RU2797114C2 - Nanoparticle and pharmaceutical composition for treatment of eye diseases or cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to ophthalmology and oncology, and is intended for the treatment of eye and oncological diseases. For the treatment of eye diseases or cancer, a nanoparticle is used, including a solid core containing a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, and when the said solid core is coated, it is coated with a non-ionic polymer. In other embodiments, the said nanoparticle and pharmaceutical compositions are provided containing a group of the said nanoparticles and an excipient, pharmaceutically acceptable carrier or excipient.

EFFECT: use of the group of inventions provides effective treatment of cancer and eye diseases achieved due to the controlled delivery of monoclonal antibodies while maintaining their integrity and activity.

25 cl, 22 dwg, 7 tbl, 14 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to nanoscale drug delivery systems, more specifically to nanoparticles containing an albumin matrix and monoclonal antibodies for use in the treatment of cancer and eye diseases.

BACKGROUND OF THE INVENTION

Monoclonal antibodies are an interesting group of glycoproteins for use in various therapeutic areas such as the treatment of cancer, autoimmune and chronic inflammatory diseases, and the treatment of transplant rejection. There are currently 30 monoclonal antibodies approved by regulatory authorities (FDA and EMEA).

One of these monoclonal antibodies is bevacizumab, immunoglobulin G (immunoglobulin G, IgG), which acts on VEGF-A (vascular endothelial growth factor) and includes four major isoforms of VEGF. It was approved by the US Food and Drug Administration (FDA) in 2004 for use as a first-line therapy for metastatic colorectal cancer and subsequently also approved for the treatment of other cancers such as non-small cell lung cancer or metastatic breast cancer. glands. Recently, bevacizumab has been introduced for the treatment of eye conditions, including corneal or retinal neovascularization, diabetic retinopathy, and age-related macular degeneration.

Topical application to the surface of the eye is a common way to administer drugs. However, defense mechanisms (lacrimation, lacrimation and lacrimation) reduce drug bioavailability by rapidly removing the composition. In the past few years, intravitreal injection of bevacizumab has been found to be a very effective treatment option for wet age-related macular degeneration, proliferative diabetic retinopathy, and choroidal neovascularization. Short-term results indicate that intravitreal bevacizumab is well tolerated and is associated with improved visual acuity, reduced retinal thickness, and angiographic bleed in most patients. However, multiple injections and follow-up visits are required to achieve and maintain improvement in visual acuity. This entails a high risk of complications such as endophthalmitis, as well as recurring pain, anxiety, and discomfort associated with inserting needles into the eye. In addition, the half-life of intravitreal bevacizumab is only about 3 days.

In the case of cancer treatment, current treatment strategies typically involve invasive procedures, including the use of chemotherapy catheters to shrink the tumor prior to surgical removal. Research programs to improve the effectiveness of cancer therapy have led to a significant improvement in patient survival, however, problems associated with toxic side effects and a poor quality of life for patients remain relevant.

Thus, there is a need to develop an efficient drug delivery method that will make the delivery of bevacizumab, as well as other monoclonal antibodies, less invasive and longer for the treatment of cancer and eye diseases.

In this regard, nanoparticles are a suitable vehicle for drug administration and show promising results in the fields of ophthalmology as well as cancer therapy. A wide range of materials can be used to prepare such nanoscale delivery systems. For example, the monoclonal antibody bevacizumab has been incorporated into PLGA nanoparticles for the treatment of age-related macular degeneration [Hao et al., American Association of Pharmaceutical Scientists (AASP) Annual Meeting and Exposition, Los Angeles, California, November 2009; Li, F. et al., The Open Ophthalmology Journal, 2012, 6, 54-58], as well as for the treatment of retinal and choroidal neovascularization [Pan CK et al., J. Ocul. Pharmacol. Ther., 2011, 27(3), 219-224; Varshochian, R. et al., European Journal of Pharmaceutical Sciences, 2013, 50, 341-352].

However, natural biopolymers are preferred over synthetic materials. In this respect, human serum albumin has been widely used to prepare nanoparticles for drug delivery due to the fact that they are biocompatible, biodegradable, non-toxic and non-immunogenic. Albumin nanoparticles have attracted considerable attention due to their high drug-binding capacity and good tolerability without serious side effects.

In recent years, a large number of different physicochemical processes have been proposed for the production of albumin nanoparticles, including thermal gelation, emulsification, and desolvation (coacervation). In any case, desolvation-based procedures seem to be the most popular due to their simplicity and reproducibility. However, newly produced nanoparticles are unstable and require an additional step of physical, chemical or enzymatic stabilization to extend the half-life in the aquatic environment and/or prevent the formation of protein macroaggregates.

In general, one of the most popular strategies for stabilizing albumin nanoparticles is albumin crosslinking. Elzoghby et al. [Journal of Controlled Release, 2012, 157, 168-182] uses various methods to obtain albumin nanoparticles and their use as active drug delivery systems. Particular attention is paid to the issue of the instability of nanoparticles in an aqueous medium, for the solution of which their crosslinking is provided using glutaraldehyde, a chemical crosslinking agent widely known in the art. Lohcharoenkal W. et al. [BioMed Research International, 2014] also considers the instability of albumin nanoparticles when they are dissolved or coagulated to form a separate phase if they are in an uncrosslinked state. In a publication by Llabot et al. [19th International Symposium on Microencapsulation, 2013] describes albumin nanoparticles crosslinked with the Gantrez polymer that envelops bevacizumab, as well as their use in corneal vascularization.

Thus, crosslinking stabilizes the albumin nanoparticles and reduces enzymatic degradation as well as delivery of the active ingredient from the nanoparticle.

However, despite the high efficiency of glutaraldehyde in stabilizing nanoparticles, its use is questionable, mainly due to toxicity, which precludes application for in vivo delivery. Thus, the most complete removal of the cross-linking agent is important. In addition, glutaraldehyde can adversely affect the stability of biomacromolecules, in particular protein drugs, antibodies, and peptides in nanoparticles, because it reacts with functional groups in macromolecules (such as primary amine residues), causing a significant loss of their activity.

To address this important shortcoming, various strategies have been proposed to strengthen or stabilize newly formed albumin nanoparticles without the need for toxic reagents. Among other things, the stabilization of nanoparticles can be achieved by heat treatment, high hydrodynamic pressure or enzymatic crosslinking with genipin or transglutaminase.

A surface coating was also used to stabilize the albumin nanoparticles. For example, cationic polymers such as polylysine or polyetheliminine have been used to coat bovine serum albumin nanoparticles to improve their stability [Wang et al., Pharm. Res., 2008, 25(12), 2896-2909],

WO 2013/042125 describes the preparation of glutaraldehyde crosslinked bovine serum albumin nanospheres comprising bevacizumab and subsequent coating of said nanospheres with an ionic PLGA layer.

WO 2011/053803 also describes drug-encapsulated polymer-coated nanoparticles, such as bevacizumab, for the treatment of eye diseases.

Considering all of the above, appropriate delivery systems are required to maintain the integrity and activity of monoclonal antibodies and control their release from nanoparticles.

DISCLOSURE OF THE INVENTION

The present inventors have found that incorporation of monoclonal antibodies such as bevacizumab into an albumin matrix provides nanoparticles with high stability in aqueous solution, and surprisingly found that these nanoparticles do not require cross-linking or stabilization by other means, thus maintaining the three-dimensional structure and biological antibody activity after delivery. On the contrary, as stated in the experimental section below, the use of glutaraldehyde (the most common cross-linking agent used in the prior art to stabilize albumin nanoparticles) inactivates antibodies, thus making it impractical to use cross-linked nanoparticles to incorporate active ingredients of this type.

The authors also tested non-crosslinked nanoparticles coated with non-ionic polymers such as hydroxypropyl methyl cellulose phthalate (HPMC-P) and polyethylene glycol 35000 (polyethylenglycol 35,000, PEG35), as well as Eudagrit® S-100, and found that antibody integrity was also is saved.

In addition, according to the method described below, albumin nanoparticles bound to monoclonal antibodies can be obtained as a dry powder, ready to be dispersed and diluted by simply adding water or an aqueous solution.

In addition, the nanoparticles of the invention allow sustained release of monoclonal antibodies and are a drug delivery system of great interest for in vivo use according to biological activity data obtained in an animal model of corneal neovascularization.

In addition, biodistribution analyzes performed using albumin nanoparticles coated with non-ionic polymers indicate that they are able to concentrate in tumor tissues, making them very promising nanoparticle systems for releasing monoclonal antibodies into diseased tissues.

In vivo experiments have shown that the nanoparticles of the invention are capable of releasing monoclonal antibodies into tumor tissue due to the presence of a reduced serum concentration of said antibodies compared to administration of the same monoclonal antibodies in aqueous solution. In addition, a significant reduction in tumor volume is achieved.

Thus, the first aspect of the present invention relates to a nanoparticle for use in medicine, in which the specified nanoparticle contains a solid core, the specified solid core containing a non-crosslinked albumin matrix and monoclonal antibodies, and in which the monoclonal antibodies are distributed throughout the volume of the albumin matrix, the specified solid core is optional coated with non-ionic polymer.

The second aspect of the invention relates to a pharmaceutical composition containing:

a plurality of nanoparticles, said nanoparticles containing a solid core, said hard core containing a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is optionally coated with a non-ionic polymer;

excipient, pharmaceutically acceptable carrier or excipient.

In another aspect, the invention relates to said pharmaceutical composition of the invention for use in medicine.

Another aspect of the invention includes a nanoparticle for use in the treatment of ocular diseases, wherein said nanoparticle comprises a solid core, said solid core containing a non-crosslinked albumin matrix and monoclonal antibodies, and wherein the monoclonal antibodies are distributed throughout the volume of the albumin matrix, said solid core is optionally coated with a non-ionic polymer .

Another aspect of the invention includes a nanoparticle for use in cancer treatment, wherein said nanoparticle comprises a solid core, said solid core containing a non-crosslinked albumin matrix and monoclonal antibodies, and wherein the monoclonal antibodies are distributed throughout the volume of the albumin matrix, said solid core is optionally coated with a non-ionic polymer .

Finally, another aspect of the invention relates to a nanoparticle containing a solid core, said solid core containing a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said solid core is coated with a non-ionic polymer.

Below is the full scope of the claims of the present invention.

According to the present invention, the use of a nanoparticle for the treatment of eye diseases or cancer is claimed, comprising a solid core containing a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, while said solid core is optionally coated with a non-ionic polymer.

According to one of the embodiments of the invention, the specified solid core can be coated, and the composition of the nanoparticle does not contain any other polymeric coatings, except for a non-ionic polymer.

According to one embodiment of the invention, the weight ratio of monoclonal antibodies/albumin is from 0.01 to 0.5.

According to one embodiment of the invention, the non-ionic polymer/albumin weight ratio is from 0.02 to 5 by weight.

In one embodiment, the albumin is human serum albumin or bovine serum albumin.

In one embodiment, the monoclonal antibodies are selected from bevacizumab, ranibizumab, trastuzumab, cetuximab, and rituximab.

According to one embodiment of the invention, the non-ionic polymer is a water-soluble cellulose selected from hydroxyethylcellulose, hydroxy-n-propylcellulose, hydroxy-n-butylcellulose, hydroxypropylmethylcellulose, phthalate

hydroxypropylmethylcellulose and ethylhydroxyethylcellulose; starch; dextran; polyvinylpyrrolidone, a polyester selected from compounds under the trademark Eudagrit, or polyalkylene glycol.

According to one of the embodiments of the invention, the specified nanoparticle is obtained by a method containing:

a) obtaining an aqueous solution of albumin and monoclonal antibodies;

b) titration of the aqueous solution obtained in step a) to a pH of 4 to 5;

c) adding a desolvating agent to the aqueous solution obtained in step b) to obtain albumin/monoclonal antibody nanoparticles;

d) further incubation of the albumin nanoparticles/monoclonal antibodies obtained in step c) with a non-ionic polymer; and additionally

e) drying the nanoparticles by vacuum drying, spray drying or freeze drying.

According to one embodiment of the invention, the eye disease is selected from macular degeneration, corneal neovascularization or angiogenesis, iris neovascularization or angiogenesis, retinal neovascularization or angiogenesis, diabetic proliferative retinopathy, non-diabetic proliferative retinopathy, glaucoma, infectious conjunctivitis, allergic conjunctivitis, ulcers. keratitis, non-ulcerative keratitis, episcleritis , scleritis, diabetic retinopathy, uveitis, endophthalmitis, infectious processes and inflammatory processes.

In one embodiment, the cancer is breast cancer, lung cancer, pancreatic cancer, multiple myeloma, renal cell carcinoma, prostate cancer, melanoma, colon cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, cancer of the liver, kidneys and gastrointestinal tract, bladder cancer or squamous cell carcinoma.

The present invention also claims a nanoparticle comprising a solid core containing a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, said solid core being coated with a non-ionic polymer.

The present invention also claims a pharmaceutical composition comprising:

- a group of nanoparticles containing a solid core, including a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, while the specified solid core is optionally coated with a non-ionic polymer;

excipient, pharmaceutically acceptable carrier or excipient.

According to one embodiment of the invention, said hard core is coated, wherein it is coated with a non-ionic polymer.

According to one embodiment of the invention, the composition is an ophthalmic or anti-cancer pharmaceutical composition.

According to one embodiment of the invention, the nanoparticles are in the form of a dry powder.

According to one embodiment of the invention, the excipient, pharmaceutically acceptable carrier or excipient is suitable for oral, topical or parenteral administration.

In one embodiment, the albumin is human serum albumin or bovine serum albumin.

In one embodiment, the monoclonal antibodies are selected from bevacizumab, ranibizumab, trastuzumab, cetuximab, and rituximab.

According to one embodiment of the invention, the non-ionic polymer is a water-soluble cellulose selected from hydroxyethylcellulose, hydroxy-n-propylcellulose, hydroxy-n-butylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, and ethylhydroxyethylcellulose; starch; dextran; polyvinylpyrrolidone, a polyester selected from compounds under the trademark Eudagrit, or polyalkylene glycol.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Effect of the bevacizumab/albumin ratio on the payload of the obtained nanoparticles. Nanoparticles were obtained after incubation of monoclonal antibodies and protein for 10 minutes. Data are expressed as mean ± SD (n=3).

Figure 2. TEM photograph of bevacizumab-loaded nanoparticles (B-NP) of albumin nanoparticles.

Figure 3. A) Fourier transform IR spectrum of human serum albumin (HSA), glutaraldehyde (glutaraldehyde, GLU), a mechanical mixture of HSA and glutaraldehyde (glutaraldehyde, HSA-GLU) and nanoparticles cross-linked with glutaraldehyde (nanoparticles cross-linked with glutaraldehyde, NP-GLU). C) Fourier transform IR spectrum of human serum albumin (HSA), bevacizumab (bevacizumab, BEVA), mechanical mixture of HSA and bevacizumab (HSA-BEVA) and bevacizumab-filled nanoparticles (B-NP).

Figure 4. X-ray spectra of bevacizumab (BEVA), bevacizumab-filled nanoparticles (B-NP) and human serum albumin (HSA).

Figure 5. DTA thermograms: A) native human serum albumin (HSA) and bevacizumab (BEVA); C) mechanical mixture (PM) of human serum albumin (HSA), bevacizumab (BEVA) and albumin nanoparticles filled with bevacizumab (B-NP); C) native human serum albumin (HSA) and glutaraldehyde (GLU); D) mechanical mixture (PM) of human serum albumin (HSA), glutaraldehyde (GLU) and albumin nanoparticles crosslinked with glutaraldehyde (NP-GLU).

Figure 6. Dynamics of changes in the average size of empty nanoparticles crosslinked with glutaraldehyde (NP-GLU) and nanoparticles filled with bevacizumab (B-NP) after their dispersion in an aqueous solution at pH 7.4. Data are expressed as mean ± SD (n=3).

Figure 7. Bevacizumab release profile from human serum albumin nanoparticles after incubation in PBS (pH 7.4). Data are expressed as mean ± SD (n=3).

Figure 8. TEM micrograph of bevacizumab-loaded albumin nanoparticles pegylated with PEG35 (B-NP-PEG35).

наночастиц, наполненных бевацизумабом (B-NP);

наночастиц, наполненных бевацизумабом и покрытых PEG35 (B-NP-PEG35); (…) наночастиц, наполненных бевацизумабом и покрытых Eudagrit® S-100 (B-NP-S-100);

наночастиц, наполненных бевацизумабом и покрытых НРМС-Р (B-NP-HPMC-P). Данные выражены в виде среднего значения ± СО (n=3).Figure 9. Release profile of bevacizumab from albumin nanoparticles after incubation in PBS (pH 7.4)

nanoparticles filled with bevacizumab (B-NP);

nanoparticles filled with bevacizumab and coated with PEG35 (B-NP-PEG35); (…) nanoparticles filled with bevacizumab and coated with Eudagrit® S-100 (B-NP-S-100);

nanoparticles filled with bevacizumab and coated with HPMC-P (B-NP-HPMC-P). Data are expressed as mean ± SD (n=3).

Figure 10. Automated electrophoresis of nanoparticles on a microfluidic chip (L - length marker; 1 - empty nanoparticles coated with PEG35 (NP-PEG35); 2 - albumin nanoparticles filled with bevacizumab (B-NP); 3 - albumin nanoparticles filled with bevacizumab and coated PEG35 (B-NP-PEG35); 4 - human serum albumin (HSA); 5 - bevacizumab).

Figure 11. Comparison of in vivo SPECT-CT images of 99mTc-labeled B-NP (top row) and 99mTc-labeled B-NP-PEG35 (bottom row) after injection into the eye of Wistar rats. The images in each row correspond to the same animals examined at the time points indicated in the figure. Activity disappears 4-8 hours after injection into the eye, while in B-NP-PEG35 it persists in the eye for at least 8 hours.

Figure 12. Curve activity - time for the dynamics of the amount of radioactivity in different areas after injection of 99mTc-B-NP nanoparticles into the eye. Volumes of interest (VOI) were obtained for the zones indicated on the graphs, and the average amounts obtained for each VOI, the data are corrected for attenuation and plotted on the graph.

Figure 13. Comparison of in vivo SPECT-CT images of 99mTc-labeled B-NP (top row) and 99mTc-labeled B-NP-PEG35 (bottom row) after intravenous administration to Wistar rats. The images in each row correspond to the same animals examined at the time points indicated in the figure.

Figure 14. Schematic representation of a schedule showing corneal cauterization at 0 hours (Day 0) and first treatment 24 hours later (Day 1).

Figure 15. Photographs of the corneas of animals treated with: (A) saline [control (-)]; (B) Avastin® (4 mg/ml bevacizumab); (C) bevacizumab-loaded albumin nanoparticles (B-NP); (D) albumin nanoparticles filled with bevacizumab and coated with PEG 35,000 (B-NP-PEG35); (E): human serum albumin (HSA) solution; (F): EYLEA® (EYLEA); (G): dexamethasone (DEXA).

Figure 16. The area of the lesion as a percentage of the area of the cornea affected by the burn. There were no statistically significant differences between lesions in different groups. Data are expressed as mean ± SD (n=9).

Figure 17. The area of invasion (invasion area, IA), the proportion of the area of the cornea with the presence of vessels. Data are expressed as mean ± SD (n=9).

* p<0.01 - results of analysis of variance using Tukey's test significantly differ from control (-).

** p<0.01 - results of analysis of variance using Tukey's test are significantly different from BEVA.

*** p<0.005 - results of analysis of variance using Tukey's test are significantly different from B-NP.

Figure 18. Area of neovascularization normalized by lesion. Data are expressed as mean ± SD (n=9).

* p<0.01 - results of analysis of variance using Tukey's test significantly differ from control (-).

** p<0.005 - results of analysis of variance using Tukey's test are significantly different from BEVA.

Figure 19. Micrographs of sections of normal and neovascularized cornea treated with bevacizumab (e - epithelial layer; s - stroma; ac - anterior chamber; v - stromal microvessels). A) Micrograph of normal rat cornea showing intact epithelium (e), stroma containing regular parallel collagen plates with flattened keratocytes in between; B) Micrograph of the cornea of a rat from the group treated with albumin nanoparticles filled with bevacizumab (B-NP). The thickness of the cornea is within the normal range, intact epithelium and a slightly fuzzy and loose stroma with little infiltration. C) Micrograph of the cornea of a rat from the group treated with albumin nanoparticles filled with bevacizumab and coated with PEG35 (B-NP-PEG35). Normal epithelium, numerous stromal microvessels (v). The thickness of the cornea is within the normal range; D and E): micrographs of the cornea of rats from the group treated with bevacizumab. Retaining the structure and hypertrophied epithelium, which is separated from the stroma. Stroma thickening with numerous and randomly arranged fibroblasts (*); F) Micrograph of the cornea of a rat from the saline treated group. Severe corneal changes, central erosion and thickening. Severe fibrosis with large, irregular fibroblasts, moderate inflammatory infiltration, and neovascularization (v). Cyst formation (c) from epithelial cells, demonstrating an attempt at abnormal repair. Scale - 200 µm, i.e. 100x magnification.

Figure 20 Tumor/normal tissue ratios for leg and neck tumors in animals treated with PEG35 coated albumin nanoparticles (NP-PEG35). Values correspond to the average value for three animals obtained after 1 hour (blue bars) and 4 hours (red bars) after intravenous (IV) administration of nanoparticles labeled with a radioactive isotope.

Figure 21. Tumor volume (mm3). Data are expressed as mean ± SD (n≥6).

* p<0.05 - results of analysis of variance using Tukey's test significantly differ from saline.

** p<0.01 - results of analysis of variance using Tukey's test significantly differ from saline.

Figure 22. Bevacizumab serum concentration (µg/me) versus time (day).

IMPLEMENTATION OF THE INVENTION

As indicated above, the first aspect of the present invention relates to a nanoparticle for use in medicine, in which the specified nanoparticle contains a solid core, the specified solid core containing a non-crosslinked albumin matrix and monoclonal antibodies, and in which the monoclonal antibodies are distributed throughout the volume of the albumin matrix, the specified solid core optionally coated with a non-ionic polymer.

In a particular embodiment, the present invention relates to a nanoparticle for use in medicine, in which the specified nanoparticle contains a solid core, the specified solid core, consisting of a non-crosslinked albumin matrix and monoclonal antibodies, and in which the monoclonal antibodies are distributed throughout the volume of the albumin matrix, the specified solid core optionally coated with a non-ionic polymer.

In yet another particular embodiment, the present invention relates to a nanoparticle for use in medicine, wherein said nanoparticle consists of a solid core, said solid core containing an uncrosslinked albumin matrix and monoclonal antibodies, and wherein the monoclonal antibodies are distributed throughout the volume of the albumin matrix, said the solid core is optionally coated with a non-ionic polymer.

In another particular embodiment, the present invention relates to a nanoparticle for use in medicine, in which the specified nanoparticle consists of a solid core, the specified solid core, consisting of a non-crosslinked albumin matrix and monoclonal antibodies, and in which the monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is optionally coated with a non-ionic polymer.

The term "nanoparticle" as used herein refers to a colloidal system having a spherical or quasi-spherical shape and an average size of less than 1 µm. In a particular embodiment, the nanoparticle has an average size in the range of 100 to 900 nm, more preferably 150 to 800 nm, even more preferably 200 to 500 nm, and most preferably 200 to 400 nm.

By "average size" is meant the average diameter of a population of nanoparticles moving together in an aquatic environment. The average size of these systems can be measured by standard methods known to those skilled in the art and described, for example, in the experimental section below.

In the context of the present invention, the term "nanoparticle" refers to a coated nanosphere or nanosphere.

By "nanosphere" is meant a solid, uncrosslinked matrix of albumin or a continuous material of albumin, in which monoclonal antibodies are distributed throughout the volume of said matrix, so that there is no pronounced core/shell structure.

By "coated nanosphere" is meant a nanosphere as defined above in which a solid non-crosslinked matrix of albumin is coated with a non-ionic polymer.

Accordingly, the nanoparticles of the invention do not contain any other polymeric coatings other than non-ionic polymers.

In a preferred embodiment of the present invention, the nanoparticle is a nanoparticle in which, when coated on a hard core, the nanoparticle contains no other polymeric coatings other than non-ionic polymers. Although the nanoparticles of the invention do not require a polymer coating, the inventors have found that in the absence of any other polymer coatings other than non-ionic polymers in the composition of the nanoparticles, these particles show a beneficial effect compared to the same particles when using an ionic coating. For example, when the nanoparticles of the invention are coated with an ionic polymer, the particles exhibit a very rapid antibody release profile (explosive release).

Thus, when the nanoparticles used in the invention are not coated with a nonionic polymer, said nanoparticles should be considered as nanospheres according to the above definition, while when the nanoparticles used in the invention are coated with a nonionic polymer, said nanoparticles should be considered as coated nanospheres according to the above definition.

Unlike the nanoparticles used in the prior art, in which the albumin matrix is cross-linked or otherwise stabilized, the nanoparticles used in the invention are characterized by the presence of a solid core of an uncrosslinked albumin matrix, which is understood as an organizational structure or configuration due to local interactions between albumin and monoclonal antibodies. Thus, within the scope of the present invention, the nanoparticles form solid matrix systems.

In this regard, the term "solid core" refers to a solid, non-crosslinked matrix-type structure in which albumin forms a continuous structure in which monoclonal antibodies are distributed, preferably evenly distributed, throughout the volume of the matrix.

So, the solid core of the nanoparticles used in the invention does not have differentiated external and internal structures, and therefore the monoclonal antibodies are distributed, more preferably evenly distributed, throughout the entire volume of the albumin matrix, and not enclosed or limited in its central cavity.

In a particular embodiment of the invention, the nanoparticle used in the invention is a nanosphere as defined above. More specifically, in said nanoparticles, the solid core is not coated with a non-ionic polymer. As described throughout this document, and also shown in the examples, the nanoparticles of the invention are stable and do not require encapsulation. In the context of the present invention, the term "stable" refers to increasing the level of stability of the particles so that the particles can be used in medicine without degradation of the material. Thus, in a specific embodiment, the nanoparticle of the present invention is a stable nanoparticle with or without an additional polymer coating.

In another particular embodiment of the invention, the nanoparticle used in the invention is a coated nanosphere according to the above definition, i.e. contains or consists of a solid core of a solid matrix of albumin or a solid material of albumin, in which monoclonal antibodies are distributed throughout the volume of said matrix and in which the solid core is coated with a non-ionic polymer.

An additional aspect of the invention relates to a nanoparticle containing a hard core, said hard core containing a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is coated with a non-ionic polymer.

More specifically, the invention also relates to a nanoparticle containing a hard core, said hard core consisting of a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is coated with a non-ionic polymer.

Also, in particular, the invention relates to a nanoparticle consisting of a solid core, said solid core containing a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said solid core is coated with a non-ionic polymer.

Even more specifically, the invention also relates to a nanoparticle consisting of a solid core, said solid core consisting of a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said solid core is coated with a non-ionic polymer.

Albumen.

As used herein, the term "albumin" refers to a family of globular, negatively charged proteins, the most common of which are serum albumins. All proteins of the albumin family are water-soluble, sparingly soluble in concentrated salt solutions, and subject to heat denaturation. Albumins are widely distributed in blood plasma and differ from other blood proteins in that they are not glycosylated.

The overall structure of albumin is characterized by several long coils, allowing them to maintain a relatively static shape, which is very important for blood pressure regulation.

In a specific embodiment, the albumin is serum albumin. Serum albumin is produced in the liver and dissolved in blood plasma, being the most abundant protein in mammals.

More preferably the serum albumin is human serum albumin (HSA) or bovine serum albumin (BSA), even more preferably the serum albumin is human serum albumin.

Human serum albumin is encoded by the ALB gene, while other mammalian forms, such as bovine serum albumin, are chemically similar.

Human serum albumin has a molecular weight of approximately 65,000 Da and consists of 585 amino acids. The amino acid sequence of HSA contains 17 disulfide bridges, one free thiol (Cys34) and one tryptophan (Trp214).

monoclonal antibodies.

The term "monoclonal antibody" (mAb or moAb) as used herein refers to antibodies or antibody fragments produced by a single clone of B-lymphocytes or a single cell, called a hybridoma, that secretes only one type of antibody molecule. Monoclonal antibodies are produced by methods known to those skilled in the art, for example by generating hybrid antibody producing cells by fusion of an antibody producing cell and a myeloma or other self-penetrating cell line.

Monoclonal antibodies have monovalent affinity in that they bind to a single epitope (the part of the antigen that is recognized by the antibodies). For almost any substance, it is possible to obtain monoclonal antibodies that specifically bind to this substance.

For the purpose of the present invention, the monoclonal antibodies to be incorporated into the nanoparticle albumin matrix must have affinity for at least one target in ocular or cancerous tissue, or must have affinity for the tissue itself. For example, the target may be a receptor associated with an eye disease or cancer, or a protein associated with an eye disease or cancer.

In a particular embodiment of the invention, the monoclonal antibodies are selected from bevacizumab (Avastin®), which inhibits the function of a natural protein called vascular endothelial growth factor (VEGF), which stimulates the formation of new blood vessels; ranibizumab (Lucentis®), which provides strong binding to VEGF-A; trastuzumab (Herceptin®), which recognizes the HER-2 receptor overexpressed in solid tumors; cetuximab (Erbitux®), which recognizes EGFR receptors, and rituximab (MabThera®), which recognizes CD20.

In a preferred embodiment of the present invention, one or more anti-VEGF antibodies (or antibody fragments) are selected to be incorporated into an albumin matrix, thus targeting vascular endothelial growth factor (VEGF) directly. Thus, in a preferred embodiment, the monoclonal antibodies are selected from bevacizumab and ranibizumab, more preferably bevacizumab.

In yet another preferred embodiment of the present invention, one or more anti-VEGF R2 antibodies (or antibody fragments) are selected to be incorporated into an albumin matrix, thereby targeting cells such as retinal pigment epithelial cells expressing vascular endothelial growth factor 2 (VEGF R2).

Examples of anti-VEGF R2 monoclonal antibodies include, but are not limited to, the Avasl2al monoclonal antibody clone and the 2C3 monoclonal antibody.

Overexpression of the VEGF receptor and VEGFR2 by epithelial cells, such as retinal pigment epithelial cells, is associated, for example, with age-related macular degeneration (AMD).

In yet another preferred embodiment of the present invention, the monoclonal antibody is a targeted ophthalmic preparation, i. antibodies specific to an antigen produced by, or associated with, eye tissue directly affected by the eye disease.

In a particular embodiment of the invention, the weight ratio of antibody/albumin is from 0.01 to 0.5, more preferably from 0.01 to 0.2. Nanoparticles with a monoclonal antibody/albumin weight ratio of less than 0.01 have not been observed to be stable over time.

non-ionic polymer.

The term "non-ionic polymer" as used herein refers to a hydrophilic polymer that does not have a net charge under the conditions of nanoparticle production. In addition, said non-ionic polymer must be biodegradable, i. degradable when used in vivo, as well as biocompatible, i.e. essentially non-toxic or not harmful to living tissues or living systems with which it comes into contact.

Examples of suitable non-ionic polymers for use in the present invention are polyvinyl alcohol; polyvinylpyrrolidone; polyallyl alcohol; polyvinyl methyl ether; polyvinyl acetal; polyalkylene alcohol; a polysaccharide optionally substituted with at least one alkyl group, hydroxyalkyl group, alkoxyalkyl group, or a combination of two or more of these groups; polyesters; polyamides, polyurethanes and polyesters.

Preferred polysaccharides include, but are not limited to, xanthan gums, guar gums, starches, cellulose, dextran, and a combination of two or more of the above.

Starches include, for example, corn starch and hydroxypropylated starch.

Cellulose includes, for example, alkylcelluloses such as C1-C6alkylcelluloses, including methylcellulose, ethylcellulose, and n-propylcellulose; substituted alkylcelluloses, including hydroxy-C1-C6-alkylcelluloses and hydroxy-C1-C6-alkyl-C1-C6-alkylcelluloses such as hydroxyethylcellulose, hydroxy-n-propylcellulose, hydroxy-n-butylcellulose, hydroxypropylmethylcellulose and ethylhydroxyethylcellulose.

In a particular embodiment of the invention, the nonionic polymer is selected from polysaccharide, polyvinylpyrrolidone, polyester and polyalkylene glycol. Preferably, the non-ionic polymer is a water-soluble cellulose selected from hydroxyethylcellulose, hydroxy-n-propylcellulose, hydroxy-n-butylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, and ethylhydroxyethylcellulose; starch; dextran; a polyester selected from compounds under the trademark Eudagrit; or a polyalkylene glycol such as polyethylene glycol or polypropylene glycol.

In yet another particular embodiment of the invention, the non-ionic polymer is selected from hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, starch, dextran 70, Eudagrit® NM; Eudagrit® NE, polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG). The polyethylene glycol is preferably PEG-10000, PEG-20000 or PEG-35000 according to its molecular weight.

It is preferred when the non-ionic polymer is selected from hydroxypropyl methylcellulose phthalate and PEG 35,000. It is preferred when the non-ionic polymer is selected from hydroxypropyl methylcellulose phthalate and PEG 35,000. Even more preferably, the non-ionic polymer is PEG 35,000.

A non-ionic polymer is used as a coating for albumin nanoparticles, giving them increased stability and generally providing an increase in the amount of monoclonal antibodies that can be loaded into the nanoparticle. It has been demonstrated that the presence of a coating does not significantly affect the physical properties of the nanoparticle. Depending on the nature of the coating, only a slight increase or decrease in size and zeta potential is possible.

In a particular embodiment of the invention, the weight ratio non-ionic polymer/albumin is from 0.02 to 5 (w/w), more preferably from 0.05 to 2 (w/w).

In yet another particular embodiment of the invention and in a further embodiment of the invention, the nanoparticles used in the present invention further comprise a compound to protect the albumin matrix during the drying of the nanoparticles or the drying of the suspension containing the nanoparticles using conventional methods, for example by spray drying, further - "protective agent". The specified protective agent is not included in the composition of the solid matrix of nanoparticles, acting as a bulk-forming agent that promotes efficient drying of nanoparticles while maintaining their structure. Almost any compound that meets these characteristics can be used as a protective agent. In a particular embodiment, said protectant is a saccharide.

Non-limiting illustrative examples of protectants that can be used in the context of the present invention include lactose, mannitol, sucrose, maltose, trehalose, maltodextrin, glucose, sorbitol, etc., as well as substances with prebiotic characteristics, such as, for example, oligofructose. , pectin, inulin, oligosaccharides (eg, galactooligosaccharides, breast milk oligosaccharides), lactulose, dietary fiber, etc., and any combination thereof. In a particular embodiment, the protectant is selected from lactose, mannitol, sucrose, maltose, trehalose, maltodextrin, glucose, sorbitol, and combinations thereof. Preferably the protectant is sucrose. If the nanoparticles used in the invention include a protective agent, the weight ratio of the albumin matrix and the protective agent can vary over a wide range; however, in a particular embodiment, the weight ratio of protectant/albumin is 1:0.1-1.5, typically 1:0.5-4, preferably about 1:1.

In some embodiments of the present invention, the nanoparticles used in the invention comprise at least one imaging agent that enables image-guided targeted drug delivery by pre-visualizing the location of the particles before, during, and after drug delivery. A wide variety of imaging agents are suitable for binding to the surface of nanoparticles, including but not limited to fluorescent imaging agents (such as indocyanine green, cyanine 5, cyanine 7, cyanine 9, fluorescein, and green fluorescent protein), radioactive isotope-labeled imaging agents (such as agents containing iodine-124, 99mTc), and magnetic resonance imaging agents (such as gadolinium contrast agents).

The nanoparticles used in the invention can deliver monoclonal antibodies in a controlled process. Such control is able to ensure the delivery of monoclonal antibodies over a long period. For example, it is contemplated that delivery may take place over a period of 1 to 30 days. In addition to being capable of delivering a drug in a controlled manner, the nanoparticles can be configured to use a variety of particle detection and imaging options before, during, and after drug delivery.

The nanoparticles used in the present invention may further contain a second monoclonal antibody or drug to provide combination therapy.

Said drug product includes, for example, other proteins such as calcitonin, insulin or cyclosporine A.

The second monoclonal antibody can be any of the above, being, for example, bevacizumab (Avastin®), ranibizumab (Lucentis®), trastuzumab (Herceptin®), cetuximab (Erbitux®) or rituximab (MabThera®).

Method for obtaining nanoparticles.

The nanoparticles used in the present invention can be obtained by precipitating proteins (albumin and monoclonal antibodies) in an aqueous medium before purification and drying.

This method contains:

a) obtaining an aqueous solution of albumin and monoclonal antibodies;

b) titration of the aqueous solution obtained in step a) to a pH of 4 to 5;

c) adding a desolvating agent to the aqueous solution obtained in step b).

This method is based on a desolvation technology in which an aqueous solution of albumin and monoclonal antibodies is slowly desolvated by slowly adding, such as dropwise addition, a desolvating agent (typically an organic solvent such as ethanol, acetone or THF) under constant stirring conditions, temperature and pH.

In a particular embodiment of the invention, the albumin used in the preparation of the aqueous solution in step a) is human serum albumin or bovine serum albumin, more preferably human serum albumin.

In yet another preferred embodiment of the present invention, the monoclonal antibodies used in preparing the aqueous solution in step a) are selected from bevacizumab (Avastin®), ranibizumab (Lucentis®), trastuzumab (Herceptin®), cetuximab (Herceptin®) and rituximab ( Mabthers®). More preferably, the monoclonal antibodies are bevacizumab or ranibizumab, even more preferably bevacizumab.

A solution of albumin and monoclonal antibodies can be obtained by conventional methods known to those skilled in the art, for example by adding albumin and monoclonal antibodies to an aqueous solution.

Albumin and monoclonal antibodies are preferably mixed at room temperature, ie. at a temperature of from 18°C to 25°C, preferably from 20°C to 22°C.

The amount of albumin that can be added to the aqueous solution can vary over a wide range of values, however, in a specific embodiment of the present invention, the amount of albumin added to said aqueous solution is from 0.1% to 10% (w/v ), preferably 0.5% to 5% (w/v), even more preferably 1% to 2% (w/v).

Similarly, the amount of monoclonal antibodies that can be added to an aqueous solution can vary over a wide range of values, however, in a specific embodiment of the present invention, the amount of monoclonal antibodies added to said aqueous solution is from 0.005% to 1% (in /o), preferably from 0.01% to 0.5% (w/o), even more preferably from 0.01% to 0.4% (w/o).

In a particular embodiment of the invention, albumin and monoclonal antibodies are added to the aqueous solution so that the weight ratio of monoclonal antibodies/albumin is from 0.01 to 0.5, more preferably from 0.01 to 0.2.

In a preferred embodiment of the present invention, the aqueous solution of albumin and monoclonal antibodies are subjected to homogenization, for example by mixing.

Step b) of the method for producing nanoparticles includes lowering the pH of an aqueous solution containing albumin and monoclonal antibodies to a slightly acidic pH. This allows the nanoparticles to be deposited in the next step of this method. This can be done by adding an acidic component to the aqueous solution obtained in step a), such as 1M HCl.

In a particular embodiment of the invention, the aqueous solution is incubated for at least 10 minutes at room temperature.

In step c) of the method for producing nanoparticles, a desolvating agent is added to the aqueous solution obtained in step b).

In a preferred embodiment of the present invention, the addition of the desolvating agent to the aqueous solution obtained in step b) is carried out with stirring.

In yet another preferred embodiment of the present invention, said desolvating agent is an organic solvent selected from ethanol and tetrahydrofuran (THF), more preferably ethanol.

The desolvating agent is slowly added to the aqueous solution with stirring. More preferably, the desolvating agent is added dropwise to the aqueous solution of albumin and monoclonal antibodies while stirring the resulting mixture.

In a preferred embodiment of the present invention, said addition of the desolvating agent is carried out under an inert gas atmosphere, such as a nitrogen atmosphere.

After adding a desolvating agent to an aqueous solution of albumin and monoclonal antibodies under the above conditions, i.e. at room temperature and stirring, spontaneously formed nanoparticles of the invention. In a particular embodiment of the invention, these nanoparticles are in suspension in the medium in which they were obtained.

Thus, the method of the invention makes it possible to obtain a homogeneous dispersion of nanoparticles using a simple desolvation process that provides solid nanospheres with a matrix type structure in which monoclonal antibodies are distributed throughout the volume of the albumin matrix.

Thus, the nanoparticles obtained by this method can be considered as self-assembling nanoparticles that form spontaneously due to local interactions between monoclonal antibodies and albumin upon the addition of a desolvating agent.

The method for producing nanoparticles may contain an additional purification step, for example using filtration, centrifugation and ultracentrifugation methods.

Similarly, said method may include an additional step of drying the obtained nanoparticles to obtain nanoparticles of the invention in the form of a powder. This form of release of these nanoparticles ensures their stability and, in particular, is additionally useful for their end use in pharmaceutical compositions.

In a preferred embodiment of the present invention, the nanoparticles obtained in step c) or after purification are subjected to drying by conventional methods, such as vacuum drying or preferably spray drying or freeze drying (lyophilization) to dry the nanoparticles.

In a particular embodiment of the invention, this drying step, in particular when carried out using spray drying or lyophilization, comprises adding a protective agent to the formed nanoparticles. This protectant protects the nanoparticle during the drying process and is, for example, a saccharide.

Non-limiting illustrative examples of saccharides that can be used as protectants in the context of the present invention include lactose, mannitol, sucrose, maltose, trehalose, maltodextrin, glucose, sorbitol, etc., as well as polysaccharides with prebiotic characteristics, such as, for example, oligofructose, pectin, inulin, oligosaccharides (eg, galactooligosaccharides, breast milk oligosaccharides), lactulose, dietary fiber, etc., and mixtures thereof. In a particular embodiment, the protectant is selected from lactose, mannitol, sucrose, maltose, trehalose, maltodextrin, glucose, sorbitol, and combinations thereof. If the nanoparticles include a protective agent, it is added in an appropriate amount; however, despite the fact that the weight ratio of the matrix of nanoparticles and the protective agent can vary over a wide range, in a specific embodiment, the weight ratio of albumin / protective agent is 1:0.1-1.5, usually 1:0.5-4 , preferably about 1:1.

Spray drying can also be used to dry nanoparticles. For this purpose, a suspension containing nanoparticles and a protective agent is placed in a spray dryer with controlled processing conditions (inlet air temperature, outlet air temperature, air pressure, sample feed rate, suction and air flow). The person skilled in the art can select the most suitable processing conditions in each case.

This method provides the possibility of obtaining nanoparticles in the form of a dry powder, which remains stable over long periods of time under controlled conditions or environmental conditions and can be easily incorporated into various final solid and liquid products.

Because the nanoparticles are prepared prior to the addition of the protectant, it does not form conjugates or complexes with the albumin matrix.

In another particular embodiment of the present invention, when the nanoparticles used in the invention are coated with a non-ionic polymer, said coated nanoparticles can be obtained by incubating albumin nanoparticles with monoclonal antibodies obtained after steps a) to c) of the method described above with a non-ionic polymer .

In a particular embodiment of the invention, the non-ionic polymer may be any of the above. It is preferred that said polymer is selected from hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, starch, dextran 70, Eudagrit® NM, Eudagrit® NE, polyvinylpyrrolidone and polyethylene glycol (PEG). More preferably, the non-ionic polymer is selected from hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and PEG 35000.

In a particular embodiment of the invention, the non-ionic polymer/albumin weight ratio is from 0.02 to 5, more preferably from 0.05 to 2.

In yet another particular embodiment of the invention, the incubation of the nanoparticles in the non-ionic polymer is less than 1 hour, more preferably less than 45 minutes.

Pharmaceutical composition.

The nanoparticles described above have the ability to retain monoclonal antibodies and protect them during processing and storage, as well as before final delivery to a given biological area. Thus, the deactivation of monoclonal antibodies after incorporation into various end products (eg, pharmaceutical compositions or cosmetic compositions) is completely prevented or significantly reduced. Experimental studies have shown that monoclonal antibodies retain their integrity in the albumin matrix.

In addition, the nanoparticles of the invention allow sustained release of monoclonal antibodies that also retain their full biological activity and thus constitute a drug delivery system of great interest for in vivo use according to animal model biological activity data. corneal neovascularization. Experiments performed in vivo have shown that albumin nanoparticles with monoclonal antibodies provide a significant reduction in the area of the eye with corneal vascularization compared with the introduction of the same monoclonal antibodies in free form.

In addition, biodistribution analyzes performed using the nanoparticles of the invention indicate that they are able to concentrate in tumor tissues, making them very promising nanoparticle systems for releasing monoclonal antibodies into affected cancer tissues.

Thus, in another aspect, the invention relates to a pharmaceutical composition containing a plurality of nanoparticles as defined above, in the form of a suspension or dry powder, and an excipient, a pharmaceutically acceptable carrier or excipient.

The characteristics of the nanoparticles have already been defined above and are incorporated herein by reference.

In a particular embodiment of the invention, the nanoparticles contained in the pharmaceutical composition of the invention are in the form of a dry powder.

While any route of administration of the pharmaceutical composition may be used in the context of the present invention, preferably the pharmaceutical composition is administered to a human or animal orally, topically or parenterally, more preferably by intravenous administration, intra-arterial administration, intra-pulmonary administration, intraocular administration, intramuscular administration, intradermal or subcutaneous administration, oral administration or inhalation.

More preferably, the pharmaceutical composition contains an excipient or carrier suitable for oral, topical or parenteral administration.

Depending on the particular route of administration, the pharmaceutical composition may be formulated as tablets, dragees, capsules, sachets, granules, powders, suspensions, emulsions, anhydrous or hydrated topical formulations and solutions.

Pharmaceutically acceptable carriers or excipients are well known to those skilled in the art and are commonly available. Preferably, the pharmaceutically acceptable carrier or excipient is chemically inert to the active composition and each of its components and is not associated with adverse side effects or toxicity under the conditions of use.

In some particular embodiments of the present invention, the pharmaceutical composition is configured to be used as a delivery system for transporting a drug for oral, topical, parenteral, or intravenous administration into a patient's circulatory system.

Compositions suitable for oral administration include liquid solutions, such as an effective amount of nanoparticles or compositions containing them, dissolved in diluents such as water or saline: capsules, sachets, tablets, lozenges, each containing a predetermined amount of nanoparticles , powders, suspensions in appropriate liquids and emulsions.

Compositions for topical application include aqueous ophthalmic solutions or suspensions, ophthalmic ointment, ophthalmic implant, or any other composition capable of delivering nanoparticles to the outer surface of the eye. Preferably, the topical composition is an ophthalmic solution or suspension containing nanoparticles for use as liquid drops. Any of the above compositions may include a suitable solvent, preservatives, and other pharmaceutically acceptable excipient commonly used in ophthalmic compositions.

Parenteral compositions typically contain from 0.5% to 25% by weight of nanoparticles in solution. Said compositions may be prepared in single-dose or multi-dose sealed containers such as ampoules and vials and may be stored in a freeze-dried (lyophilized) state requiring the addition of a sterile liquid carrier such as water for injection just prior to use.

Diseases to be treated.

As stated above, albumin and monoclonal antibody nanoparticles have been shown to be a very promising drug delivery system for the treatment of eye diseases and cancer.

Thus, another aspect of the present invention relates to nanoparticles or compositions as defined above for the treatment of eye diseases.

In a particular embodiment of this aspect of the invention, the nanoparticle contains a hard core, said hard core containing a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is not coated with a polymer. More specifically, said hard core is not coated with a non-ionic polymer.

In another particular embodiment of this aspect of the invention, the nanoparticle contains a hard core, said hard core, consisting of a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is not coated with a polymer. More specifically, said hard core is not coated with a non-ionic polymer.

In another particular embodiment of this aspect of the invention, the nanoparticle contains a solid core, said solid core consists of a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said solid core is not coated with a polymer. More specifically, said hard core is not coated with a non-ionic polymer.

In another particular embodiment of this aspect of the invention, the nanoparticle contains a hard core, said hard core, consisting of a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is not coated with a polymer. More specifically, said hard core is not coated with a non-ionic polymer.

In another aspect of the present invention, it also relates to a method for treating an eye disease, said method comprising administering to a patient in need of such treatment nanoparticles or a composition containing nanoparticles as described above.

In another aspect, the present invention also relates to the use of nanoparticles or compositions containing nanoparticles, as described above, for the preparation of a drug for the treatment of eye diseases.

The specified composition can be administered to the patient orally, topically or by intraocular injection. In a preferred embodiment, the composition is administered topically, for example by ocular mucosal application or intravitreal injection.

Thus, in a preferred embodiment of the present invention, when nanoparticles are administered for the treatment of an eye disease, said nanoparticles are administered as part of a topical or injectable pharmaceutical composition such as described above.

In another particular embodiment of the invention, the eye disease to be treated is selected from macular degeneration, corneal neovascularization or angiogenesis, iris neovascularization or angiogenesis, retinal neovascularization or angiogenesis, diabetic proliferative retinopathy, non-diabetic proliferative retinopathy, glaucoma, infectious conjunctivitis, allergic conjunctivitis, ulcerative keratitis, non-ulcerative keratitis, episcleritis, scleritis, diabetic retinopathy, uveitis, endophthalmitis, infectious processes and inflammatory processes.

Another aspect of the present invention relates to nanoparticles or compositions as defined above for the treatment of cancer.

In a particular embodiment of this aspect of the present invention, the nanoparticle contains a hard core, said hard core containing a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is coated with a non-ionic polymer.

In another particular embodiment of this aspect of the present invention, the nanoparticle contains a hard core, said hard core, consisting of a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is coated with a non-ionic polymer.

In another particular embodiment of this aspect of the present invention, the nanoparticle contains a solid core, said solid core consists of a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said solid core is coated with a non-ionic polymer.

In another particular embodiment of this aspect of the present invention, the nanoparticle contains a hard core, said hard core, consisting of a non-crosslinked albumin matrix and monoclonal antibodies, in which monoclonal antibodies are distributed throughout the volume of the albumin matrix, said hard core is coated with a non-ionic polymer.

In another aspect of the present invention, it also relates to a method of treating cancer, said method comprising administering to a patient in need of such treatment nanoparticles or a composition containing nanoparticles as described above.

In another aspect of the present invention, it also relates to the use of nanoparticles or compositions containing nanoparticles, as described above, for the preparation of a drug for the treatment of cancer.

In a preferred embodiment of the invention, said composition is administered to the patient parenterally, for example by intravenous, intra-arterial, intramuscular or subcutaneous injection.

Thus, in a preferred embodiment of the invention, when nanoparticles are administered for cancer treatment, said nanoparticles are administered as part of a parenteral composition such as described above.

In yet another particular embodiment of the invention, the cancer to be treated includes, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Examples of cancers to be treated by administering the nanoparticles include, for example, breast cancer, lung cancer, pancreatic cancer, multiple myeloma, renal cell carcinoma, prostate cancer, melanoma, colon cancer, colorectal cancer, kidney cancer, cervical cancer. uterus, ovarian cancer, liver, kidney and gastrointestinal cancer, bladder cancer or squamous cell carcinoma.

In some embodiments of the invention, the nanoparticles used in the present invention, or compositions containing them, can be administered with a second therapeutic composition and/or a second drug for the treatment of eye diseases or cancer.

The frequency of administration of the composition and the second composition or the second drug may be adjusted during the course of treatment. In some embodiments of the invention, the first and second drugs are administered simultaneously, sequentially or in parallel. When administered separately, the nanoparticle composition and the second composition may be administered at different frequencies or intervals.

Alternatively, the nanoparticles used in the present invention may contain a second monoclonal antibody or drug to provide combination therapy.

Said drug includes, for example, other proteins such as calcitonin, insulin, or cyclosporin A.

The second monoclonal antibody can be any of the above, being, for example, bevacizumab (Avastin®), ranibizumab (Lucentis®), trastuzumab (Herceptin®), cetuximab (Erbitux®) or rituximab (MabThera®).

Examples.

The following abbreviations are used in the examples below.

BEVA - bevacizumab.

B-NP - albumin nanoparticles filled with bevacizumab.

HSA - human serum albumin.

PM - mechanical mixture.

NP-Glu - albumin nanoparticles cross-linked with glutaraldehyde.

B-NP-GLU - albumin nanoparticles cross-linked with glutaraldehyde and filled with bevacizumab.

NP-PEG35 - albumin nanoparticles coated with polyethylene glycol 35000.

B-NP-PEG35 - albumin nanoparticles filled with bevacizumab and coated with polyethylene glycol 35000.

B-NP-HPMC-P - albumin nanoparticles filled with bevacizumab and coated with hydroxypropyl methylcellulose phthalate.

B-NP-S100 - albumin nanoparticles filled with bevacizumab and coated with Eudagrit S-100.

Materials.

Human serum albumin or HSA (fraction V, 96-99% pure), polyethylene glycol 35000 (PEG35) and 25% aqueous glutaraldehyde (GLU) were obtained from Sigma (Madrid, Spain).

Bevacizumab (Avastin®) was purchased from Roche (Spain). Hydroxypropyl methylcellulose K100 LV (HPMC; MM 164000) manufactured by Ashland Chemical Hispania (Spain). Avastin® is supplied as a concentrate for solution for infusion in single-use vials with a nominal content of 100 mg of bevacizumab in a 4 ml vial or 400 mg of bevacizumab in a 16 ml vial (25 mg/ml concentration). Hydroxypropylmethylcellulose phthalate (HPMC-R) was purchased from Acros Organic (Spain). The Micro BCA protein assay kit was purchased from Pierce (Thermo Fisher Scientific Inc., Illinois, USA). The Shikari Q-beva enzyme immunoassay kit used to detect bevacizumab was purchased from Matriks Biotech (Turkey). Acetone was purchased from Prolabo, VWR International Ltd (England) and stannous chloride dihydrate and pure alcohol were purchased from Rapgeas Pharma (Spain). Isoflurane was purchased from Braun and T-69 Intervet Painless Killer from Schering-Plough Animal Health. The technetium-99 t pertechnetate eluate was obtained from a Drytec® 99Mo-99mTc generator purchased from General Electric.

The following instruments were used for physicochemical studies: Thermo / Nicolet 360FT-IR spectrometer (E.S.P. Thermo Fisher Scientific, USA), Bruker Axs D8 Advance diffractometer (Germany), Mettler calorimeter was used for thermogravimetric analysis (TG) and differential scanning colorimetry (DSC). Toledo dsc822e with Mettler Toledo TSO 801 RO sampling robot and Julabo FT900 cooler, and for elemental analysis - LECO CHN-900 elemental analyzer (Michigan, USA).

For studies using radiolabeling and biodistribution studies, the following instruments were used: Symbia SPECT-CT, Siemens Medical Systems (Germany), AtomLab 500 activity calibrator, Biodex (USA), LKB Pharmacia gamma counter.

Physicochemical characteristics of nanoparticles (size, zeta potential and morphology).

The size and zeta potential of nanoparticles were determined using a Zeta Plus instrument (Brookhaven Inst. Corp., USA). The diameter of the nanoparticles was determined after dispersion in ultrapure water (1/10) and measured at 25°C by dynamic light scattering at an angle of 90°C. The zeta potential was determined as follows: 200 μl of the sample was dissolved in 2 ml of a solution of 1 mm KCl with pH adjusted to 7.4.

The morphological characteristics of the nanoparticles were studied using scanning electron microscopy (SEM) under a Zeiss DSM940 digital scanning electron microscope (Oberkochen, Germany). For this purpose, the samples were dispersed in water and centrifuged at 27,000 g for 20 minutes at 4°C to remove the cryoprotectant. Then the microspheres were placed on glass plates glued to metal rods with double-sided adhesive tape and dried. They were sputtered with a 4 nm thick platinum/palladium layer using a Cressington 208HR planetary/tilt stage ion sputterer equipped with an MTM-20 thickness control. SEM was performed using a LEO 1530 instrument (LEO Electron Microscopy Inc, Thornwood, NY, USA) at a voltage of 1-3 kV and a filament current of about 0.5 mA.

Exit.

The amount of HSA converted to nanoparticles (yield) was determined by counting HSA forming nanoparticles using a Micro BCA kit. Briefly, 10 mg of nanoparticles were weighed and dispersed in 10 ml of ultrapure water and centrifuged at 15,000 rpm for 15 minutes at 4° C. (Rotor 3336, Biorage Heraeus, Hanau, Germany). Then the microspheres were ground with 1 ml of 0.02 N NaOH, and 200 μl of this solution was transferred into a 96-well microplate with further use for analysis of Micro BCA protein in a spectrophotometer at 562 nm.

The selectivity of the kit was determined using controls containing other excipients and preparations (glutaraldehyde, PEG 35,000, HPMC or bevacizumab) to determine possible distortions in the determination of albumin. Data analysis was performed using the following formula:

where Wliof is the amount of HSA converted into nanoparticles,

a Wix is the amount of HSA used to obtain nanoparticles.

Calculation of drug payload in nanoparticles.

The amount of antibodies contained in the albumin nanoparticles was assessed using enzyme immunoassay (Shikari Q-BEVA). For this purpose, 10 mg of nanoparticles were weighed and dispersed in 1 ml of water. The suspension was centrifuged for 10 minutes at 10,000 rpm (Rotor 3336, Biofuge Heraeus, Hanau, Germany). The supernatant was removed. Then the nanoparticles were dissolved in 1 ml of 0.02 N NaOH. 200 μl of the resulting solution was transferred to a 96-well microplate coated with human vascular endothelial growth factor (VEGF) and specific ELISA for bevacizumab was performed (test procedure Q-Beva, Shikari Q-Beva, Matriks Biotek).

Each sample was analyzed in triplicate and calculated using standard curves ranging from 0.1 to 100 μg/ml (r2>0.993). The limits of detection and quantification were 0.1 µg/mL and 100 µg/mL, respectively (r2>0.993).

Bevacizumab loading (DL) and incorporation efficiency (EE) were calculated using the following formulas:

where Won is the amount of included bevacizumab,

Wtot - the total amount of the drug used,

a Wnch is the mass of nanoparticles.

IR-Fourier spectroscopy.

The molecular structure of the HSA nanoparticles was studied using FT-IR spectroscopy. IR spectra of samples dispersed in 1% sample in KBr disks were recorded on a NICOLET Fourier IR spectrometer (Thermo / Nicolet 360FT-IR E.S.P. Thermo Fisher Scientific, USA). The samples were scanned in the range from 4000 to 400 cm-1. The shooting conditions were as follows: 8.0 resolution and 40x sample scan. Data were analyzed using OMNIC software (Thermo Fisher Scientific, USA).

X-ray studies.

X-ray studies were performed to study the distribution of crystallographic planes and the variability of the degree of crystallinity of the polymer matrix in different samples of nanoparticles. For this purpose, samples in the form of a powder on a metal plate were placed in a diffractometer (Bruker Axs D8 Advance, Germany) and measured in the range of angles up to 360° at room temperature. The diffraction patterns were analyzed using the Diffrac.Suite program.

Thermal analysis.

The response of various nanoparticles to temperature changes was investigated using thermal analysis (thermogravimetric analysis (TGA) combined with differential thermal analysis (DTA)). Behavioral changes were analyzed upon thermal exposure of HSA functional groups during the formation of nanoparticles. Thermal analyzes were performed on a TGA/sDTA 85 le Mettler Toledo thermogravimetric and differential thermal analysis analyzer. Thermograms were obtained by heating about 5-10 mg of the sample in a perforated aluminum crucible with a scanning frequency of 10°C/min from 25 to 250°C. Thermal analyzes were performed under still air using N2 (20 mL-min-1) as purge gas. Measurements were performed in triplicate.

elemental analysis.

Elemental analysis (C, H, O, and N) of nanoparticles was performed to confirm the relationship of various stabilizing additives in HSA nanoparticles on a LECO CHN-900 elemental analyzer (Michigan, USA). Briefly, 1 mg of each sample was tested in triplicate, expressed as a percentage (% by weight) with SD ± 0.4. This method demonstrates changes in the composition of oxygen, hydrogen or nitrogen of albumin (HSA) when bound to other components (glutaraldehyde, PEG35, HPMC-P or bevacizumab).

In vitro release study.

In vitro release studies of bevacizumab-loaded albumin nanoparticles were performed in PBS (pH 7.4). Eppendorf tubes containing 10 mg of each nanoparticle composition were dispersed in a total volume of 1 ml of PBS placed in Eppendorf tubes and placed in a shaking bath at 37°C with constant stirring at a frequency of 60 cycles/min (Unitronic 320 OR, Selecta, Madrid, Spain). Eppendorf tubes were taken at various time intervals and centrifuged for 10 minutes at 10,000 rpm (Rotor 3336, Biofuge Heraeus, Hanau, Germany). Supernatants were analyzed for bevacizumab content using a specific ELISA (Shikari Q-Beva, Matriks Biotek). Release profiles were expressed as cumulative release as a percentage and plotted over time.

In addition, based on the release profiles, the kinetics were evaluated using an exponential model based on the Korsmeier-Peppas equation (equation 4):

where Q is the percentage of the release of the drug at the point in time

t,

K is a constant that takes into account the structural and geometric characteristics of the device under study, n is the diffusion exponent, which is usually used as an indicator of the drug transport mechanism in the dosage form.

A value of n≤0.43 indicates that the release of the drug occurs according to Fickian diffusion, while a value of n≥0.85 indicates that the release of the drug is dominated by the erosion mechanism. For values of 0.43<n<0.85, release is designated as abnormal, implying that drug release is due to a combination of diffusion and erosion [Gao Y., et al., In Vitro Release Kinetics of Antituberculosis Drugs from Nanoparticles Assessed Using a Modified Dissolution Apparatus . Biomed Research International 2013].

Example 1 Preparation of human serum albumin nanoparticles filled with bevacizumab. Effect of the bevacizumab/HSA ratio on the physicochemical properties of the obtained nanoparticles.

Bevacizumab-loaded nanoparticles were prepared using an aqueous protein precipitation procedure prior to purification and drying.

For this purpose, 100 mg of HSA and a variable amount of bevacizumab (BEVA) (1-20 mg) were dissolved in 5-10 ml of water for injection, and then the solution was titrated to pH 4.1-4.4 using 1 M HCL. The mixture was incubated at room temperature for 10 minutes. Nanoparticles were obtained by continuous addition of 16 ml of ethanol used as a desolvatant with constant stirring (500 rpm) at room temperature. The obtained nanoparticles were purified by two different methods - ultracentrifugation and ultrafiltration. In the first, the nanoparticles were purified twice by centrifugation at 21,000 g for 20 minutes at 4°C (Sigma 3K30, Osterode am Harz, Germany) and redispersion of the microspheres in the original volume in water. In the second, the nanoparticles were purified by ultrafiltration through a polysulfone membrane filter element with a pore size of 50 kDa (Medica SPA, Italy). The nanoparticles were then lyophilized in a Genesis 12EL (Virtis, NY, USA) after redispersion or 5% sucrose aqueous solution. These compositions are human serum albumin nanoparticles filled with bevacizumab particles without any additional stabilization, hereinafter referred to as B-NP compositions.

To incorporate the monoclonal antibodies into the nanoparticles, two key parameters were determined, the bevacizumab/albumin ratio and the incubation time for both formulations before nanoparticle formation. Summary Table 1 shows the main physicochemical properties of the obtained nanoparticles when changing the ratio of monoclonal antibodies/protein. At a low bevacizumab/albumin ratio (eg 0.01), the nanoparticles were not stable over time. For ratios above 0.01, the resulting nanoparticles were stable with an average size close to 300 nm and a negative surface charge of about -15 mV.

Similarly, the estimated yield of the process was about 80%. The figure 1 shows the effect of the ratio of bevacizumab/albumin on the content of monoclonal antibodies.

Consistent with these results, the amount of bevacizumab contained in the nanoparticles increased with increasing BEVA/HSA ratio. All of these experiments were performed after 10 minutes of incubation between monoclonal antibodies and protein. It is noteworthy that no significant differences in the physicochemical properties of nanoparticles were observed with an increase in this parameter.

Table 1. Effect of the bevacizumab/albumin ratio on the physicochemical properties of the obtained nanoparticles. The incubation time between bevacizumab and albumin is 10 minutes. Data are expressed as mean ± SD (n=3).

Example 2. Effect of cross-linking of nanoparticles filled with bevacizumab with glutaraldehyde on their physicochemical properties.

Due to the fact that human serum albumin nanoparticles in the absence of bevacizumab did not remain stable and disappeared immediately after formation, control nanoparticles filled with bevacizumab were obtained by cross-linking with 12.5 μg of glutaraldehyde in ethanol (300 μl), hereinafter - compositions B-NP-GLU. For this purpose, newly formed nanoparticles filled with bevacizumab were incubated for 5 minutes with glutaraldehyde before purification and lyophilization.

Summary Table 2 shows the main physicochemical properties of untreated HSA nanoparticles (without additional stabilization procedures) and control nanoparticles obtained by crosslinking with glutaraldehyde. By incorporating bevacizumab into albumin nanoparticles, stable nanoparticles with a high antibody content were obtained. It is noteworthy that the incorporation efficiency, calculated as the content of active monoclonal antibodies in nanoparticles, was close to 90% at a bevacizumab content of about 13%. When cross-linking nanoparticles filled with bevacizumab with glutaraldehyde, the obtained nanoparticles were slightly smaller than those obtained without a chemical cross-linking agent. However, treatment of the nanoparticles with glutaraldehyde inactivated the monoclonal antibodies and ELISA quantitation showed very low antibody levels.

Example 3. Characterization of nanoparticles filled with bevacizumab.

TEM.

In FIG. 2 shows the morphology of bevacizumab-filled (B-NP) nanoparticles that are spherical in shape and have an uneven surface.

IR-Fourier spectroscopy.

IR spectroscopy makes it possible to evaluate the manifestation of conformational changes in the secondary structure of a protein. In FIG. 3 shows the FT-IR spectra of bevacizumab-filled nanoparticles compared to individual spectra of monoclonal antibodies, protein, and their mechanical mixture.

The IR spectra of proteins show a large number of amide bonds, which reflect various vibrations of peptide groups. Such signals: amide band I in the range from 1600 to 1700 cm-1 (mainly the C=O region) and amide band II at 1550 cm-1 (C-N region associated with N-H bending vibrations) - were used as confirmation of the presence of such a chemical connection and its direct relation to the secondary structure of the protein. However, amide band I is more sensitive to changes in protein secondary structure than amide II. Thus, changes in the frequency and strength of these signals are confirmation of the interaction with the protein.

In this case, and due to the fact that the nanoparticles are formed by two different proteins (albumin and bevacizumab), slight changes in signal frequency were observed corresponding to the amide I peak (1642 for HSA and 1645 cm-1 for B-NP).

As a control, nanoparticles crosslinked with glutaraldehyde were also studied. In this case, a slight frequency shift of amide I (1642-1645 cm-1) was also found due to the interaction between glutaraldehyde and albumin.

X-ray studies.

In FIG. 4 shows X-ray spectra of human serum albumin, bevacizumab, and bevacizumab-loaded nanoparticles. In all cases, such spectra show an amorphous structure.

Thermal analysis.

Thermal analyzes were performed to determine the reaction between the functional groups of HSA and bevacizumab. In FIG. Figure 5 shows thermograms of native albumin (HSA) and bevacizumab (BEVA) (A), nanoparticles with bevacizumab (B-NP) and mechanical mixture (P.M.) of albumin and bevacizumab (B), native albumin (HSA) and glutaraldehyde (GLU) (C ), nanoparticles cross-linked with glutaraldehyde (NP-GLU), and a mechanical mixture (P.M.) of albumin and glutaraldehyde (D).

Thermograms show that native albumin has an exothermic effect around 30° C., which corresponds to a reversible transition, and a second thermal effect associated with an endothermic glass transition.

The absence of an exothermic signal in nanoparticles corresponding to albumin may be due to the combination of albumin with protein (BEVA) and a crosslinking agent (glutaraldehyde). Thus, bevacizumab and albumin form a complex.

elemental analysis.

Table 3 shows elemental analysis data for human serum albumin, bevacizumab, albumin nanoparticles crosslinked with glutaraldehyde, and albumin nanoparticles filled with bevacizumab. Bevacizumab exhibits significantly lower carbon and nitrogen content compared to human serum albumin. On the contrary, the oxygen content in monoclonal antibodies is about two times higher than in albumin. Similarly, bevacizumab-filled (B-NP) nanoparticles exhibited a lower nitrogen content and a higher oxygen content compared to native albumin.

Example 4. Stability of nanoparticles filled with bevacizumab.

The stability of nanoparticles was evaluated in ultrapure water. Samples were dispersed in purified water and stored at room temperature for 3 days. Stability was evaluated at various time intervals by measuring the size, polydispersity coefficient, and zeta potential of the nanoparticles.

After dispersion in water (at pH adjusted to 7.4), the bevacizumab-filled nanoparticles were stable for at least 24 hours (FIG. 6). Their behavior was similar to that observed for empty nanoparticles crosslinked with glutaraldehyde (Fig. 6). Similarly, the experiment did not affect the polydispersity coefficient (PC) of B-NP. So, at t=0, the CP was 0.19±0.01, and after 24 hours this parameter was 0.16±0.03 (data not shown).

Example 5 In vitro release of bevacizumab from nanoparticles.

In FIG. 7 shows the in vitro release profile of bevacizumab from albumin nanoparticles in PBS. This profile was characterized by an initial burst of release of about 23% of incorporated antibodies in the first 5 minutes, followed by a slow controlled release over the next 24 hours. At the end of the experiment, a release of about 40% of the included bevacizumab was achieved. The explosive release may be associated with antibodies adsorbed on the surface of the nanoparticles.

Example 6 Preparation and characterization of coated albumin nanoparticles filled with bevacizumab.

Incorporation of bevacizumab into human serum albumin nanoparticles coated with various compounds was performed using a four-step procedure. In particular, non-ionic HPMC-P and PEG35 were selected to evaluate their ability to bind to bevacizumab-filled nanoparticles. Eudragit S-100 ionic coating was also used.

The first stage was to obtain nanoparticles in an aqueous medium. Thereafter, the nanoparticles were coated by simple incubation in an aqueous medium. The third stage was used to purify the obtained nanoparticles, which were dried at the final stage.

First stage. 100 mg HSA and a variable amount of bevacizumab (1-20 mg) were dissolved in 5-10 ml water for injection, and then the solution was titrated to pH 4.1-4.4 using 1 M HCl. The mixture was incubated at room temperature for 10 minutes. Nanoparticles were obtained by continuous addition of 16 ml of ethanol used as a desolvatant with constant stirring (500 rpm) at room temperature.

Second phase. To coat the newly obtained nanoparticles filled with bevacizumab, one of the following compounds was added: PEG 35000, hydroxymethylpropyl cellulose phthalate, or Eudragit S-100.

As a control, bevacizumab-loaded nanoparticles were stabilized by cross-linking with 12.5 μg of glutaraldehyde in ethanol (300 μl) for 5 minutes as described above.

Third stage. The resulting nanoparticles were purified. Used two different procedures - ultracentrifugation and ultrafiltration. In the first, the nanoparticles were purified twice by centrifugation at 21,000 g for 20 minutes at 4°C (Sigma 3K30, Osterode am Harz, Germany) and redispersion of the microspheres in the original volume in water. In the second, the nanoparticles were purified by ultrafiltration through a polysulfone membrane filter element with a pore size of 50 kDa (Medica SPA, Italy).

Fourth stage. At the final stage, the nanoparticles were lyophilized in a Genesis 12EL device (Virtis, NY, USA). When used as a purification method by ultracentrifugation, the microspheres after the final centrifugation step were dispersed in a 5% aqueous sucrose solution. When using ultrafiltration, the microspheres were also redispersed in a 5% aqueous sucrose solution.

Table 4 summarizes the main physicochemical properties of these nanoparticles. In general, the amount of bevacizumab contained was similar in all cases and close to 14%. However, upon incubation of bevacizumab-filled nanoparticles with various coating auxiliaries, nanoparticles with altered physicochemical properties were obtained. Thus, bevacizumab-filled and PEG35-coated (B-NP-PEG35) nanoparticles exhibited medium sizes and a negative zeta potential similar to untreated bevacizumab-filled (B-NP) nanoparticles. On the contrary, when B-NP was incubated with HPMC-P, the average size of the resulting nanoparticles increased significantly compared to B-NP. When incubated with ionic Eudragit® S-100, the resulting nanoparticles showed smaller sizes and an increased negative zeta potential compared to B-NP. Using SEM, it was found that albumin nanoparticles have a spherical shape and a smooth surface.

Table 4 Physicochemical characteristics of bevacizumab incorporated into coated albumin nanoparticles. Data are expressed as mean ± SD (n=3). KP - coefficient of polydispersity. Coating agents: S-100 (ionic Eudragit® SI00), HPMC-P (hydroxypropyl methylcellulose phthalate), PEG35 (polyethylene glycol 35000). SA/protein ratio - coating substance/albumin ratio. B-NP-GLU - albumin nanoparticles cross-linked with glutaraldehyde and filled with bevacizumab. B-NP - raw albumin nanoparticles filled with bevacizumab.

Morphological examination (Fig. 8) of the nanoparticles filled with bevacizumab and coated with PEG35 (B-NP-PEG35) shows that they have a spherical shape with an uneven surface and a uniform distribution.

Example 7 In Vitro Release of Bevacizumab from Coated Nanoparticles.

In FIG. 9 shows the in vitro release profile of bevacizumab from albumin nanoparticles coated with two non-ionic polymers (PEG35 and HPMC-P) and ionic Eudragit® SI00 in PBS at pH 7.4. For PEG35 coated nanoparticles (B-NP-PEG35), the profile was similar to that observed for untreated nanoparticles (B-NP) with the difference that at the end of the experiment the amount of bevacizumab released was higher than for B-NP. In any event, these PEGylated nanoparticles exhibited a biphasic release profile characterized by an initial burst during the first 5 minutes of about 22% followed by a more sustained and slower release step lasting at least 24 hours. The burst release was about 22% and may be associated with antibodies adsorbed on the surface of the nanoparticles. The biphasic process, ignoring the first 5 minutes of explosive release, was adjusted using the Korsmeier-Peppas equation to a diffusion profile (n=0.54; R2=0.994). During the diffusion phase, bevacizumab release plateaued at 48% after the first two hours.

For nanoparticles coated with HPMC-P (B-NP-HPMC-P), the amount of bevacizumab released during the first 60 minutes was about 40%. Thereafter, a constant rate of release of the remaining antibodies was observed. However, in this case, the release rate of bevacizumab was higher than for B-NP or B-NP-PEG35. So, after 8 hours of incubation, HPMC-R coated nanoparticles released about 100% of the bevacizumab contained in them.

Unlike non-ionic polymer coated nanoparticles, Eudragit® S-100 (B-NP-S-100) coated nanoparticles showed an immediate release profile.

Example 8 Integrity of bevacizumab after incorporation into albumin nanoparticles.

To confirm the results obtained using the ELISA kit for quantitative determination of the content of bevacizumab in albumin nanoparticles, the integrity of antibodies (bevacizumab) included in various nanoparticles was analyzed using an automated electrophoresis system on a microfluidic chip Experion™ (Bio Rad, USA). Samples were evaluated under non-reducing conditions and under reducing conditions using 2-mercaptoethanol. The obtained data were processed using the Experion System software.

Nanoparticles were weighed and ground with 1 ml of 0.005 N NaOH. The concentration of various solutions was about 400 ng protein/µl (linear dynamic range of the test - 5-2000 ng/µl). Free protein samples (albumin and bevacizumab) were used as controls. All of these samples were evaluated as received or after treatment with β-mercaptoethanol and heating. The samples were then processed according to the protocol of the Experion System Pro260 assay kit (Bio-Rad Lab., USA). After loading the samples and controls into the chip, they were analyzed using the Experion™ automated electrophoresis system (Bio Rad, USA).

Results were obtained as densitometric bands on a simulated gel electrophoretic image. Each band corresponds to a separate sample. The Experion software identifies the various size peaks and expresses them in kilodaltons (kDa) on the system control band.

The analysis results are shown in FIG. 10. In lane 5, the bevacizumab band is most pronounced around 150 kDa. A similar band was observed for lanes 2 (B-NP) and 3 (B-NP-PEG35). Similarly, lanes 1-3 also show clear bands corresponding to albumin (lane 4).

Example 9 Study of biodistribution of nanoparticles administered through the eyes to Wistar rats.

Biodistribution study of radiolabeled nanoparticles in Wistar rats for in vivo SPECT-CT visualization.

Nanoparticles were radiolabeled with 99mTc by reduction of 99mTc-pertechnate with tin chloride in a manner described elsewhere. Briefly, 20 μl of a solution of stannous chloride dihydrate in water for injection with a final tin concentration of 0.02 mg/ml was added to 9 mg of lyophilized nanoparticles, followed by the addition of 60 μl of the 99mTcO4 eluate to the pre-reduced tin. 4 µl of a radiolabeled suspension of nanoparticles (5 MBq) was mixed with 0.6 mg of the composition of unlabeled nanoparticles, the resulting mixture was carefully injected into the right eye of Wistar rats anesthetized with isoflurane.

Ophthalmic administration to Wistar rats for in vivo SPECT-CT imaging.

Animals were anesthetized for one hour to avoid active removal of the suspension from the eye, then awakened and SPECT-CT images were taken at six different time points from 05:00 to 17:30. since the introduction of nanoparticles.

For imaging studies, animals were anesthetized prior to each study with isoflurane and placed in the prone position on a Symbia T2 Truepoint SPECT-CT system (Siemens). Images were obtained using a matrix of 128×128, 7 images/s; CT was used at 110 mAs and 130 kV, 130 images 3 mm thick. Image fusion was performed using Syngo MI Applications TrueD software. The images were processed and quantified using the built-in software package. Quantitative values were obtained by automatically constructing an isocontour in three planes for selected zones to obtain volumetric regions of interest (VOI), for which the average values of the quantities were determined.

In FIG. 11 and 12 show the biodistribution of B-NP and B-NP-PEG35 after administration to the eye as eye drops. The radioactivity associated with the nanoparticles persisted in the eye for at least 4 hours for B-NP and 8 hours for B-NP-PEG35, although it slowly disappeared from the injection site, moving into the gastrointestinal tract. The movement of radiolabeled nanoparticles through the pharynx of the animal is observed in the upper leftmost image in FIG. 11. Intensity of SPECT images in FIG. 11 was adjusted to the maximum intensity of each individual image in order to improve the accuracy of estimating the position of radioactivity in the animal body.

A semi-quantitative assessment of radioactivity over time, corrected for attenuation, is shown graphically in FIG. 12 for B-NP. The results for B-NP-PEG35 were very similar, but the elimination of these particles took longer because they remain in the eye longer.

Example 10. Biodistribution of nanoparticles after intravenous administration to male Wistar rats

For in vivo biodistribution imaging experiments, Wistar rats were injected with 99mTc labeled HSA nanoparticles loaded with bevacizumab (B-NP) and HSA nanoparticles filled with bevacizumab and coated with PEG35 (B-NP-PEG35). A single dose of 5 mg bevacizumab/kg body weight was administered intravenously and images were taken every two hours for up to ten hours from the time of administration.

1 hour after intravenous administration, radioactivity associated with injection of B-NP and B-NP-PEG35 was observed in the liver and kidneys, as shown in FIG. 13. In addition, it should be noted that B-NP-PEG35 is less absorbed by the liver. B-NP and B-NP-PEG35 do not accumulate in any organs.

Example 11 Effect of bevacizumab-filled albumin nanoparticles on corneal neovascularization.

Male Wistar rats weighing approximately 200 g were obtained from Harlan to analyze the efficacy of bevacizumab-loaded nanoparticles in a rat model of corneal neovascularization. The studies were approved by the ethics committee of the organization in the field of animal research (protocol number 172-14) in accordance with European legislation on animal experiments.

Animals were maintained sedated after intraperitoneal administration of 100 μl of a 5 mg/kg xylazine solution (Xilagesic, Calier Laboratory) and 200 μl of a 40 mg/kg ketamine solution (IMALGENE, Merial). Then, one drop of mydriatic eye drops (Coliricusi Tropicamida, 10 mg/ml, Alcon) was injected into each eye of the rats. After 5 minutes, the corneas of the rats were cauterized by applying a lapis pencil (Argepenal, Braun) to the surface of the eyes for 5 seconds. Finally, the eyes were washed with sterile 0.9% by volume NaCl solution.

Twelve hours later, the animals were anesthetized with isoflurane (Isovet, Spain) and divided into separate groups. The following drugs were administered to the eyes of the animals in the form of eye drops: (i) 10 µl of an aqueous solution of 4 mg/ml of bevacizumab (Avastin®) every 12 hours for 7 days, (ii) 10 µl of an aqueous solution of 4 mg/ml of aflibercept ( Eilea®) every 12 hours for 7 days, (iii) 10 µl of an aqueous solution of 0.1% dexamethasone phosphate (Coliriculi dexametasona®) every 12 hours for 7 days, (iv) albumin nanoparticles filled with bevacizumab (B-NP ; 10 µl suspension containing 40 µg bevacizumab) once a day for one week, (v) albumin nanoparticles filled with bevacizumab and coated with PEG35 (B-NP-PEG35; 10 µl suspension containing 40 µg bevacizumab) once a day for 1 week. As controls, one group of animals was injected with saline (PBS), and the other group of animals was injected with human serum albumin dissolved in PBS (HSA), in an amount similar to that administered with B-NP-PEG35.

In FIG. 14 is a schematic representation of the schedule showing corneal cauterization at 2 pm (day 0) and the first treatment 24 hours later (day 1).

For calculations, digital images of the corneas were obtained and analyzed using ImageJ software (public domain, http://rsb.info.nih.gov/ij/). The images were analyzed in binary mode with conversion to black and white. From these images, the total area of the cornea as well as the surface of the cornea affected by the silver nitrate burn (lesion) was determined, and the area with observed neovascularization (corneal neovascularization) was calculated by counting the pixels. These parameters were used to determine the area of invasion (IA) and CNV (neovascularization of the cornea, normalized by the area of the lesion):

Summary Table 5 summarizes the efficacy of various bevacizumab formulations as a result of the reduction in ocular surface area affected by lesion-induced neovascularization (FIG. 15). In all cases, the lesion in the eyes of the animals was similar, no statistically significant differences in the area of the lesion were found between the four groups (p>0.05; Fig. 16).

The group of animals treated with bevacizumab solution (BEVA) showed a lower area of neovascularization of the eye compared to animals treated with PBS (negative control). On the other hand, in animals treated with bevacizumab (B-NP) nanoparticles, the area affected by corneal neovascularization was 2.7 times lower than in animals treated with Avastin® (BEVA) (Figs. 17 and 18) . When animals were treated with PEGylated nanoparticles filled with bevacizumab, the area affected by neovascularization was approximately 1.4 times lower than in animals treated with Avastin®. Importantly, nanoparticle-treated animals received 50% less bevacizumab compared to Avastin®-treated animals. Another important observation was that under the chosen experimental conditions, neither dexamethasone nor Eylea® showed a positive effect on neovascularization (Table 9, Fig. 15).

Table 5. Effect of bevacizumab compositions on corneal neovascularization caused by pencil burn. BEVA - bevacizumab solution (Avastin® 4 mg/ml, two doses per day for 7 days); B-NP - albumin nanoparticles filled with bevacizumab (4 mg/ml, one dose per day for 7 days); B-NP-PEG35 - albumin nanoparticles filled with bevacizumab and coated with PEG35 (4 mg/ml, one dose per day for 7 days); HSA - human serum albumin; dexamethasone - 0.1% solution twice a day for 7 days; Eilea - aflibercept solution (Eilea® 4 mg/ml, two doses per day for 7 days); control - PBS. IA - area of invasion. CNV - the area of neovascularization of the cornea, normalized by the area of the lesion. Data are expressed as mean ± SD, n=9.

b p < 0.05 - results of analysis of variance using Tukey's test significantly differ from control (-)

p <0.01 - results of analysis of variance using Tukey's test significantly differ from control (-)

d p < 0.005 - results of analysis of variance using Tukey's test significantly differ from control (-)

e p < 0.001 - results of analysis of variance using Tukey's test significantly differ from control (-)

Histology.

For histological examination of eyes with corneal neovascularization at the end of treatment, two eyes were removed from each group. The surface of the eye was washed with saline and the anterior pole was separated from the posterior one. The corneas were laid on a flat surface, fixed with 4% paraformaldehyde for 24 hours, and then each sample was washed several times with PBS. The corneas were stored in 70% methanol for subsequent sections and analysis.

For analysis of the corneas, they were embedded in paraffin and sections were made 4 μm thick from the center of the cornea and the zone of neovascularization. They were then stained with hematoxylin-eosin and analyzed using optical microscopy. When evaluating sections, the intensity of neovascularization, the intensity of inflammation, fibrosis, edema, and the average thickness of the cornea were assessed. The study was performed by an investigator with treatment masking. Images were taken under a Nikon Eclipse Ci microscope equipped with a Nikon DS-Ri 1 digital camera. Images were analyzed using a calibrated Nikon NIS-elements digital image analysis system.

To assess the intensity of neovascularization, the following scale was used: 0 - no neovascularization; 1 - minimal or close to absent vascularization; 2 - easy vascularization; 3 - limited or focal vascularization in the subepithelial and prestromal areas (moderate neovascularization); 4 - very frequent or intense; 5 - diffuse and intense vascularization. The intensity of inflammation was assessed in a similar way: 0 - no inflammation; 1 - minimal or close to absent inflammation; 2 - focal inflammation, low number of mixed inflammatory cells, such as lymphocytes, neutrophilic leukocytes and eosinophilic leukocytes; 3 - moderate inflammation; 4 - very frequent or intense; 5 - intense, diffuse inflammation, inflammatory cells of mixed type. To assess the activity of fibroblasts, the following scale was used: 0 - no activity of fibroblasts; 1 - minimal or close to absent fibroblast activity; 2 - focal activity of fibroblasts; 3 - moderate activity of fibroblasts; 4 - very frequent; 5 - diffuse and intense activity of fibroblasts. Finally, the following scale was used to classify edema: 0, no edema; 1 - minimal or close to absent edema; 2 - mild edema; 3 - moderate swelling; 4 - very frequent or intense; 5 - diffuse and intense edema.

In FIG. 19 shows histological examinations of the eyes of the animals included in the study. Corneal lesions treated with B-NP (FIG. 19B) and B-NP-PEG35 (FIG. 19C) are in the recovery phase and do not cause temporary visual impairment as they did initially. Thus, the damage is reversible. In contrast, corneal lesions treated with saline very seriously impair vision and appear to be irreversible (FIG. 19F).

Summary Table 6 shows the histopathological scores in the different groups. Corneas treated with saline showed a thickness 1.4 times that of corneas treated with bevacizumab and 2.7 times the thickness of corneas treated with B-NP. On the other hand, corneas treated with Avastin® (BEVA) were twice as thick as B-NP treated corneas and 1.3 times thicker than B-NP treated corneas. -PEG35. Similarly, corneas treated with B-NP showed improved symptoms with low fibrosis, low vascularity, and no edema.

Table 6. Histopathological evaluations of animal eye specimens. Control - animals treated with saline; BEVA - animals treated with Avastin®; B-NP - animals treated with nanoparticles filled with bevacizumab; B-NP-PEG35 - Animals treated with PEGylated nanoparticles filled with bevacizumab.

Example 12. Biodistribution of nanoparticles after intravenous administration to tumor-bearing athymic mice.

For tumor imaging experiments in mice, human hepatocarcinoma (HepG2) cells were cultured under standard conditions, harvested one week after seeding, and suspended in PBS. Tumors were induced in athymic mice by subcutaneous injection of 5x105 HepG2 cells at two different sites, the right limb and upper back. Tumor growth was monitored for 12-15 days until it was clearly visible, then the animals were used for in vivo SPECT-CT imaging experiments after intravenous injection of 99mTc-labeled HSA nanoparticles coated with PEG35 (NP-PEG35). Animals were sacrificed 1 and 4 hours after i.v. administration, and both tumors and part of the muscle tissue of the contralateral leg (without tumor) were excised. The samples were counted on a gamma counter calibrated at 99mTc, corrected for sample weight and attenuation, and the tumor/normal tissue ratio was calculated using the muscle tissue of the contralateral leg as a basis.

In tumor bearing mice, the radioactivity associated with intravenously administered NP-PEG35 is concentrated in the tumors (compared to normal tissue) as seen in FIG. 20. This phenomenon seems to be time-dependent, since 4 hours after the administration of the nanoparticles, the amount in the tumors decreased, although it remained high (tumor/normal tissue ratio >6).

Example 13 Effect of bevacizumab-loaded albumin nanoparticles on a mouse model of colorectal cancer.

The studies were approved by the ethics committee of the organization in the field of animal research (protocol number 107-16) in accordance with European legislation on animal experiments. For experiments at Harlan Sprague Dawley, Inc. Forty-two male athymic mice weighing about 20 grams and 3 weeks old were purchased. Mice were kept under controlled conditions in accordance with the rules of the organization. Food and water were provided without restrictions.

Human colon cancer cells (HT-29) were cultured under standard conditions, harvested one week after seeding, and suspended in PBS. For tumor induction, mice were anesthetized with isoflurane by inhalation, and 100 µl of a suspension containing 2-3×106 HT-29 tumor cells (bevacizumab sensitive human colon cancer cell line) was injected subcutaneously into the right flank of each animal. Tumor growth was monitored for 12-15 days until it became clearly visible (diameter 0.4-0.6 cm), after which treatment was started.

Mice were randomized into six groups of seven each: (i) bevacizumab aqueous solution (Avastin), (ii) bevacizumab-loaded albumin nanoparticles (B-NP), (iii) bevacizumab-filled albumin nanoparticles coated with PEG35,000 (B -NP-PEG35), (iv) saline (PBS), (v) blank nanoparticles coated with PEG 35000 (NP-PEG35) in an amount similar to that administered with B-NP-PEG35, (vi) an aqueous HSA solution containing an amount albumin, similar to that administered in the group treated with B-NP. In all cases, 150-200 μl of preparations containing 5 mg of bevacizumab/kg of body weight were administered intravenously twice a week. The control group received intravenous administration of 0.9% saline at the same time points.

On the 0th day (before the first injection), the 15th day, the 22nd day and the 26th day from the moment of the first injection, blood samples were taken. A specific enzyme immunoassay (Shikari Q-Beva) was used to measure the serum concentration of bevacizumab.

The volume and weight of tumors were recorded 1-2 times a week. For tumors (V), two dimensions were measured with a caliper: width (W) and length (L) - and the volume was calculated using the following formula (Eq. 5):

In FIG. 21 shows the tumor volume as a function of time. Tumors of the same size developed in each group, no statistically significant differences between the volume of tumors between the six groups at the beginning of the experiment were found (p>0.05).

On day 12, the group treated with B-NP-PEG35 showed a significant reduction in tumor volume (p<0.05) compared to the other groups. By day 14, the groups treated with BEVA and B-NP-PEG35 showed a significant reduction in tumor volume (p<0.05) compared to the rest of the groups. On day 22, the untreated groups showed increased tumor volume compared to those treated with bevacizumab-filled nanoparticles (B-NP), bevacizumab (BEVA) and bevacizumab-filled pegylated nanoparticles (B-NP-PEG35). It should be noted that by the end of the experiment, half of the animals in the group that did not receive bevacizumab developed ulcers at the sites of tumors.

In FIG. 22 shows serum levels of bevacizumab. Samples were taken on the following days: 0 (before injection), 15, 22, and 26 after the first injection. Of note is an increase in serum levels of bevacizumab on day 22, which correspond to the next day after weekly dosing. In FIG. 22 shows that the serum levels of bevacizumab in mice treated with unbound drug are up to six times higher than those in treated nanoparticles (B-NP and B-NP-PEG35).

The benefit of administering bevacizumab incorporated into nanoparticles is lower serum levels of bevacizumab. After intravenous administration of unbound bevacizumab, high concentrations in the blood occur, which can lead to side effects. This concentration gradually decreases until a plateau is reached. However, after the introduction of a new dose, the concentration of bevacizumab in the blood increases (see Fig. 22), thus increasing the likelihood of side effects.

On the other hand, after intravenous administration of bevacizumab nanoparticles, serum levels of the drug gradually increase until a plateau is reached. This concentration is six times lower than achieved with the introduction of unbound bevacizumab and remains constant. In addition, each time a new dose is administered, the peak blood concentration of bevacizumab is much lower. This reduces the chance of side effects.

In addition, polyethylene glycol forms a steric barrier on the surface of the nanoparticles, preventing opsonization, which is the main mechanism for losing the administered dose within a few hours after IV administration.

PET imaging.

At the end of the experiment, three mice were selected for 18F-FDG-PET imaging from the groups treated with bevacizumab, pegylated nanoparticles filled with bevacizumab, and saline. For this, mice were not fed at night, but were allowed to drink water without restrictions. One day later, mice were anesthetized with 2% isoflurane in 100% O2 gas and immobilized for the 18F-FDG-PET procedure. Forty minutes before the scan, 18F-FDG (10 MBq ± 2 in 80-100 µl) was injected into the tail vein. PET imaging was performed on a dedicated small animal scanner Philips Mosaic (Cleveland, Ohio) with a resolution of 2 mm, an axial field of view (FOV) of 11.9 cm, and a transverse FOV of 12.8 cm. Anesthetized mice were placed in a horizontal position on the PET table -scanner to obtain a static image (sinogram) within 15 minutes. The images were reconstructed using a 3D RAMLA (true 3D reconstruction) algorithm with two iterations and a relaxation parameter of 0.024 into a 128×128 matrix with a 1 mm voxel size and corrected for dead time, attenuation, random coincidences and scattering. To evaluate tumor uptake of 18F-FDG, all studies were exported and analyzed using PMOD software (PMOD Technologies Ltd., Adliswil, Switzerland). Regions of interest (ROI) were built on frontal PET images for small animals with a thickness of 1 mm on consecutive sections, including the tumor as a whole. Finally, the Maximum Standardized Uptake Value (SUV) was calculated for each tumor using the formula:

Summary Table 7 shows the results of PET imaging in different groups. With respect to SUVmax (maximum tumor uptake), the lowest value was in the B-NP-PEG35 treated group, while the highest was in the saline treated group.

In terms of volume, the lowest values belonged to the groups treated with B-NP-PEG35 and HSA. However, the individuals selected from the HSA group were not representative as they were the only ones that did not have ulcers. The highest value was again recorded in the group that received no treatment (saline). The B-NP-PEG35 group showed three times less volume than the saline group and 1.5 times less than the BEVA group.

Finally, for TLG (total focal glycolysis), the lowest value was recorded for the B-NP-PEG35 treated group, which was 1.5 times less than in the BEVA group and up to 3.5 times less than in the BEVA group. group receiving saline.

Example 14 Preparation of human serum albumin nanoparticles filled with ranibizumab.

Ranibizumab-loaded HSA nanoparticles were prepared using the same procedure as described in Example 1 for bevacizumab-loaded albumin nanoparticles.

For nanoparticles filled with ranibizumab, the optimal antibody/albumin ratio was 0.15. The nanoparticles with ranibizumab showed a particle size of about 210 nm with an EC below 0.3 and a zeta potential of 15 mV.

Claims (34)

1. The use of a nanoparticle for the treatment of eye diseases, comprising a solid core containing a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, wherein, when said solid core is coated, it is coated with a non-ionic polymer. 2. The use of a nanoparticle for cancer treatment, comprising a solid core containing a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, wherein, when said solid core is coated, it is coated with a non-ionic polymer. 3. Use according to claim 1 or 2, wherein the monoclonal antibody/albumin weight ratio is between 0.01 and 0.5. 4. Use according to claim 1 or 2, wherein the non-ionic polymer/albumin weight ratio is from 0.02 to 5 by weight. 5. Use according to claim 1 or 2, wherein the albumin is human serum albumin or bovine serum albumin. 6. Use according to claim 1 or 2, wherein the monoclonal antibodies are selected from bevacizumab, ranibizumab, trastuzumab, cetuximab and rituximab. 7. Use according to claim 1 or 2, wherein the non-ionic polymer is a water-soluble cellulose selected from hydroxyethylcellulose, hydroxy-n-propylcellulose, hydroxy-n-butylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate and ethylhydroxyethylcellulose; starch; dextran; polyvinylpyrrolidone, a polyester selected from compounds under the trademark Eudagrit, or polyalkylene glycol. 8. Use according to claim 1 or 2, wherein the non-ionic polymer is selected from hydroxypropyl methylcellulose phthalate and polyethylene glycol. 9. Use according to claim 1 or 2, wherein the monoclonal antibody is selected from bevacizumab and trastuzumab and the non-ionic polymer is selected from hydroxypropyl methylcellulose phthalate and polyethylene glycol. 10. Use according to claim 1 or 2, according to which the specified nanoparticle is obtained by a method containing: a) obtaining an aqueous solution of albumin and monoclonal antibodies; b) titration of the aqueous solution obtained in step a) to a pH of 4 to 5; c) adding a desolvating agent to the aqueous solution obtained in step b) to obtain albumin/monoclonal antibody nanoparticles; d) optionally incubating the albumin nanoparticles/monoclonal antibodies obtained in step c) with a non-ionic polymer and optionally e) drying the nanoparticles by vacuum drying, spray drying or freeze drying. 11. Use according to claim 1, wherein the ocular disease is selected from macular degeneration, corneal neovascularization or angiogenesis, iris neovascularization or angiogenesis, retinal neovascularization or angiogenesis, diabetic proliferative retinopathy, non-diabetic proliferative retinopathy, glaucoma, infectious conjunctivitis, allergic conjunctivitis, ulcerative keratitis , non-ulcerative keratitis, episcleritis, scleritis, diabetic retinopathy, uveitis, endophthalmitis, infectious processes and inflammatory processes. 12. Use according to claim 2, wherein the cancer is breast cancer, lung cancer, pancreatic cancer, multiple myeloma, renal cell carcinoma, prostate cancer, melanoma, colon cancer, colorectal cancer, kidney cancer, cervical cancer uterus, ovarian cancer, liver, kidney and gastrointestinal cancer, bladder cancer or squamous cell carcinoma. 13. A nanoparticle for the treatment of ophthalmic diseases, comprising a solid core containing a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, said solid core being coated with a non-ionic polymer. 14. A nanoparticle for cancer treatment, comprising a solid core containing a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, said solid core being coated with a non-ionic polymer. 15. A nanoparticle according to claim 13 or 14, wherein the non-ionic polymer is selected from hydroxypropyl methylcellulose phthalate and polyethylene glycol. 16. A nanoparticle according to claim 13 or 14, wherein the monoclonal antibody is selected from bevacizumab and trastuzumab and the non-ionic polymer is selected from hydroxypropyl methylcellulose phthalate and polyethylene glycol. 17. Ophthalmic pharmaceutical composition for the treatment of eye diseases, including: - a group of nanoparticles containing a solid core, including a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, wherein, when said solid core is coated, it is coated with a non-ionic polymer; excipient, pharmaceutically acceptable carrier or excipient. 18. An anticancer pharmaceutical composition for the treatment of cancer, comprising: - a group of nanoparticles containing a solid core, including a non-crosslinked albumin matrix and monoclonal antibodies distributed throughout the volume of the albumin matrix, wherein, when said solid core is coated, it is coated with a non-ionic polymer; excipient, pharmaceutically acceptable carrier or excipient. 19. The composition according to claim 17 or 18, in which the nanoparticles are in the form of a dry powder. 20. A composition according to claim 17 or 18 wherein the excipient, pharmaceutically acceptable carrier or excipient is suitable for oral, topical or parenteral administration. 21. A composition according to claim 17 or 18, wherein the albumin is human serum albumin or bovine serum albumin. 22. A composition according to claim 17 or 18, wherein the monoclonal antibody is selected from bevacizumab, ranibizumab, trastuzumab, cetuximab, and rituximab. 23. The composition according to claim 17 or 18, in which the non-ionic polymer is a water-soluble cellulose selected from hydroxyethylcellulose, hydroxy-n-propylcellulose, hydroxy-n-butylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate and ethylhydroxyethylcellulose; starch; dextran; polyvinylpyrrolidone, a polyester selected from compounds under the trademark Eudagrit, or polyalkylene glycol. 24. A composition according to 17 or 18 wherein the non-ionic polymer is selected from hydroxypropyl methylcellulose phthalate and polyethylene glycol. 25. A composition according to 17 or 18 wherein the monoclonal antibody is selected from bevacizumab and trastuzumab and the non-ionic polymer is selected from hydroxypropyl methylcellulose phthalate and polyethylene glycol.

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