Nanoparticles for Delivery of Therapeutics in CNS Infection. J Neuroimmune Pharmacol. 2017. Vol. 12(1 ): 31-50; Zhou Y, Penga Z, Sevena ES, Leblanc RM. Crossing the blood-brain barrier with nanoparticles. J. Control. Release. 2018. Vol. 270:290-303). Additionally, it must be understood that the term “nanoparticle” includes any conformation thereof, for example, nanocapsule or nanosphere.

It must be understood by“immunomodulator”, a molecule with biological activity that regulates the immune system, either by inducing, increasing or decreasing the immune response to different stimuli. These immunomodulators include cytokines such as interleukins, lymphokines, interferons and hematopoietic growth factors.

It must be understood by the term“pharmaceutical composition” a mixture or association of substances comprising at least one active ingredient, namely, a compound having a biologically active activity and exerting an effect on the applied organism and an inactive medium where said active ingredient, also named excipient, is incorporated.

It must be understood by the term“excipient”, inert or inactive carriers that allow to administrate the active ingredient(s) to an organism. Said excipients will depend directly on the administration route used and on the desired pharmacodynamic and pharmacokinetic profile. As such, an appropriate excipient may be a binder, diluent, disintegrant, lubricant, a coating compound, sweeteners, flavorings, colorants, etc. In the present invention, the following acronyms will be used to refer to:

PBS: Phosphate Buffered Saline

CS: Chitosan

SBE-P-CD: sulfobutylether-p-cyclodextrin

NPs: Nanoparticles.

IFN-b: Interferon beta.

NP/IFN-b: Nanoformulation consisting of NPs of chitosan and sulfobutylether- -cyclodextrin loaded with IFN-b in: intranasal.

ip: intraperitoneal. inNP/IFN-b: NPs of chitosan and sulfobutylether- -cyclodextrin loaded with

IFN-b and administered by intranasal route. inNP: Blank NPs of chitosan and sulfobutylether- -cyclodextrin (without IFN- b) and administered by intranasal route.

inlFN-b: Naked IFN-b administered by nasal route

iplFN-b: Naked IFN-b administered by intraperitoneal route

ipPBS: Phosphate buffered saline administered by intraperitoneal route

A first object of the present invention refers to a NP that comprises an immunomodulating molecule and biocompatible excipients, said immunomodulating molecule being an interferon and said biocompatible excipients the CS and the CD. The immunomodulating molecule is preferably an interferon (IFN) that may be any of its classifications: type I, type II, type III (also named interferon type cytokines). Interferon type I comprises interferon alpha (IFN-a) and the subtypes interferon alpha 1/13 (IFN-a1 , IFN-a13), interferon alpha 2a (IFN-a2a), interferon alpha 2b (IFN-a2b), interferon alpha 2c (IFN-a2c), interferon alpha 4a (IFN-a4a), interferon alpha 4b (IFN-a4b), interferon alpha G (IFN-aG), interferon alpha 5 (IFN- a5), interferon alpha 61 (IFN-a61 ), interferon alpha K (IFN-aK), interferon alpha 6 (IFN-a6), interferon alpha 54 (IFN-a54), interferon alpha J (IFN-aJ), interferon alpha J1 (IFN-aJ1 ), interferon alpha 7 (IFN-a7), interferon alpha B2 (IFN-aB2), interferon alpha B (IFN-aB), interferon alpha 8 (IFN-a8), interferon alpha C (IFN-aC), interferon alpha 6L (IFN-a6L), interferon alpha H (IFN-aH), interferon alpha H1 (IFN- aFI1 ), interferon alpha 14 (IFN-a14), interferon alpha WA (IFN-aWA), interferon alpha 16 (IFN-a16), interferon alpha O (IFN-aO), interferon alpha I (IFN-al), interferon alpha 17 (IFN-a17), interferon alpha 88 (IFN-a88), interferon alpha F (IFN-aF), interferon alpha 21 (IFN-a21 ), interferon beta (IFN-b), interferon delta (IFN-d), interferon epsilon (IFN-e), interferon kappa (I FN-K), interferon tau (IFN-t) and interferon omega (IFN-co); type II interferon comprises interferon gamma (IFN- g); and type III interferon or cytokines interferon type comprises interferon lambda 2 (IRN-l2 o IL-28A), interferon lambda 3 (IRN-l3 o IL-28B) and interferon lambda 1 (IFN-lI o IL-29). It may also be an interferon that has undergone chemical modifications, for example, combination with polyethylene glycol (PEG) to obtain pegylated interferon or peginterferon. In a particular execution of the present invention, the NP comprises interferon beta (IFN-b). Thus as used herein, the term “an interferon” encompasses pharmaceutically active derivatives of any of the above interferons, including e.g. pegylated forms.

The NP of the present invention comprises, further, CS and CD. The CS is a linear cationic polysaccharide composed of b-(1 -4) D-glucosamine and N-acetyl-D- glucosamine. In the present invention CS of any molecular weight and deacetylation degrees may be used, although preferably CS of 50 to 190 KDa molecular weight and 75 to 85% of deacetylation is used. CS modifications with positive charges may also be used (for example, thiolated CS). The CS may be derived from animals (for example, from crustaceans or insects), by chitin deacetylation or be produced under controlled conditions (for example, by fungi fermentation). On the other hand, the CD is a cyclic oligosaccharide that is composed of 6, 7 or 8 units of D-glucopyranose and is produced by the enzymatic conversion of starch. In the present invention it may be used alpha-cyclodextrin (ct-CD), beta-cyclodextrin (b-CD), gamma- cyclodextrin (g-CD) and delta-cyclodextrin (d-CD), and any chemically modified or combined derivative thereof. Preferably, sulfobutylether- -cyclodextrin (SBE- -CD) is used in the present invention, which corresponds to b-CD substituted with 6 to 7 sulfobutyl groups ((CFh)4 S03-Na) per molecule of b-CD. Thus as used herein, the term“a cyclodextrin” includes derivatives of any of the above-mentioned derivatives, e.g. SBE^-CD.

A preferred modality of the present invention considers that the proportion CS and CD in the NP is defined by the ratio of electrical charges between their components (the CS presents positive charges and the SBE- -CD presents negative charges). Preferably, the range of electrical charge ratio is between CS and CD is between 0.25 and 1 .40, more preferably between 0.70 and 1 .30, and even more preferably between 0.75 and 1 .25.

It is important to consider that proportions between CS and CS for the present invention make sense if said proportion is defined according to their electrical charge ratio. We affirm this because for a same mass ratio of CS/CD, a different electrical charge ratio could result depending on the type of CD used. In an even more preferred execution of the invention, the electrical charge ratio of CS/ SBE- -CD is 0.75.

It is known that the size of the NP is a relevant characteristic in order to enhance the delivery of biologically active molecules contained in them. In general, NPs present a high value in the ratio of superficial area and volume. This means that the smaller the size of the NP, the total contact surface is significantly greater. Thus, in a preferred embodiment of the present invention, the NP has a size range between 150 to 300 nm, which would give a better bioavailability of the interferon when administered to a patient.

A second object of the present invention corresponds to a pharmaceutical composition of IFN, which comprises NPs containing encapsulated IFN in a matrix of CS and CD, and a pharmaceutically acceptable excipient. Said NPs consist of the NPs previously described, including all the mentioned variants but without limiting to those. In a preferred modality of the present invention the IFN contained in the NP is selected from the group consisting of type I IFN, type II IFN and type III IFN, preferably IFN-beta (interferon-b), encapsulated in a mixture of CS and CD, where the range of electrical charge ratio CS/CD is preferably between 0.25 and 1 .40; more preferably between 0.70 and 1 .30, and even more preferably between 0.75 and 1.25. In a preferred embodiment of this invention, the CD is sulfobutylether- -cyclodextrin (SBE- -CD).

Preferably, the NP contained in the pharmaceutical composition has a size between 150 to 300 nm. Preferably, the NP has an approximate size of 200 nm. This size is particularly desired for pharmaceutical compositions whose administration route is nasal, however, the size of the NPs in the pharmaceutical composition may vary within the mentioned range depending on the administration route for which said composition is designed.

The preferred administration route of the pharmaceutical composition of the present invention is intranasal (transmucosal) but it is not limited to this, being possible to formulate a pharmaceutical composition containing the NPs with IFN for any administration route.

This preferred administration route is particularly relevant for the treatment of diseases that require that IFN being delivered to the central nervous system more directly. The NPs of the present invention contained in the pharmaceutical composition allow a slow and sustained diffusion of the IFN towards the neural axons of the olfactory bulb or the trigeminal pathway, thus, optimizing the current treatments that use this drug by systemic administration.

The pharmaceutical composition also comprises a pharmaceutically acceptable excipient according to the definition previously described. Since one of the preferred routes of administration of the present invention is by intranasal route, the excipient used is any that a person with knowledge in the technical field will recognize as appropriate for the formulation of said composition. For example, in a particular execution, the excipient may be water or PBS, where the pharmaceutical form of the composition of the present invention may be in spray or for use in nebulizers.

In some embodiments, the pharmaceutical composition may comprise one or more further active pharmaceutical ingredients (i.e. in addition to the interferon). For instance, the pharmaceutical composition may further comprise any therapeutic agent that is typically used in combination with interferons, e.g. ribavirin, boceprevir and/or telaprevir, amongst others. The interferon-containing pharmaceutical compositions of the present invention may be used as fixed or free combinations with such additional therapeutic agents. Thus in a further aspect the present invention provides a pharmaceutical combination comprising the pharmaceutical composition as defined above and a further therapeutic agent, for simultaneous, separate or sequential administration to a subject.

The pharmaceutical compositions and combinations as described herein may be used to treat any disease or condition typically treated with interferons, optionally in combination with further therapeutic agents. For example, the disease may be an infection (e.g. an infection associated with chronic granulomatous disease); malignant or severe osteoporosis; multiple sclerosis; hairy cell leukemia, malignant melanoma, follicular lymphoma, condyloma acuminatum, Kaposi Sarcoma related to AIDS, chronic hepatitis C or chronic hepatitis B. In some embodiments, the pharmaceutical composition or combination is used to treat an autoimmune disorder (e.g. multiple sclerosis), a cancer (e.g. leukemia) or an infectious disease (e.g. hepatitis A, B or C).

The following examples are aimed to illustrate the invention and its preferred modalities, but under no circumstances they should be considered to restrict the scope of the invention, which will be defined by the tenor of the claims attached hereto.

EXAMPLES

Example 1. Synthesis of NPs loaded with IFM-b (NP/IFN-b) by ionic interaction.

The synthesis of NP was carried out in an aqueous medium. IFN-b is first mixed with SBE-p-CD by stirring and then CS was added. Under these conditions, the ionic interaction allowed the generation of NPs with a density of positive charge on the external surface.

Several CS and SBE-p-CD combinations were previously assessed based on the electrical charge associated with each polymer (Table 1 ). In the preparation of NP/IFN-b, the procedure was carried out at room temperature and IFN-b was incubated under constant stirring for 15 min in the solution of SBE-p-CD. Then, the CS solution was added and the whole system was kept under stirring for another 5 min. The CS solution used in the synthesis of NPs is prepared from a stock solution of CS (molecular weight between 50,000 to 190,000 Da; 75-85% deacetylated, Sigma Aldrich^®), dissolved in 1 % acetic acid (HAc). Ultra-pure water is used in all solution preparations (resistivity of 18,2 MW-cm in ultrafiltration system, Milli Q^®).

Finally, the obtained NPs were washed twice in ultra-pure water by centrifugation at 7,500 x g for 20 min and were immediately used for the treatment of animals or for its physicochemical characterization. Nevertheless, they may also be kept suspended in water at 4°C for at least a month or they may be lyophilized and reconstituted, without altering their physicochemical properties.

Table 1. Proportion of concentrations of CS and SBE-p-CD for preparing blank NPs at different charge ratios

Example 2. Physicochemical parameters and characterization of blank NP and NP/IFN-b.

The visual aspect of the resulting solution after the ionic interaction process was characterized for each CS/CD charge ratio (Rc) in terms of the presence of solids precipitation, the presence of an opalescent suspension or a homogeneous solution without precipitation or opalescence (Table 2). Likewise, the solution/suspension resulting from the ionic interaction was characterized in its parameters of hydrodynamic volume, polydispersity (PDI) and zeta potential (surface charge density) by dynamic light scattering and laser doppler anemometry (DLS and LDA, respectively) (Table 2). To evaluate these 3 parameters in the same sample, a volume of 0.70 ml of each solution was arranged in a DTS1070 capillary cell and was analyzed for each parameter in cycles of 15 readings each in a Zetasizer nano-ZS equipment (Malvern Instruments).

From the results presented in Table 2, it is highlighted that the presence of opalescence reveals the formation of NP in the solution product of the ionic interaction. Only three Rc of CS/SBE- -CD of 0.75, 1.0 and 1.25 showed the effective formation of NP with hydrodynamic volumes (sizes) between 201.6 and 279.5 nm. The polydispersity of the NP varied between 0.11 and 0.20, which represents an homogeneity greater than 80% in the hydrodynamic volume measured for NPs in each Rc. Whereas, the positive Zeta potential between 19.6 and 27.4 mV indicates the charge density due to the coating with the CS polycation on the surface of the NP in each Rc.

Table 2. Physicochemical characteristics of blank NP of QS/SBE-p-CD that were prepared at different charge ratios.

In the therapeutic evaluation results described in the examples, the evaluated treatments involve only NP obtained at a CS/CD charge ratio of 0.75 due to their smaller sizes and polydispersity values, which implies homogeneous suspensions of NP with spherical characteristics. In fact, the blank NP and NP loaded with 50,000 IU of IFN-b formed to Rc 0.75 were analyzed by transmission electron microscopy (60-OOOx, 30.000 kV), observing spherical structures with no apparent differences in shape and size due to the association with the IFN-b in agreement with the measurements in DLS (approximately 200 nm) (Fig. 1).

Example 3. Characterization of NPs loaded with mouse and human interferons

The physicochemical parameters of hydrodynamic volume, polydispersity and zeta potential were evaluated in NPs with CS/CD charge ratio 0.75. It was observed that said parameters remain similar when loaded with either mouse IFN (IFN-g and IFN-b) or human IFN-b (Table 3). The efficiency of association of IFNs to the system of NPs reached between 70-90% with all the evaluated interferons (Table 3), which shows that the NP composed of CS and CD represents an appropriate nanostructure for the encapsulation of the interferon.

Table 3. Physicochemical characteristics of NPs of CS/SBE-p-CD with charges ratio 0.75 loaded with mouse and human interferons.

*The association efficiency corresponds to the percentage of macromolecules encapsulated in NPs. This value was estimated by the difference between the total mass of interferon added during the reaction of synthesis of NPs and the free or non-associated mass remaining in the supernatant obtained after the centrifugation step. The levels of murine or human interferons in the supernatant were determined by a commercial ELISA kit. Nd: not determined.

Example 4. Association and functional assays of IFN-b loaded in the NPs of CS/SBE- -CD (NP/IFN-b)

Assays of association, release and functionality with mouse interferon beta 1a (IFN- ia) were carried out in the NPs system of QS/SBE-P-CD (NP/IFN-b). The amount of IFNs associated with NPs was estimated by the difference between total IFN added in each reaction before the formation of NPs and the free IFN found in the supernatant after the ionic interaction with CS occurred. The levels of IFN in the supernatant were determined by a commercial ELISA kit. As is observed in Table 3, the association efficiency of murine and human IFN-b to the NPs was over 85%. Additionally, the functionality of IFN-b released from the NPs was evaluated by the induction of the expression of MHC class I (MHC-I) molecules on the surface of mouse fibrosarcoma cell line (L(tk-) cells). The expression of MHC-I is induced by IFN-b when is released from the NP and interacts with its specific receptor on the membrane of L(tk-) cells. The induction of the expression of MHC-I molecules in response to released IFN-b from NPs of QS/SBE^-CD (Rc 0,75) is shown in FIG. 2. The NPs loaded with 50 ng/mL of IFN-b were incubated with L(tk-) cells for up to 120 h and the expression of MHC-I molecules induced by IFN-b was analyzed by flow cytometry. IFN-b is naked interferon beta; NP/IFN-b is NP loaded with IFN-b; NP is blank nanoparticle (without IFN-b). Each point represents the average of three independent measurements with their respective standard deviation. The results showed that IFN-b was released in a slow and long-lasting way to induce the expression of MHC-I molecules up to 5 days (120 h) (FIG 2). In contrast, the naked IFN-b reached a response maximum on the expression of MHC-I at 90 h of incubation. Importantly, blank NPs were unable to induce the expression of MHC-I (FIG. 2).

Example 5. Effect of NP/IFN-b on cell viability

In order to evaluate the toxicity potential of NPs and NP/IFN-b, the viability of cell cultures exposed to said NPs was evaluated by flow cytometry (FIG. 3). For that purpose, it was analyzed the binding of a viability dye (eFluor780) to cells that have been incubated for 94 h in the presence of blank NPs, different amounts of naked IFN-b, or with NP/IFN-b.

After the incubation with 25,000, 50,000 or 125,000 IU of IFN-b either naked or associated in NP (NP/IFN-b), cell viability of L(tk-) cells declined only in 20% with the greater concentration of IFN-b evaluated. This effect was mainly observed in the incubation of cells with NP/IFN-b, whereas in the incubation with blank NP or naked IFN-b, the cell viability was close to the control without treatment (FIG. 3A).

In addition, the viability of a non-immortalized cell culture (primary cell culture) was evaluated, which could represent a better model to determine toxicological sensitivity. To do that, a suspension of mouse splenocytes was incubated incubated with NP/IFN-b that contained 25,000, 50,000 or 125,000 IU of IFN-b for 94 h. As expected, the culture of untreated splenocytes showed lower viability than L(tk-) cells (-80%). The treatment with NP/IFN-b reduced viability up to 25% in relation to the untreated control. Likewise, treatment with blank NP or naked IFN-b produced a minimal or null reduction of cell viability in relation to the control without treatment (FIG. 3B).

Example 6. Evaluation of the therapeutic effect of NP/IFN-b

In the experiments described in this example, the therapeutic effectiveness of NPs containing commercially available human IFN-b (NP/IFN-b) was evaluated for the treatment of multiple sclerosis (MS). The effectiveness of this composition was evaluated in mice induced with experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS.

Induction of EAE in mice

EAE was induced in male C57BL/6J mice of 8-12 weeks old by subcutaneous administration in the dorsal region of an emulsion containing: 2.5 mg/mL of Mycobacterium tuberculosis, H37Ra (Difco), 0.75 pg/pL of neuro-antigen derived from myelin (MOGp, peptide (35-55) of glycoprotein of oligodendrocyte myelin) and 50% of the total volume of Freund’s complete adjuvant (IFA; Difco). In addition, at day 0 and day 2 of postimmunization, an aqueous solution of 2.5 mg/mL (200 pL) of Pertussis toxin was administered by ip route.

Therapeutic evaluation of NP/IFN-B in mice induced with EAE

Mice induced with EAE reaching the peak of the disease and at the beginning of the chronic phase (day 14-15 postimmunization) were treated daily with NP/IFN- b during two weeks. The experimental design is shown in FIG. 4 and considers 5 experimental groups: mice induced with EAE receiving intranasal (in) treatment with NP/IFN-b (in NP/IFN-b), with naked IFN-b (inlFN-b), or with blank NPs (inNP); and EAE mice receiving systemic treatment by ip route with naked IFN-b (iplFN-b) or with PBS (vehicle of systemic IFN-b) (ipPBS). In FIG. 4 each mouse represents a group of at least 10 mice. The evaluation of therapeutic effect of NP/IFN-b was made with a dose of IFN-b of 50.000 IU for any administration route or formulation. Since the association efficiency of human IFN-b to the system of NPs composed of CS/SBE^-CD is at least 85% (Table 3), the synthesis of NP/IFN-b was performed with a volume containing 57.500 IU of human IFN-b (15% excess) to ensure the effective association of 50.000 IU of the cytokine to NPs. The excess, not associated, was eliminated by NPs washing. For nasal administrations, a volume of 20 mI_ was used and for the ip administration a volume of 200 mI_.

The therapeutic effectiveness of NP/IFN-b and the specific administration route was evaluated by the recovery of the clinical severity of EAE mice during two weeks of treatment, according the following clinical score:

0= no clinical symptoms

1= partial loss of tail tone

2= total loss of tail tone (flaccid tail) 3 = partial paralysis of the hind legs

4 = total paralysis of the hind legs

5 = moribund (immobile animal, it does not consume water or food, weight loss over 20%, breathing difficulties) and must to undergo euthanasia

6 = death The therapeutic effect NP/IFN-b containing 50,000 IU IFN-b and daily administrated to EAE mice was evaluated. The FIG. 5 shows the progression of the daily clinical score during 2 weeks of intranasal treatment with: NP/IFN-b (inNP/IFN- b), naked IFN-b (inlFN-b), blank NPs (inNP), or with systemic treatment by intraperitoneal route of naked IFN-b (iplFN-b) or PBS (ipPBS). The treatment with ipPBS, iplFN-b, inNP, inlFN-b and inNP/IFN-b included 17, 12, 20, 16 and 18 animals, respectively. Each symbol of FIG. 5 represents the average ± standard error of three series of independent experiments. Table 4 shows values and statistical significance of p-value for the comparison between inNP/IFN-b group and the rest of the other treated groups shown in FIG. 5. The Mann Whitney U test nonparametric (two-tail) was used.

Table 4. P-values for the comparison between the EAE group mice daily treated with inNP/IFN-b and other treatments

The results shown in FIG. 5 and Table 4 indicate that the treatment with inNP/IFN-b is significantly better than any of the other treatments. Treatment with inNP/IFN-b results in a marked and sustained drop of the initial clinical score, which at the end of the treatment reaches an average of 57% of recovery of clinical severity (FIG. 5). Comparatively, the systemic (iplFN-b) or nasal (inlFN-b) administration of IFN-b decreased the initial clinical score in 18% and 29%, respectively. By contrast, the ip administration of PBS reduces the initial clinical score by only 2.34%, which represents the spontaneous variation of remission due to immunization conditions. Statistical analyzes show that the reduction in clinical score observed with the treatment of inNP/IFN-b is significantly greater when is compared individually with any other treatment (Table 4).

Taken together, these results suggest that the greater effectiveness of the NP/IFN-b would be due to: 1 ) the selected administration route; 2) the protection offered by the NP of CS/CD; and 3) an optimal release of the active.

Example 7. Analysis of NP/IFN-b posology on the therapeutic response in EAE mice

The therapeutic effect of the decrease in the frequency and dose of administration of inNP/IFN-b was determined hereunder. The experimental groups are the same as those shown in figure 4 and the procedure is the same described for figure 5. In some experiments the concentration of 50,000 IU of IFN-b was maintained and the frequency of in or ip administration was diminished from 24 h to 48 h or 72 h. In another experiment the dose decreased to 25,000 IU of IFN-b every 72 h and in another condition the dose of IFN-b decreased to 5,000 of IFN-b every 24 h (Table 5).

Table 5: Scheme of dose and frequency of NP/IFN-b administration in EAE mice

According to the different administration scheme (5/24, 25/72, 50/72, 50/48 and 50/24 IU x 1000/h), it is estimated that the total administered dose of IFN-b per week in treated animals may reach a decrease of up to 90% in relation to a daily treatment of 50,000 IU (Table 6).

Table 6: Total administered dose of Interferon b per week of treatment

The therapeutic improvement induced by the different dose and dosing frequencies of inNP/IFN-b and its controls, either by nasal or systemic route, are shown in FIG. 6. This figure shows the progression of the daily clinical score of EAE mice during two weeks of intranasal treatment with: NP/IFN-b (inNP/IFN-b), naked IFN-b (inlFN-b), blank NPs (inNP), or with systemic treatment by intraperitoneal route of naked IFN-b (iplFN-b) or PBS (ipPBS). Treatments were administered in a dose/frequency scheme (IU x 1000/hrs.) of 5/24 (FIG. 6A), 25/72 (FIG. 6B), 50/72 (FIG. 6C) y 50/48 (FIG. 6D). As a reference, the results from FIG. 6 obtained with an administration scheme 50/24 (FIG. 6E) have been included. Each symbol in the curves of the figure represents the average of 8-12 animals per group and their respective standard errors in two series of independent experiments. Additionally, associated with each graphic of clinical progression, P-value and statistical significance is attached for the comparison between the inlFN-b group and the remaining treatment groups. A nonparametric Mann Whitney U test two-tailed was used.

The obtained results show that at the end of the treatment period, the administration of inNP/IFN-b produces a significant disease amelioration reaching 42%, 52%, 53% and 56% of clinical recovery upon administration scheme of 25/72, 50/72, 50/48 and 50/24 (IU x 1000/hr.), respectively (FIG. 6, B-E). The results of the treatment with inNP/IFN-b show that it is still possible to keep a significant therapeutic effect in a 25/72 scheme; i.e., even though the dose of IFN-b is reduced by 50% and the administration frequency declines up to 33% in relation to the daily treatment of 50,000 IU of IFN-b.

Only in the treatment with a daily dose of 5,000 IU of inNP/IFN-b significant therapeutic effects were not observed compared to IFN-b administered in naked form by ip or in route (FIG. 6A).

In addition, it was observed that in any administration scheme of 25/72, 50/72, 50/48 or 50/24 (IU x 1000/hr.) the therapeutic effect is significantly better than naked IFN-b administered by ip or in route (FIG. 6, B-E). In FIG. 7 is shown the analysis of the variation of cumulative clinical score in response to the treatment with inNP/IFN-b at the end of the two weeks of treatment (FIG. 7 A) or per each week of treatment (FIG. 7B). Each bar represents the average value and its respective standard error in the variation of the clinical score of 8 to 12 animals. Letters a and b over each column indicate groups with statistical difference (a and b) with a value of P<0.05 (^*) or P<0.01 (^**), after using a nonparametric Mann Whitney U test (two-tail).

The results show that there is a tendency to enhance the clinical recovery as the dose of encapsulated IFN-b increases. The best therapeutic effect was observed with inNP/IFN-b treatment of 50,000 IU every 48 h (FIG 7 A), which is equivalent to weekly administration of 200 IU IFN-b (Table 6). However, a reduction of about 38% of weekly administration of the amount of cytokine (scheme of 25,000 IU every 72 h, Table 6,) was enough to produce similar therapeutic effects (FIG. 7A).

The therapeutic effect of the treatment with inNP/IFN-b is expressed mainly during the first week of administration, with a decrease in the effect of clinical recovery during the second week. This decrease was only significant for the condition 5/24h. This effect was not observed in the 50/24h treatments (FIG. 7B).

The results exposed herein show that it is possible to improve the current conditions of the treatment with IFN-b by using nanoencapsulated IFN-b in a structure of CS and CD, in terms of producing a significant therapeutic effect with a lower dose of the immunomodulatory active and a lower frequency of administration. Furthermore, these characteristics would make easier to design treatments with a lower cost and higher satisfaction for the patient by reducing the discomfort related to a frequent systemic administration of this therapeutic active.

Claims

1. A nanoparticle containing an interferon, wherein the nanoparticle comprises said interferon encapsulated in chitosan and a cyclodextrin.

2. The nanoparticle according to claim 1 , wherein the interferon is selected from the group consisting of interferon type I, interferon type II and interferon type III.

3. The nanoparticle according to claim 2, wherein the interferon is interferon beta (IFN-b).

4. The nanoparticle according to any preceding claim, wherein the cyclodextrin has negative charge and the electrical charges ratio between chitosan and the cyclodextrin is between 0.25 and 1.40.

5. The nanoparticle according to claim 4, wherein the electrical charges ratio between chitosan and the cyclodextrin is between 0.70 and 1.30.

6. The nanoparticle according to claim 5, wherein the electrical charges ratio between chitosan and the cyclodextrin is between 0.75 and 1.25.

7. The nanoparticle according to any preceding claim, wherein the cyclodextrin is sulfobutylether- -cyclodextrin.

8. The nanoparticle according to any preceding claim, wherein said nanoparticle has a size between 150 to 300 nm.

9. The nanoparticle according to any preceding claim, wherein the interferon is pegylated.

10. A pharmaceutical composition comprising an interferon, wherein the composition comprises nanoparticles as defined in any preceding claim and a pharmaceutically acceptable excipient.

1 1 . The pharmaceutical composition according to claim 10, wherein the composition is formulated for intranasal administration.

12. The pharmaceutical composition according to claim 10 or claim 1 1 , wherein the composition comprises a further active therapeutic agent.

13. The pharmaceutical composition according to claim 12, wherein the active therapeutic agent comprises ribavirin, boceprevir and/or telaprevir.

14. The pharmaceutical composition according to any of claims 10 to 13, for use in medicine.

15. The pharmaceutical composition according to any of claims 10 to 13, for use in treatment of an autoimmune disorder, cancer or an infectious disease.

16. The pharmaceutical composition according to any of claim 10 to 15, for use in intranasal administration to a subject.

17. An inhaler, nebulizer or spray device comprising the pharmaceutical composition according to any of claims 10 to 16.

18. A pharmaceutical combination, comprising (i) the pharmaceutical composition according to claim 10 or claim 1 1 and (ii) a further active therapeutic agent, for simultaneous, separate or sequential administration to a subject.

19. The pharmaceutical combination according to claim 18, wherein the active therapeutic agent comprises ribavirin, boceprevir and/or telaprevir.

20. The pharmaceutical combination according to claim 18 or claim 19, for use in treatment of an autoimmune disorder, cancer or an infectious disease.

21. A method of treating an autoimmune disorder, cancer or an infectious disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a pharmaceutical composition according to any of claims 1 to 13.

22. A method according to claim 21 , wherein the autoimmune disorder is multiple sclerosis.

23. A method according to claim 21 , wherein the cancer is leukemia.

24. A method according to claim 21 , wherein the infectious disease is hepatitis A, B or C.