Albumin nanoparticles for the treatment of cancer and eye diseases

Abstract

The present invention relates to nanoparticles comprising a core, said core consisting of a non-crosslinked albumin matrix and a monoclonal antibody optionally coated with a non-ionic polymer for use as a medicine, to a pharmaceutical composition comprising said nanoparticles, as well as their use in the treatment of cancer and eye diseases.

Description

1 / 78 Albumin nanoparticles for the treatment of cancer and eye diseases. Field of the invention.

[0001] The present invention relates to nanometer-sized drug delivery systems and, more particularly, to nanoparticles containing an albumin matrix and a monoclonal antibody to be used in the treatment of cancer and eye diseases. BACKGROUND

[0002] Monoclonal antibodies have emerged as an interesting group of glycoproteins for use in different therapeutic areas, such as cancer, autoimmune and chronic inflammatory diseases, as well as the treatment of transplant rejection. Currently, there are approximately 30 monoclonal antibodies approved by regulatory agencies (FDA and EMEA).

[0003] One of these monoclonal antibodies is Bevacizumab, an immunoglobulin G (IgG) that targets VEGF-A (vascular endothelial growth factor) and includes four of the major VEGF isoforms. It was approved by the FDA in 2004 for first-line treatment of metastatic colorectal cancer and was later also approved for other types of cancer, such as non-small cell lung cancer or metastatic breast cancer. Recently, bevacizumab has begun to be used in the treatment of eye diseases, including corneal or retinal neovascularization, diabetic retinopathy, and age-related macular degeneration.

[0004] Topical application to the surface of the eye is a common route for administering medication. In Petition 870200019339, dated 10 / 02 / 2020, page 5 / 100 2 / 78 However, protective mechanisms (slowness, tearing, and drainage) decrease the bioavailability of the drug, rapidly removing the formulation. In recent years, intravitreal injection of bevacizumab has been found to be a very effective treatment for the wet form of age-related macular degeneration, proliferative diabetic retinopathy, and choroidal neovascularization. Short-term results suggested that intravitreal bevacizumab was well tolerated and associated with improved visual acuity, decreased retinal thickness, and reduced angiographic leakage in most patients. However, achieving and maintaining visual improvement requires repeated injections and follow-up visits. This implies a high risk of complications such as endophthalmitis, as well as repeated pain, apprehension, and distress associated with needle insertion into the eyes. Furthermore, the intravitreal half-life of injected bevacizumab is only approximately 3 days.

[0005] In the case of cancer therapies, current treatment strategies often involve invasive procedures, including the application of catheters for chemotherapy to shrink the tumor before its surgical removal. Research efforts to improve the effectiveness of cancer therapy have led to a substantial improvement in patient survival; however, problems associated with toxic side effects and poor patient quality of life remain a significant issue.

[0006] Therefore, an effective method of drug administration needs to be developed for Petition 870200019339, dated 10 / 02 / 2020, page 6 / 100 3 / 78 to make bevacizumab, as well as other monoclonal antibodies, less invasive and longer-lasting for the treatment of cancer and eye diseases.

[0007] In this sense, nanoparticles have emerged as a suitable vehicle for drug delivery and have produced promising results in the ophthalmic field and in cancer therapies. There is a wide variety of materials that can be used to prepare these nano-sized 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 treatments 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].

[0008] However, natural biopolymers are preferred over synthetic materials. In this regard, 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 gained considerable attention due to their high binding capacity for various drugs and because they are well tolerated without serious side effects. Petition 870200019339, dated 10 / 02 / 2020, page 7 / 100 4 / 78

[0009] In recent years, a wide variety of physicochemical processes for the preparation of albumin nanoparticles have been proposed, including thermal gelation, emulsification, and desolvation (coacervation). In any case, desolvation-based procedures appear to be the most popular due to their simplicity and repeatability. However, the newly obtained nanoparticles are unstable, and a supplementary physical, chemical, or enzymatic stabilization step must be performed to prolong their half-life in an aqueous environment and / or prevent the formation of protein macroaggregates.

[0010] In general, cross-linking of albumin is one of the most popular strategies for stabilizing albumin nanoparticles. Elzoghby et al. [Journal of Controlled Release, 2012, 157, 168-182] compile different methods for preparing albumin nanoparticles and their use as active drug delivery systems. Particular mention is made of the instability of nanoparticles in aqueous media requiring cross-linking, citing glutaraldehyde as the common chemical cross-linking agent used in the technique. Lohcharoenkal, W. et al. [BioMed Research International, 2014] also refer to the instability of albumin nanoparticles as they dissolve or coalesce to form a separate phase if not cross-linked. Llabot et al. [19th International Symposium on Microencapsulation, 2013] describe Gantrez-crosslinked albumin nanoparticles encapsulating bevacizumab, as well as their use in corneal vascularization. Petition 870200019339, dated 10 / 02 / 2020, page 8 / 100 5 / 78

[0011] Thus, crosslinking stabilizes the albumin nanoparticles and reduces enzymatic degradation, as well as the release of the active ingredient from the nanoparticle.

[0012] However, although glutaraldehyde is highly effective at stabilizing nanoparticles, its use is questionable mainly due to its toxicity, which hinders its use for in vivo release. Therefore, it is essential to remove the crosslinker as completely as possible. In addition, glutaraldehyde can affect the stability of biomacromolecules, more particularly protein drugs, antibodies, and peptides, in nanoparticles, since it reacts with functional groups present in macromolecules (such as primary amine residues), resulting in a significant loss of activity.

[0013] In order to resolve this important drawback, different strategies have been proposed to harden or stabilize newly formed albumin nanoparticles without the need to use toxic reagents. Among others, stabilization of the nanoparticles can be achieved by heat treatment, high hydrodynamic pressure, or enzymatic crosslinking with genipin or transglutaminase.

[0014] Surface coating has also been used to stabilize albumin nanoparticles. For example, cationic polymers, such as polylysine or polyethyleneimine, have been used to coat bovine serum albumin nanoparticles to improve their stability [Wang et al., Pharm. Res., 2008, 25 (12), 2896-2909].

[0015] Document WO2013 / 042125 describes the Petition 870200019339, dated 10 / 02 / 2020, page 9 / 100 6 / 78 preparation of glutaraldehyde-crosslinked bovine serum albumin nanospheres incorporating bevacizumab and subsequent encapsulation of said nanospheres in an ionic PLGA coating.

[0016] Document WO2011 / 053803 also refers to nanoparticles with a polymeric shell that encapsulates a therapeutic agent, such as bevacizumab, for the treatment of eye diseases.

[0017] In view of the above, suitable delivery systems are needed that preserve the integrity and activity of monoclonal antibodies and control their release from nanoparticles. BRIEF DESCRIPTION OF THE INVENTION

[0018] The authors of the present invention have discovered that trapping a monoclonal antibody, such as bevacizumab, in an albumin matrix provides the nanoparticles with high stability in aqueous solution and, surprisingly, said nanoparticles do not need to be cross-linked or stabilized by other means, thus allowing the maintenance of the 3D structure as well as the biological activity of the antibody once released. Conversely, and as shown in the experimental part below, the use of glutaraldehyde (the most common cross-linker used in the prior art to stabilize albumin nanoparticles) inactivates the antibody, making the use of cross-linked nanoparticles for encapsulating this type of active ingredient unfeasible.

[0019] The authors also tested non-crosslinked nanoparticles decorated with nonionic polymers, such as hydroxypropyl methylcellulose phthalate (HPMC-P) and Petition 870200019339, dated 10 / 02 / 2020, page 10 / 100 7 / 78 polyethylene glycol 35,000 (PEG35), as well as Eudagrit® S-100, and observed that antibody integrity is also maintained.

[0020] Furthermore, following the method described below in this document, monoclonal-loaded albumin nanoparticles can be manufactured as a dry powder ready to disperse and reconstitute by simply adding water or an aqueous solution.

[0021] Furthermore, the nanoparticles of the invention allow for sustained release of the monoclonal antibody and constitute a drug delivery system of great interest for in vivo applications, as indicated by biological activity data obtained in an animal model of corneal neovascularization.

[0022] Furthermore, biodistribution assays performed with albumin nanoparticles coated with non-ionic polymers indicate that they are able to concentrate in tumor tissues, making them very promising nanoparticulate systems for releasing monoclonal antibodies in affected tissues.

[0023] In fact, in vivo experiments have shown that the nanoparticles of the invention are capable of releasing the monoclonal antibody into the tumor tissue, since a lower concentration of said antibody is present in the serum when compared with the administration of the same monoclonal antibody in aqueous solution. Furthermore, the tumor volume is significantly reduced.

[0024] Thus, a first aspect of the present invention relates to a nanoparticle for use in medicine, wherein said nanoparticle comprises a Petition 870200019339, dated 10 / 02 / 2020, p. 11 / 100 8 / 78 solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core optionally being coated with a non-ionic polymer.

[0025] A second aspect of the invention relates to a pharmaceutical composition comprising: - a plurality of nanoparticles, said nanoparticles comprising a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core optionally being coated with a non-ionic polymer; and - a pharmaceutically acceptable excipient, carrier or vehicle.

[0026] In another aspect, the invention relates to the aforementioned pharmaceutical composition of the invention for use in medicine.

[0027] Another aspect of the invention is a nanoparticle for use in the treatment of eye diseases, wherein said nanoparticle comprises a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core optionally being coated with a non-ionic polymer.

[0028] Another aspect of the invention is a Petition 870200019339, dated 10 / 02 / 2020, p. 12 / 100 9 / 78 nanoparticle for use in cancer treatment, wherein said nanoparticle comprises a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core optionally being coated with a non-ionic polymer.

[0029] Finally, another aspect of the invention relates to a nanoparticle comprising a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core being coated with a non-ionic polymer. DESCRIPTION OF THE FIGURES

[0030] Figure 1. Influence of the bevacizumab / albumin ratio on the payload of the resulting nanoparticles. The nanoparticles were prepared after incubation for 10 min. of the monoclonal antibody and the protein. Data expressed as mean ± SD (n = 3).

[0031] Figure 2. TEM photograph of albumin nanoparticles loaded with bevacizumab (B-NP).

[0032] Figure 3. A) FT-IR spectrum of human serum albumin (HSA), glutaraldehyde (GLU), physical mixture of HSA and glutaraldehyde (HSA-GLU) and glutaraldehyde-crosslinked nanoparticles (NP-GLU). B) FTIR spectrum of human serum albumin (HSA), bevacizumab (BEVA), physical mixture of HSA and bevacizumab (HSA-BEVA) and bevacizumab-loaded nanoparticles (B-NP).

[0033] Figure 4. X-ray spectra of Petition 870200019339, dated 10 / 02 / 2020, p. 13 / 100 10 / 78 bevacizumab (BEVA), bevacizumab-loaded nanoparticles (B-NP), and human serum albumin (HSA).

[0034] Figure 5. DTA thermograms of: A) native human serum albumin (HSA) and bevacizumab (BEVA); B) physical mixture (PM) between human serum albumin (HSA) and bevacizumab (BEVA) and bevacizumab-loaded albumin nanoparticles (B-NP); C) native human serum albumin (HSA) and glutaraldehyde (GLU); D) physical mixture (PM) between human serum albumin (HSA) and glutaraldehyde (GLU) and glutaraldehyde-crosslinked albumin nanoparticles (NP-GLU).

[0035] Figure 6. Evolution of the average size of glutaraldehyde-crosslinked empty nanoparticles (NPGLU) and bevacizumab-loaded nanoparticles (B-NP) after dispersion in aqueous solution at pH 7.4. Data expressed as mean ± SD (n = 3).

[0036] Figure 7. Release profile of bevacizumab from human serum albumin nanoparticles after incubation in PBS (pH 7.4). Data expressed as mean ± SD (n = 3).

[0037] Figure 8. TEM micrograph of bevacizumab-loaded albumin nanoparticles pegylated with PEG35 (B-NP-PEG35).

[0038] Figure 9. Bevacizumab release profile from albumin nanoparticles after incubation in PBS (pH 7.4) (...---) Bevacizumab-loaded NPs (B-NP); (-) Bevacizumab-loaded NPs coated with PEG35 (B-NP-PEG35); bevacizumab-loaded NPs coated with Eudagrit® S-100 (B-NP-S-100); bevacizumab-loaded NPs coated with HPMC-P (B-NP-HPMC-P). Data Petition 870200019339, dated 10 / 02 / 2020, page 14 / 100 11 / 78 expressed as mean ± SD (n = 3).

[0039] Figure 10. Automated microfluidic-based electrophoresis of nanoparticles (L: Ladder; 1: PEG35-coated empty nanoparticles (NP-PEG35); 2: 1. Albumin nanoparticles loaded with bevacizumab (BNP); 2. Albumin nanoparticles loaded with bevacizumab (BNP); 3. Bevacizumab nanoparticles coated with PEG35 (B-NP-PEG35); 4. Human serum albumin (HSA); 5. Bevacizumab.

[0040] Figure 11. In vivo SPECT-CT images of 99mTc-labeled BNP (top row) compared with 99mTc-labeled BNP-PEG35 (bottom row) after ocular administration to Wistar rats. The images in each row correspond to the same animal studied at the times indicated in the figure. Activity disappears between 4h and 8h after ocular administration, while B-NP-PEG35 remains in the eye for at least 8h.

[0041] Figure 12. Temporal activity curves for the evolution of the amount of radioactivity in different regions after ocular administration of 99mTc-B-NP nanoparticles. Volumes of interest (VOIs) were drawn over the areas indicated in the graphs and the average counts of values ​​obtained from each VOI, data corrected for decay and plotted.

[0042] Figure 13. In vivo SPECT-CT images of 99mTc-labeled BNP (top row) compared with 99mTc-labeled BNP-PEG35 (bottom row) after intravenous administration in Wistar rats. The images in each row correspond to the same animal studied at the times indicated in the figure.

[0043] Figure 14. Schematic diagram of the row Petition 870200019339, dated 10 / 02 / 2020, page 15 / 100 12 / 78 of the timeline showing corneal cauterization occurring at 0h (day 0) and the first treatment at 24h (day 1).

[0044] Figure 15. Photographs of the corneas of animals treated with: (A) physiological saline [Control (-)]; (B) Avastin® (4 mg / mL bevacizumab); (C) bevacizumab-loaded albumin nanoparticles (B-NP); (D) PEG 35,000-coated bevacizumab-loaded albumin nanoparticles (B-NP-PEG35); (E) human serum albumin solution (HSA); (F) Eylea® (EYLEA); (G) dexamethasone (DEXA).

[0045] Figure 16. Area of ​​the lesion expressed as a percentage of the corneal area affected by the burn. No statistically significant differences were found between the lesions of the different groups. Data are shown as mean ± SD (n = 9).

[0046] Figure 17. Area of ​​invasion (AI), fraction of the corneal area in which vessels are present. Data are shown as mean ± SD (n = 9). * p <0.01 ANOVA followed by Tukey's test significantly different from the control (-) * * p <0.01 ANOVA followed by Tukey's test significantly different from BEVA * ** p <0.005 ANOVA followed by Tukey's test significantly different from B-NP

[0047] Figure 18. Area of ​​neovascularization normalized by the lesion. Data are shown as mean ± SD (n = 9). * p <0.01 ANOVA followed by Tukey's test significantly different from the control (-) * * p <0.005 ANOVA followed by Tukey's test Petition 870200019339, dated 10 / 02 / 2020, p. 16 / 100 13 / 78 significantly different from BEVA

[0048] Figure 19. Photomicrographs of sections of normal and neovascularized corneas treated with bevacizumab (e, epithelial layer; s, stroma; ac, anterior chamber; v, stromal microvessels). A) A photomicrograph of a normal rat cornea showing intact epithelium (e), the stroma containing regular lamellae of parallel collagen with flattened keratocytes in the middle; B) A photomicrograph of a rat cornea from the group treated with bevacizumab-loaded albumin nanoparticles (B-NP). Corneal thickness within the normal limit, intact epithelium and slightly disorganized and relaxed stroma, with very discreet infiltration. C) Photomicrographs of a rat cornea from the group treated with bevacizumab-loaded albumin nanoparticles coated with PEG35 (B-NP-PEG35). Normal epithelium, numerous stromal microvessels (v). Corneal thickness within normal values; D & E): Photomicrographs of rat cornea from the group treated with bevacizumab.Preserved and hypertrophic epithelium separating from the stroma. Thickening of the stroma with numerous disorganized fibroblasts, intense cellular infiltration and edema (*); F) Photomicrograph of the rat cornea from the group treated with physiological saline. Severe alterations in the cornea, central erosion and increased thickness. Intense fibrosis with large disorganized fibroblasts, moderate inflammatory infiltration and neovascularization (v). Formation of a cyst (c) from epithelial cells showing an attempt at abnormal repair. Scale bar, 200 μm, HE X100.

[0049] Figure 20. Tumor / non-tumor rates for leg and neck tumors in animals treated with Petition 870200019339, dated 10 / 02 / 2020, page 17 / 100 14 / 78 PEG35-coated albumin nanoparticles (NP-PEG35). The values ​​correspond to the average value of three animals obtained 1 hour (blue bars) and 4 hours (red bars) after intravenous (iv) administration of radiolabeled nanoparticles.

[0050] Figure 21. Tumor volume (mm3). Data are shown as mean ± SD (n ^ 6). *p <0.05 ANOVA followed by Tukey's test showed a significant difference from normal saline solution. **p <0.01 ANOVA followed by Tukey's test showed a significantly different result from normal saline solution.

[0051] Figure 22. Serum concentration of bevacizumab (pg / mL) versus time (day). DETAILED DESCRIPTION OF THE INVENTION

[0052] As mentioned previously, a first aspect of the present invention relates to a nanoparticle for use in medicine, wherein said nanoparticle comprises a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core optionally being coated with a non-ionic polymer. In a specific embodiment, the present invention relates to a nanoparticle for use in medicine, wherein said nanoparticle comprises a solid core, said solid core consisting of a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core optionally being coated with a non-ionic polymer. Petition 870200019339, dated 10 / 02 / 2020, p. 18 / 100 15 / 78

[0053] In a specific embodiment, the present invention relates to a nanoparticle for use in medicine, wherein said nanoparticle consists of a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core optionally being coated with a non-ionic polymer.

[0054] In another specific embodiment, the present invention relates to a nanoparticle for use in medicine, wherein said nanoparticle consists of a solid core, said solid core consists of a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core optionally being coated with a non-ionic polymer.

[0055] As used in this document, the term nanoparticle refers to a colloidal system with a spherical or nearly spherical shape and an average size smaller than 1 μm. In a particular embodiment, the nanoparticle has an average size ranging from 100 to 900 nm, more preferably from 150 to 800 nm, even more preferably from 200 to 500 nm, and most preferably from 200 to 400 nm.

[0056] Average size is understood to be the average diameter of the population of nanoparticles moving together in an aqueous medium. The average size of these systems can be measured by standard methods known to those skilled in the art and are described, for example, in the experimental section below. In the context of the present invention, the Petition 870200019339, dated 10 / 02 / 2020, page 19 / 100 16 / 78 The term nanoparticle refers to a nanosphere or a decorated nanosphere.

[0057] The term nanosphere should be understood as a solid matrix of non-crosslinked albumin or a continuous albumin material, in which the monoclonal antibody is distributed throughout said matrix, therefore not presenting a distinct core / shell structure.

[0058] The term decorated nanosphere should be understood as a nanosphere as defined above, in which the non-crosslinked solid albumin matrix is ​​coated or decorated with a non-ionic polymer.

[0059] Therefore, the nanoparticles of the invention are free of any other polymeric coating other than a non-ionic polymer.

[0060] In a preferred embodiment, the nanoparticle of the invention is a nanoparticle in which, when the solid core is coated, the nanoparticle is devoid of any other polymeric coating other than a non-ionic polymer. Although the nanoparticles of the invention do not require a coating polymer, the inventors have found that, when the nanoparticle is devoid of any other polymeric coating other than a non-ionic polymer, said particles exhibit an advantageous effect over the same particles when an ionic coating material is used. For example, when the nanoparticle of the invention is coated with an ionic polymer, the particles show a very rapid antibody release profile (explosive release).

[0061] Thus, when the nanoparticles used in Petition 870200019339, dated 10 / 02 / 2020, page 20 / 100 17 / 78 invention are not coated with a non-ionic polymer, said nanoparticles should be considered as nanospheres according to the definition given above, whereas when the nanoparticles used in the invention are coated with a non-ionic polymer, the nanoparticles should be considered decorated nanospheres according to the definition given above.

[0062] In contrast to the nanoparticles used in the prior art, in which the albumin matrix is ​​cross-linked or stabilized by other means, the nanoparticles used in the invention are characterized by having a solid core of a non-cross-linked albumin matrix, understood as such an organized structure or pattern resulting from local interactions between albumin and monoclonal antibody. Thus, within the scope of the present invention, the nanoparticles are forming solid matrix systems.

[0063] Therefore, the term solid core refers to a solid, non-crosslinked matrix-type structure in which albumin forms a continuous structure in which the monoclonal antibody is distributed, preferably homogeneously distributed, throughout the matrix.

[0064] Thus, the solid core of the nanoparticles used in the invention does not differentiate between external and internal structural structures and, therefore, the monoclonal antibody is distributed, more preferably homogeneously distributed, throughout the albumin matrix, but not encapsulated or confined to a central cavity thereof. Petition 870200019339, dated 10 / 02 / 2020, p. 21 / 100 18 / 78

[0065] In a particular embodiment, the nanoparticle used in the invention is a nanosphere as defined above. More particularly, in said nanoparticles, the solid core is not coated with a non-ionic polymer. As described throughout the text, and also shown in the examples, the nanoparticles of the invention are stable and do not require any encapsulation. In the context of the present invention, the term stable refers to the increased stability of the particles, so that the particles can be used in medicine without any material disintegration. Thus, in a particular embodiment, the nanoparticle of the present invention is a stable nanoparticle, with or without the presence of an optional coating polymer.

[0066] In another specific embodiment, the nanoparticle used in the invention is a decorated nanosphere as defined above, i.e., it comprises or consists of a solid core of a solid albumin matrix or a continuous albumin material, in which the monoclonal antibody is distributed throughout the matrix, and in which the solid core is coated with a nonionic polymer.

[0067] In fact, a further aspect of the invention relates to a nanoparticle comprising a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core being coated with a non-ionic polymer.

[0068] More particularly, the invention also relates to a nanoparticle comprising a solid core, Petition 870200019339, dated 10 / 02 / 2020, p. 22 / 100 19 / 78 the said solid core consisting of a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, the said solid core being coated with a non-ionic polymer.

[0069] Also particularly, the invention relates to a nanoparticle consisting of a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core being coated with a non-ionic polymer.

[0070] Even more particularly, the invention also relates to a nanoparticle consisting of a solid core, said solid core consisting of a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core being coated with a non-ionic polymer. Albumin

[0071] As used in this document, the term albumin refers to a family of negatively charged globular proteins, the most common being serum albumins. All proteins in the albumin family are water-soluble, moderately soluble in concentrated saline solutions, and are subject to heat denaturation. Albumins are commonly found in blood plasma and differ from other blood proteins in that they are not glycosylated.

[0072] The general structure of albumin is Petition 870200019339, dated 10 / 02 / 2020, page 23 / 100 20 / 78 characterized by several long helices, allowing it to maintain a relatively static shape, essential for regulating blood pressure.

[0073] In one particular embodiment, albumin is serum albumin. Serum albumin is produced in the liver and dissolved in blood plasma, being the most abundant protein in mammals.

[0074] More preferably, serum albumin is human serum albumin (HSA) or bovine serum albumin (BSA), even more preferably serum albumin is human serum albumin.

[0075] Human serum albumin is encoded by the ALB gene, while other mammalian forms, such as bovine serum albumin, are chemically similar.

[0076] 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 a total of 17 disulfide bridges, one free thiol (Cys34), and a single tryptophan (Trp214). Monoclonal antibody

[0077] The term monoclonal antibody (mAb or moAb), as used in this document, refers to an antibody or antibody fragment produced by a single clone of B lymphocytes or by 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 producing antibody-forming hybrid cells from a fusion of an antibody-producing cell and a myeloma or other self-penetrating cell line. Petition 870200019339, dated 10 / 02 / 2020, p. 24 / 100 21 / 78

[0078] Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope (the part of an antigen that is recognized by the antibody). Given almost any substance, it is possible to produce monoclonal antibodies that bind specifically to that substance.

[0079] For the purposes of the present invention, the monoclonal antibody to be incorporated into the nanoparticle albumin matrix must have an affinity for at least one target in ocular tissue or within a cancerous tissue, or it must have an affinity for the tissue itself. For example, the target may be a receptor associated with an ocular disorder or cancer, or a protein associated with an ocular disorder or cancer.

[0080] In one particular embodiment, the monoclonal antibody is selected from bevacizumab (Avastin®) which inhibits the function of a natural protein called vascular endothelial growth factor (VEGF) that stimulates the formation of new blood vessels; ranibizumab (Lucentis®) which provides strong binding to VEGFA; trastuzumab (Herceptin®) which recognizes the HER-2 receptor overexpressed in solid tumors; cetuximab (Erbitux®) which recognizes EGFR receptors and rituximab (Mabthera®) which recognizes CD20.

[0081] In a preferred embodiment, one or more anti-VEGF antibodies (or fragments thereof) are selected to be incorporated into the albumin matrix, thus allowing targeting of vascular endothelial growth factor (VEGF) itself. Thus, in a preferred embodiment, the monoclonal antibody is Petition 870200019339, dated 10 / 02 / 2020, page 25 / 100 22 / 78 selected from bevacizumab and ranibizumab, most preferably bevacizumab. In another preferred embodiment, one or more anti-VEGF R2 antibodies (or fragments thereof) are selected for incorporation into the albumin matrix, thus enabling targeting of cells, such as retinal pigment epithelial cells, expressing vascular endothelial growth factor receptor 2 (VEGF R2). Examples of anti-VEGF R2 monoclonal antibodies include, but are not limited to, the mAb Avasl2al and mAb 2C3 clones. Overexpression of VEGF and VEGFR2 receptors by epithelial cells, such as retinal pigment epithelial cells, is associated, for example, with age-related macular degeneration (AMD).

[0082] In another preferred embodiment, the monoclonal antibody is an eye-targeting agent, that is, an antibody specific for an antigen produced by or associated with ocular tissue implicated in an ocular disorder. In a particular embodiment, the monoclonal antibody / albumin weight ratio ranges from 0.01 to 0.5, more preferably from 0.01 to 0.2. Nanoparticles having a monoclonal antibody / albumin weight ratio of less than 0.01 have been observed to be unstable over time. Non-ionic polymer

[0083] The term nonionic polymer, as used in this document, refers to a hydrophilic polymer that, under the preparative conditions of the nanoparticles, does not exhibit a net charge. Furthermore, said nonionic polymer must be biodegradable, or Petition 870200019339, dated 10 / 02 / 2020, page 26 / 100 23 / 78 In other words, they degrade during in vivo use, as well as being biocompatible, meaning they are substantially non-toxic or have a detrimental impact on the living tissues or living systems with which they come into contact.

[0084] Examples of suitable nonionic polymers for use in the present invention are polyvinyl alcohol; polyvinylpyrrolidone; polyalic 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 polyethers.

[0085] Preferred polysaccharides include, without limitation, xanthan gum, guar gum, starches, cellulose, dextran, and a combination of two or more of the foregoing.

[0086] Starches include, for example, corn starch and hydroxypropyl starch.

[0087] Cellulose includes, for example, alkylcelluloses, such as C1-C6 alkylcelluloses, including methylcellulose, ethylcellulose and n-propylcellulose; substituted alkylcelluloses, including C1-C6 hydroxyalkylcelluloses and C1-C6 hydroxyalkylcelluloses, such as hydroxyethylcellulose, hydroxy-n-propylcellulose, hydroxy-n-butylcellulose, hydroxypropylmethylcellulose and ethylhydroxyethylcellulose.

[0088] In a particular embodiment, the nonionic polymer is selected from a polysaccharide, polyvinylpyrrolidone, a polyester, and a polyalkylene glycol. Preferably, the nonionic polymer Petition 870200019339, dated 10 / 02 / 2020, page 27 / 100 24 / 78 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 with the trade name Eudagrit; or a polyacylene glycol, such as polyethylene glycol or polypropylene glycol.

[0089] In another particular embodiment, the nonionic 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-10,000, PEG-20,000 or PEG-35,000 according to its molecular weight.

[0090] Preferably, the nonionic polymer is selected from hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and PEG 35000. More preferably, the nonionic polymer is selected from hydroxypropyl methylcellulose phthalate and PEG 35000. Even more preferably, the nonionic polymer is PEG 35000.

[0091] The nonionic polymer acts as a coating for the albumin nanoparticles, providing greater stability and generally allowing an increase in the amount of monoclonal antibody that can be loaded onto the nanoparticle. It has been shown that the presence of the coating does not significantly affect the physical properties of the nanoparticle. Only depending on the nature of the coating can the size and zeta potential be affected. Petition 870200019339, dated 10 / 02 / 2020, page 28 / 100 25 / 78 slightly increased or decreased.

[0092] In a particular embodiment, the nonionic polymer / albumin ratio ranges from 0.02 to 5 (w / w), more preferably from 0.05 to 2 (w / w).

[0093] In another particular and optional embodiment, the nanoparticles used in the present invention further comprise a compound for protecting the albumin matrix during the drying process of the nanoparticles or the drying of the suspension containing the nanoparticles by conventional methods, for example, by spray drying, hereinafter referred to as the protective agent. Said protective agent is not part of the solid matrix of the nanoparticles, but acts as a bulking agent to facilitate the efficient drying of the nanoparticles so that their structure is maintained. Virtually any compound that meets these characteristics can be used as a protective agent. In one particular embodiment, said protective agent is a saccharide.

[0094] Illustrative but not limiting examples of protective agents that may 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 (e.g., galacto-oligosaccharides, human milk oligosaccharides), lactulose, dietary fiber, etc., and any combination thereof. In one particular embodiment, the protective agent is Petition 870200019339, dated 10 / 02 / 2020, page 29 / 100 26 / 78 selected from lactose, mannitol, sucrose, maltose, trehalose, maltodextrin, glucose, sorbitol, and combinations thereof. Preferably, the protective agent is sucrose. If the nanoparticles used in the invention include a protective agent, the weight ratio of the albumin matrix and the protective agent may vary within a wide range; however, in one specific embodiment, the albumin:protective agent ratio by weight is 1:0.1-5, typically 1:0.5-4, preferably about 1:1.

[0095] In some embodiments, the nanoparticles used in the invention comprise at least one imaging agent that allows for image-guided targeted release of the therapeutic agent, enabling the location of the particles to be visualized before, during, or after therapeutic release. A variety of imaging agents are suitable for coupling to the surface of the nanoparticles, including, but not limited to, fluorescence imaging agents (such as indocyanine green, cyanine 5, cyanine 7, cyanine 9, fluorescein, and green fluorescent protein), radionuclide-labeled imaging agents (such as agents comprising iodine-124,99mTc), and magnetic resonance imaging agents (such as gadolinium contrast agents).

[0096] The nanoparticles used in the invention can release the monoclonal antibody in a controllable process. This control can allow the release of the monoclonal antibody for an extended period of time. For example, it is contemplated that the release can occur over a period of 1 to 30 days. In addition to being adapted for Petition 870200019339, dated 10 / 02 / 2020, page 30 / 100 27 / 78 To deliver the therapeutic agent in a controllable manner, nanoparticles can be adapted to provide a variety of options for detecting and imaging the particles before, during, and after the delivery of the therapeutic agent.

[0097] The nanoparticles used in the present invention may optionally comprise a second monoclonal antibody or therapeutic agent in order to also provide a combination therapy.

[0098] The aforementioned therapeutic agent includes, for example, other proteins, such as calcitonin, insulin or cyclosporine A.

[0099] The second monoclonal antibody may be any of those mentioned above, such as bevacizumab (Avastin®), ranibizumab (Lucentis®), trastuzumab (Herceptin®), cetuximab (Erbitux®) or rituximab (Mabthera®). Process for the preparation of nanoparticles

[00100] The nanoparticles used in the present invention can be prepared by precipitating the proteins (albumin and monoclonal antibody) in an aqueous environment before purification and drying.

[00101] This process includes: a) preparation of an aqueous solution of albumin and a monoclonal antibody; b) titration of the aqueous solution from step a) to a pH between 4 and 5; c) addition of a desolvent to the aqueous solution from step b).

[00102] This method is based on a process of Petition 870200019339, dated 10 / 02 / 2020, p. 31 / 100 28 / 78 desolvation, in which an aqueous solution of albumin and the monoclonal antibody are slowly desolvated by adding, such as dropwise addition, a desolvating agent (usually an organic solvent such as ethanol, acetone or THF), under constant stirring, temperature and pH conditions.

[00103] In a particular embodiment, 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.

[00104] In another specific embodiment, the monoclonal antibody used in the preparation of the aqueous solution in step a) is selected from bevacizumab (Avastin®), ranibizumab (Lucentis®), trastuzumab (Herceptin®), cetuximab (Erbitux®) and rituximab (Mabthera®). More preferably, the monoclonal antibody is bevacizumab or ranibizumab, even more preferably it is bevacizumab.

[00105] The albumin and monoclonal antibody solution can be prepared by conventional methods known to those skilled in the art, for example, by adding the albumin and monoclonal antibody to the aqueous solution.

[00106] Albumin and monoclonal antibody are preferably mixed at room temperature, i.e., at a temperature between 18°C ​​and 25°C, preferably between 20°C and 22°C.

[00107] The amount of albumin that can be added to the aqueous solution can vary within a wide range; however, in one specific embodiment, the Petition 870200019339, dated 10 / 02 / 2020, p. 32 / 100 29 / 78 The amount added to the said aqueous solution is between 0.1% and 10% (w / v), preferably between 0.5% and 5% (w / v), even more preferably between 1% and 2%> (p / v).

[00108] Similarly, the amount of monoclonal antibody that can be added to the aqueous solution can vary within a wide range, however, in a specific embodiment, the amount added to said aqueous solution is comprised between 0.005% and 1% (w / v), preferably between 0.01% and 0.5% (w / v), even more preferably between 0.01% and 0.4% (w / v).

[00109] In a particular embodiment, albumin and monoclonal antibody are added to the aqueous solution, such that the weight ratio of monoclonal antibody:albumin ranges from 0.01 to 0.5, more preferably from 0.01 to 0.2.

[00110] In a preferred embodiment, the aqueous solution of albumin and the monoclonal antibody are homogenized by means of, for example, agitation.

[00111] Step b) of the nanoparticle preparation process involves reducing the pH of the aqueous solution containing the albumin and monoclonal antibody to a slightly acidic pH. This allows the nanoparticles to precipitate in the subsequent step of this process. This can be done by adding an acidic component to the aqueous solution obtained after performing step a), such as 1M HCl.

[00112] In one particular embodiment, the aqueous solution is incubated for at least 10 minutes at room temperature. Petition 870200019339, dated 10 / 02 / 2020, page 33 / 100 30 / 78

[00113] In step c) of the nanoparticle preparation process, a desolvating agent is added to the aqueous solution obtained after step b).

[00114] In a preferred embodiment, the desolvating agent is added to the aqueous solution obtained after performing step b) under stirring.

[00115] In another preferred embodiment, said desolvating agent is an organic solvent selected from ethanol and tetrahydrofuran (THF), most preferably ethanol.

[00116] The desolvating agent is added slowly to the aqueous solution under stirring. More preferably, the desolvating agent is added dropwise to the aqueous solution of albumin and monoclonal antibody while stirring the resulting mixture.

[00117] In a preferred embodiment, said addition is carried out under an inert atmosphere, such as under a nitrogen atmosphere.

[00118] After adding the desolvating agent to the aqueous solution of albumin and monoclonal antibody under the conditions mentioned above, i.e., at room temperature and under agitation, the nanoparticles of the invention are formed spontaneously. In a particular embodiment, said nanoparticles are in suspension in the medium in which they were obtained.

[00119] Thus, the invention process allows the formation of a uniform dispersion of nanoparticles through a simple desolvation process, leading to solid nanospheres with a matrix-like structure in which the monoclonal antibody is distributed throughout the Petition 870200019339, dated 10 / 02 / 2020, page 34 / 100 31 / 78 albumin matrix.

[00120] Therefore, the nanoparticles obtained by this process can be considered self-assembling nanoparticles that are spontaneously formed through local interactions between the monoclonal antibody and albumin after the addition of a desolvating agent.

[00121] The process for producing nanoparticles may include an additional purification step, for example, by means of filtration, centrifugation or ultracentrifugation techniques.

[00122] Similarly, the aforementioned process may include an additional drying step of the formed nanoparticles in order to obtain the nanoparticles of the invention in powder form. This form of presentation of said nanoparticles contributes to their stability and is also particularly useful for their eventual application in pharmaceutical products.

[00123] In a preferred embodiment, the nanoparticles obtained after performing step c), or after being purified, are subjected to a drying treatment by conventional methods, for example, vacuum drying or, advantageously, by spray drying or lyophilization (freeze-drying), in order to dry the nanoparticles.

[00124] In a particular embodiment, this drying treatment, particularly when carried out by spray drying or freeze-drying, comprises adding a protective agent to the nanoparticles once they have been formed. This protective agent protects the nanoparticles during the process of Petition 870200019339, dated 10 / 02 / 2020, page 35 / 100 32 / 78 drying of the same, such as, for example, a saccharide.

[00125] Illustrative but not limiting examples of saccharides that can be used as protective agents 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 (e.g., galacto-oligosaccharides, human milk oligosaccharides), lactulose, dietary fiber, etc., and mixtures thereof. In a particular embodiment, the protective agent is selected from lactose, mannitol, sucrose, maltose, trehalose, maltodextrin, glucose, sorbitol, and combinations thereof.If the nanoparticles include a protective agent, this is added in the appropriate quantity; even though the weight ratio of the nanoparticle matrix and the protective agent may vary within a wide range, in one specific embodiment, the albumin:protective agent ratio by weight is 1:0.1-5, typically 1:0.5-4, preferably about 1:1.

[00126] Nanoparticles can also be dried by spray drying. For this purpose, the suspension containing the nanoparticles and the protective agent is introduced into a spray dryer and the processing conditions [air inlet temperature, air outlet temperature, air pressure, sample pumping rate, suction and airflow] are controlled. Those skilled in the art can determine the most suitable processing conditions for each case.

[00127] This method allows obtaining Petition 870200019339, dated 10 / 02 / 2020, page 36 / 100 33 / 78 nanoparticles in the form of dry powder, which contributes to its stability during long periods of storage under controlled or environmental conditions and can also be easily incorporated into different desired solid and liquid products.

[00128] Since the nanoparticles are formed prior to the addition of the protective agent, this does not form any conjugate or complex with the albumin matrix.

[00129] In another particular embodiment, when the nanoparticles to be used in the invention are coated with a nonionic polymer, said coated nanoparticles can be obtained by incubating already formed monoclonal albumin nanoparticles, following steps a) ac) of the process as defined above, with the nonionic polymer.

[00130] In a particular embodiment, the nonionic polymer may be any of those described above. Preferably, said nonionic polymer is selected from hydroxypropyl methylcellulose, hydroxypropyl methyl cellulose phthalate, starch, dextran 70, Eudagrit® NM, Eudagrit® NE, polyvinylpyrrolidone and polyethylene glycol (PEG). More preferably, the nonionic polymer is selected from hydroxypropyl methylcellulose, hydroxypropyl methyl cellulose phthalate and PEG 35,000.

[00131] In one particular embodiment, the non-ionic polymer / albumin ratio ranges from 0.02 to 5, more preferably from 0.05 to 2.

[00132] In another particular embodiment, the incubation of nanoparticles in the nonionic polymer is Petition 870200019339, dated 10 / 02 / 2020, page 37 / 100 34 / 78 performed in less than 1 hour, more preferably in less than 45 minutes. Pharmaceutical composition

[00133] The nanoparticles described above have the ability to trap a monoclonal antibody and protect it during processing and storage, as well as until its final release to the biological site of interest. The deactivation of the monoclonal antibody after incorporation into the different intended products (e.g., pharmaceutical or cosmetic compositions) is thus prevented or substantially reduced. In fact, experimental tests have shown that the monoclonal antibody maintains its integrity in the albumin matrix.

[00134] Furthermore, the nanoparticles of the invention allow for a sustained release of the monoclonal antibody that also maintains its biological activity in its entirety, thus constituting a drug delivery system of great interest for in vivo applications, as indicated by the biological activity data obtained in an animal model of corneal neovascularization. In fact, the in vivo experiments performed showed that the albumin and monoclonal antibody nanoparticles provide a significant reduction in the ocular surface area affected by corneal vascularization when compared to the administration of the same monoclonal antibody in free form.

[00135] Furthermore, biodistribution assays performed with the nanoparticles of the invention indicate that they are capable of concentrating in tumor tissues, thus making them very promising nanoparticulate systems for releasing monoclonal antibodies into tissues. Petition 870200019339, dated 10 / 02 / 2020, page 38 / 100 35 / 78 carcinogens affected.

[00136] Therefore, in another aspect, the invention relates to a pharmaceutical composition comprising a plurality of nanoparticles as defined above, in the form of a suspension or in the form of a dry powder, and a pharmaceutically acceptable excipient, carrier or vehicle.

[00137] The characteristics of nanoparticles have already been defined above and are incorporated into this document by reference.

[00138] In one particular embodiment, the nanoparticles contained in the pharmaceutical composition of the invention are in the form of a dry powder.

[00139] Although any suitable means of administering the pharmaceutical composition may be used within the context of the present invention, preferably the pharmaceutical composition is administered to humans or animals orally, topically or parenterally, most preferably via intravenous administration, intra-arterial administration, intrapulmonary administration, intraocular administration, intramuscular administration, transdermal or subcutaneous administration, oral administration or inhalation.

[00140] More preferably, the pharmaceutical composition comprises a vehicle or vehicle suitable for oral, topical or parenteral administration.

[00141] Based on the particular method of administration, the pharmaceutical composition may be formulated as tablets, pills, capsules, sachets, granules, powders, suspensions, emulsions, formulations and topical solutions. Petition 870200019339, dated 10 / 02 / 2020, page 39 / 100 36 / 78 anhydrous or hydrated.

[00142] Acceptable pharmaceutical carriers or vehicles are well known to those skilled in the art and are readily available to the public. It is preferable that the pharmaceutically acceptable carrier or vehicle be chemically inert to the active formulation and to each of its components and one that has no adverse side effects or toxicities under conditions of use.

[00143] In some embodiments, the pharmaceutical composition is adapted as a delivery system to transport the therapeutic agent orally, topically, parenterally, or intravenously into an individual's circulatory system.

[00144] Suitable formulations for oral administration include liquid solutions, such as an effective amount of nanoparticles, or a composition comprising them, dissolved in diluents such as water or saline solution; capsules, sachets, tablets, lozenges, each containing a predetermined amount of nanoparticles; powders; suspensions in an appropriate liquid; and emulsions.

[00145] Topical formulations include aqueous ophthalmic solutions or suspensions, ophthalmic ointment, ocular insert, or any other formulation capable of delivering the nanoparticles to the external surface of the eye. Preferably, the topical formulation is an ophthalmic solution or suspension containing the nanoparticles for application as a liquid drop. Any of the formulations mentioned above may include a solvent. Petition 870200019339, dated 10 / 02 / 2020, page 40 / 100 37 / 78 suitable, preservatives and other pharmaceutically acceptable excipients commonly found in ocular formulations.

[00146] Parenteral formulations will typically contain 0.5 to 25% by weight of nanoparticles in solution. These formulations may be presented in sealed containers for single or multiple doses, such as ampoules and vials, and may be stored in lyophilized (freeze-dried) conditions requiring only the addition of a sterile liquid vehicle, for example, water for injections, immediately before use. Diseases to be treated

[00147] As mentioned above, albumin and monoclonal antibody nanoparticles have shown to be a very promising drug delivery system for the treatment of eye diseases and cancer.

[00148] Therefore, another aspect of the present invention relates to a nanoparticle or composition as defined above for use in the treatment of eye diseases.

[00149] In a specific embodiment of this aspect, the nanoparticle comprises a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core is not coated with any polymer. More particularly, said solid core is not coated with a non-ionic polymer.

[00150] In another specific embodiment of this aspect, the nanoparticle comprises a solid core, said solid core consisting of a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the Petition 870200019339, dated 10 / 02 / 2020, p. 41 / 100 38 / 78 monoclonal antibody is distributed throughout the albumin matrix; said solid core is not coated with any polymer. More specifically, said solid core is not coated with a non-ionic polymer.

[00151] In another specific embodiment of this aspect, the nanoparticle consists of a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core is not coated with any polymer. More particularly, said solid core is not coated with a non-ionic polymer.

[00152] In another specific embodiment of this aspect, the nanoparticle consists of a solid core, said solid core consists of a non-crosslinked albumin matrix and a monoclonal antibody, and wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core is not coated with any polymer. More particularly, said solid core is not coated with a non-ionic polymer.

[00153] In another aspect, the invention also relates to a method for treating an eye disease, said method comprising administering to an individual in need of such treatment a nanoparticle or composition comprising nanoparticles as described above.

[00154] In yet another aspect, the invention also relates to the use of a nanoparticle or composition comprising nanoparticles as described above for the manufacture of a medicament for the treatment of diseases. Petition 870200019339, dated 10 / 02 / 2020, page 42 / 100 39 / 78 eyepieces.

[00155] The aforementioned composition can be administered to the individual orally, topically, or by intraocular injection. In a preferred embodiment, the composition is administered topically, for example, via a route to the ocular mucosa or by intravitreal injection.

[00156] Thus, in a preferred embodiment, when nanoparticles are administered for the treatment of an eye disease, said nanoparticles are administered in a topical or injectable pharmaceutical composition, such as those described above in this document.

[00157] In another particular embodiment, the eye disease to be treated is selected from macular degeneration, neovascularization or angiogenesis of the cornea, neovascularization or angiogenesis of the iris, neovascularization or angiogenesis of the retina, diabetic proliferative retinopathy, non-diabetic proliferative retinopathy, glaucoma, infectious conjunctivitis, allergic conjunctivitis, ulcerative keratitis, non-ulcerative keratitis, episcleritis, scleritis, diabetic retinopathy, uveitis, endophthalmitis, infectious conditions and inflammatory conditions.

[00158] Another aspect of the present invention relates to a nanoparticle or composition as defined above for use in cancer treatment.

[00159] In one particular embodiment of this aspect, the nanoparticle comprises a solid core, Petition 870200019339, dated 10 / 02 / 2020, p. 43 / 100 40 / 78 said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core being coated with a non-ionic polymer.

[00160] In another particular embodiment of this aspect, the nanoparticle comprises a solid core, said solid core consisting of a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core being coated with a non-ionic polymer.

[00161] In another specific embodiment of this aspect, the nanoparticle consists of a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core being coated with a non-ionic polymer.

[00162] In another particular embodiment of this aspect, the nanoparticle consists of a solid core, said solid core consisting of a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, said solid core being coated with a non-ionic polymer.

[00163] In another aspect, the invention also relates to a method for the treatment of cancer, said method comprising administering to an individual in need of such treatment a nanoparticle or Petition 870200019339, dated 10 / 02 / 2020, page 44 / 100 41 / 78 composition comprising the nanoparticles as described above. In yet another aspect, the invention also relates to the use of a nanoparticle or composition comprising nanoparticles, as described above, for the manufacture of a medicament for the treatment of cancer.

[00164] In a preferred embodiment, the said composition is administered parenterally to the individual, for example, by intravenous, intra-arterial, intramuscular or subcutaneous administration.

[00165] Thus, in a preferred embodiment, when nanoparticles are administered for the treatment of cancer, said nanoparticles are administered in a parenteral formulation, such as those described above in this document.

[00166] In another particular embodiment, the cancer to be treated includes, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Examples of cancers to be treated by nanoparticle administration 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, ovarian cancer, liver cancer, renal and gastric cancer, bladder cancer, or squamous cell carcinoma.

[00167] In some embodiments, the nanoparticles used in the present invention, or compositions containing them, can be administered with a second therapeutic compound and / or second therapy, whether for the treatment of eye diseases or cancer.

[00168] The dosage frequency of the composition and the Petition 870200019339, dated 10 / 02 / 2020, page 45 / 100 42 / 78 The second compound or second therapy may be adjusted over the course of treatment. In some embodiments, the first and second therapies are administered simultaneously, sequentially, or concurrently. When administered separately, the nanoparticle composition and the second compound may be given at different frequencies or dosing intervals.

[00169] Alternatively, the nanoparticles used in the present invention may comprise a second monoclonal antibody or therapeutic agent, in order to also provide a combination therapy.

[00170] The aforementioned therapeutic agent includes, for example, other proteins, such as calcitonin, insulin or cyclosporine A.

[00171] The second monoclonal antibody may be any of those mentioned above, such as bevacizumab (Avastin®), ranibizumab (Lucentis®), trastuzumab (Herceptin®), cetuximab (Erbitux®) or rituximab (Mabthera®). Examples

[00172] In the examples provided below, the following abbreviations are used: BEVA: Bevacizumab B-NP: Albumin nanoparticles loaded with bevacizumab HSA: Human serum albumin PM: physical mixture NP-Glu: Albumin nanoparticles cross-linked with glutaraldehyde. B-NP-GLU: Charged albumin nanoparticles Petition 870200019339, dated 10 / 02 / 2020, page 46 / 100 43 / 78 with bevacizumab cross-linked with glutaraldehyde NP-PEG35: Albumin nanoparticles coated with polyethylene glycol 35,000 B-NP-PEG35: Albumin nanoparticles loaded with bevacizumab, coated with polyethylene glycol 35,000 B-NP-HPMC-P: Albumin nanoparticles loaded with bevacizumab and coated with hydroxypropylmethylcellulose phthalate. B-NP-S100: Albumin nanoparticles loaded with bevacizumab coated with Eudagrit S-100 Materials:

[00173] Human serum albumin or HSA (fraction V, purity 96-99%), polyethylene glycol 35,000 (PEG35) and 25% aqueous glutaraldehyde (GLU) were obtained from Sigma (Madrid, Spain).

[00174] Bevacizumab (Avastin®) was purchased from Roche (Spain). Hydroxypropylmethylcellulose K100 LV (HPMC; MW 164,000) from Ashland Chemical Hispania (Spain). Avastin® is supplied as a concentrate for solution for infusion in a single-use vial containing a nominal amount of 100 mg of bevacizumab in 4 mL or 400 mg of bevacizumab in 16 mL (concentration of 25 mg / mL). Hydroxypropylmethylcellulose phthalate (HPMC-P) was purchased from Acros Organic (Spain). The Micro BCA protein test kit was purchased from Pierce (Thermo Fisher Scientific Inc. (Illinois, USA)). The Shikari Qbeva Enzyme immunoassay used for bevacizumab detection was purchased from Matriks Biotech (Turkey). Acetone was purchased from Prolabo, VWR International Ltd (England) and tin chloride dihydrate and absolute ethanol were Petition 870200019339, dated 10 / 02 / 2020, page 47 / 100 44 / 78 acquired from Panreac Pharma (Spain). The isofluorine was from Braun and the euthanasia agent T-69 Intervet from Schering-Plough Animal Health. The technetium-99m pertechnetate eluate was obtained from a Drytec®99Mo-99mTc generator acquired from General Electric.

[00175] For the physicochemical studies, the following devices were used: Thermo / Nicolet 360FTIR (ESP Thermo Fisher Scientific, USA), a Bruker Axs D8 Advance diffractometer (Germany), for thermogravimetric (TG) analysis and differential scanning calorimetry (DSC) a Mettler Toledo dsc822e was used with the Mettler Toledo TSO 801RO sample robot and the Julabo FT900 chiller, and for elemental analysis a LECO CHN-900 elemental analyzer (Michigan, USA).

[00176] For the radiolabeling and biodistribution studies, the following devices were used: Symbia SPECT / CT, Siemens Medical Systems, Germany, Ativimeter AtomLab 500, Biodex, USA, Gamma counter, LKB Pharmacia. Physicochemical characterization of nanoparticles (size, zeta potential and morphology)

[00177] The particle size and zeta potential of the 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 using a dynamic light scattering angle of 90°C. The zeta potential was determined as follows: 200 pL of the samples were diluted in 2 mL of a 1 mM KCl solution adjusted to pH 7.4.

[00178] The morphological characteristics of Petition 870200019339, dated 10 / 02 / 2020, page 48 / 100 45 / 78 nanoparticles were studied by scanning electron microscopy (SEM) using a Zeiss DSM940 digital scanning electron microscope (Oberkochen, Germany). For this purpose, the samples were dispersed in water and centrifuged at 27,000 xg for 20 minutes at 4°C to eliminate the cryoprotectant. The pellets were then mounted on glass plates adhered with double-sided tape onto metal stubs and dried. They were coated with a 4 nm palladium-platinum layer using a Cressington 208HR spray applicator with a rotary-planetary tilt stage, equipped with an MTM-20 thickness controller. SEM was performed using a LEO 1530 instrument (LEO Electron Microscopy Inc, Thornwood, NY) operating between 1 and 3 kV with a filament current of approximately 0.5 mA. Production

[00179] The amount of HSA transformed into nanoparticles (yield) was determined by quantifying the HSA that forms the nanoparticles using a Micro BCA. Briefly, 10 mg of nanoparticles were weighed and dispersed in 10 mL of ultrapure water and centrifuged at 15,000 rpm for 15 min at 4°C (Rotor 3336, Biofuge Heraeus, Hanau, Germany). Then, the pellet was broken up with 1 mL of 0.02 N NaOH and 200 µL of this solution were transferred to a 96-well microplate and followed a specific micro-BCA protein assay kit in a spectrophotometer at 562 nm.

[00180] The selectivity of the kit was determined using controls containing the other excipients and drugs (glutaraldehyde, PEG 35,000, HPMC or bevacizumab) in order to Petition 870200019339, dated 10 / 02 / 2020, page 49 / 100 46 / 78 detect any possible interference in the albumin determinations. Data analysis was performed using the following equation: Yield (%) = (Wlyop / Winitial) x 100 [eq. 1] where Wlyop was the HSA that was transformed into nanoparticles and Winitial was the amount of HSA used to prepare the nanoparticles. Quantification of drug payload in nanoparticles

[00181] The amount of antibody carried on the albumin nanoparticles was estimated by enzyme immunoassay (Shikari Q-BEVA). For this purpose, 10 mg of the nanoparticles were weighed and dispersed in 1 mL of water. The suspension was centrifuged for 10 min at 10,000 rpm (Rotor 3336, Biofuge Heraeus, Hanau, Germany). The supernatant was removed. Then, the nanoparticles were broken up with 1 mL of 0.02N NaOH. A 200 pL amount of the resulting solution was transferred to a 96-well microplate coated with human vascular endothelial growth factor (VEGF) and subjected to a bevacizumab-specific ELISA (Q-Beva test procedure, Shikari Q-Beva, Matriks Biotek).

[00182] Each sample was tested in triplicate and calculations were performed using standard curves in the range of 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).

[00183] The loading of bevacizumab (DL) and its encapsulation efficiency (EE) were calculated according to the following equations: Petition 870200019339, dated 10 / 02 / 2020, page 50 / 100 47 / 78 DL = [Wencap / Wnp] [eq.2] EE = [Wencap / Wtotal] xlOO [eq. 3] where Wencap was the amount of bevacizumab encapsulated, Wtotal was the total amount of drug used, and Wnp was the weight of the nanoparticles. FT-IR determinations

[00184] The molecular structure of HSA nanoparticles was investigated by FTIR spectroscopy. Infrared spectra of samples dispersed at 1% of the sample in KBr disks were recorded on a NICOLET FTIR spectrometer (Thermo / Nicolet 360FT-IR ESP Thermo Fisher Scientific, USA). Samples were scanned from 4,000 to 400 cm⁻¹. The recording conditions were: resolution of 8.0 and sample scan of 40. The data were analyzed using OMNIC software (Thermo Fisher Scientific, USA). Radiographic studies

[00185] Radiological studies were conducted to study the distribution of crystallographic planes and the variability of the crystallinity of the polymer matrix in the different nanoparticle samples. For this purpose, the samples were placed in powder form on a metal plate in a diffractometer (Bruker Axs D8 Advance, Germany) - and measurements above 360° were performed at room temperature. The diffractograms were analyzed using the Diffrac.Suite.Thermal Analysis program.

[00186] The response of different nanoparticles to temperature changes was studied by thermal analysis (thermogravimetric analysis TGA coupled with thermal analysis). Petition 870200019339, dated 10 / 02 / 2020, page 51 / 100 48 / 78 differential DTA). The variations in the thermal behavior of the HSA functional groups were analyzed when it reacts to form nanoparticles. Thermal studies were performed using a Mettler Toledo TGA / sDTA 85le simultaneous thermal analyzer. Thermograms were obtained by heating approximately 5-10 mg of the sample in a perforated aluminum crucible at a scan rate of 10°C / min. from 25 to 250°C. Thermal analyses were performed under a static air atmosphere and N2 (20 mL min.-1) as purge gas. Measurements were made in triplicate. Elemental Analysis

[00187] Elemental analysis (C, H, O, and N) of the nanoparticles was performed to confirm the association of the different stabilizing agents using the LECO CHN-900 HSA nanoparticle elemental analyzer (Michigan, USA). Briefly, 1 mg of each sample was tested in triplicate and the results were expressed as a percentage (% w / w) of SD ± 0.4. This technique shows changes in the oxygen, hydrogen, or nitrogen composition of albumin (HSA) when associated with other components (glutaraldehyde, PEG35, HPMC-P, or bevacizumab). In vitro release study

[00188] In vitro release studies of bevacizumab-loaded albumin nanoparticles in PBS (pH 7.4) were performed. Eppendorf tubes containing 10 mg of each nanoparticle formulation were dispersed in a total volume of 1 mL of PBS, distributed into Eppendorf tubes, and placed in a stirring bath at 37°C with constant agitation of 60 movements / min (Unitronic 320 OR, Selecta, Madrid, Spain). At different intervals of Petition 870200019339, dated 10 / 02 / 2020, page 52 / 100 49 / 78 time, the Eppendorf tubes were taken and centrifuged for 10 min. at 10,000 rpm (Rotor 3336, Biofuge Heraeus, Hanau, Germany). The supernatants were analyzed for bevacizumab content using the specific ELISA test (Shikari QBeva, Matriks Biotek). Release profiles were expressed in terms of cumulative release as a percentage and plotted as a function of time.

[00189] Furthermore, based on the release profiles, the kinetics were examined using the exponential model of the Korsmeyer-Peppas equation (eq.4): Q = Ktn [eq. 4] where Q is the percentage of drug released at time te, K is a constant incorporating the structural and geometric characteristics of the device under investigation, and en is the diffusional exponent, which is normally used as an indicator of the drug transport mechanism from the dosage form.

[00190] An n value <0.43 indicates that drug release is controlled by Fickian diffusion, while an n value ≥ 0.85 suggests that drug release is dominated by an erosion mechanism. For values ​​0.43 <n <0,85, a liberação é descrita como anômala, implicando que uma combinação de difusão e erosão contribui para o controle da liberação do fármaco [Gao Y. e outros, In Vitro Release Kinetcs of Antituberculosis Drugs from Nanoparticles Assessed Using a Modified Dissolution Apparatus. Example 1. Preparation of human serum albumin nanoparticles loaded with bevacizumab. Influence of the bevacizumab / HSA ratio on the physicochemical properties of the nanoparticles. Petition 870200019339, dated 10 / 02 / 2020, page 53 / 100 50 / 78 resulting nanoparticles

[00191] Nanoparticles loaded with bevacizumab were prepared by a procedure in which the nanoparticles were obtained by precipitating the proteins in an aqueous environment before purification and drying.

[00192] 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 with 1 M HCl. The mixture was incubated at room temperature for 10 minutes. Nanoparticles were obtained by the continuous addition of 16 mL of ethanol used as a desolvent under continuous stirring (500 rpm) at room temperature. The resulting nanoparticles were purified by two different procedures: ultracentrifugation and ultrafiltration. In the first, the nanoparticles were purified twice by centrifugation at 21,000 x g for 20 min at 4°C (Sigma 3K30, Osterodeam Harz, Germany) and redispersion of the microsphere in the original volume in water. In the latter, the nanoparticles were purified by ultrafiltration through a polysulfone membrane cartridge with a pore size of 50 kDa (Medica SPA, Italy). Finally, the nanoparticles were lyophilized in a Genesis 12EL apparatus (Virtis, New York, USA) after redispersion or addition of a 5% aqueous sucrose solution. These formulations are human serum albumin nanoparticles loaded with bevacizumab, without any additional stabilization, hereinafter referred to as B-NP formulations.

[00193] For antibody encapsulation Petition 870200019339, dated 10 / 02 / 2020, page 54 / 100 51 / 78 monoclonal antibodies in nanoparticles, two main parameters were identified: the bevacizumab / albumin ratio and the incubation time between the two compounds before nanoparticle formation. Table 1 summarizes the main physicochemical properties of the resulting nanoparticles varying the monoclonal antibody / protein ratio. When the bevacizumab / albumin ratio was low (e.g., 0.01), nanoparticles did not exhibit stability over time. For ratios greater than 0.01, the resulting nanoparticles were stable with an average size close to 300 nm and a negative surface charge of approximately -15 mV.

[00194] Similarly, the process yield was calculated at approximately 80%. Figure 1 shows the effect of the bevacizumab / albumin ratio on the monoclonal antibody load. According to these results, the amount of bevacizumab loaded onto the nanoparticles increased with increasing BEVA / HSA ratio. All these experiments were performed after 10 min. of incubation between the monoclonal antibody and the protein. Interestingly, no significant differences were observed in the physicochemical properties of the nanoparticles when this parameter was increased. Table 1. Influence of the bevacizumab / albumin ratio on the physicochemical properties of the resulting nanoparticles. Incubation time between bevacizumab and albumin: 10 min. Data expressed as mean ± SD (n = 3). Petition 870200019339, dated 10 / 02 / 2020, page 55 / 100 52 / 78 BEVA / HSA Rate Average Size (nm) PDI Zeta Potential (mV) Yield (%) 0.01 240 ± 3 0.27 ± 0.01 -18.9 ± 0.9 75 0.03 326 ± 7 0.21 ± 0.03 14.1 ± 0.3 78 0.05 353 ± 6 0.26 ± 0.01 -11.7 ± 0.2 78 0.08 304 ± 4 0.16 ± 0.03 -15.5 ± 0.3 80 0.15 306 ± 4 0.22 ± 0.01 -16.1 ± 0.3 85 Example 2 - Influence of crosslinking bevacizumab-loaded nanoparticles with glutaraldehyde on their physicochemical properties.

[00195] Because human serum albumin nanoparticles in the absence of bevacizumab are not stable and disappear soon after formation, control nanoparticles loaded with bevacizumab were obtained after cross-linking with 12.5 μg of glutaraldehyde in ethanol (300 μL), hereinafter referred to as B-NP Formulations of GLU. For this purpose, the newly formed bevacizumab-loaded nanoparticles were incubated for 5 min. with glutaraldehyde before purification and lyophilization.

[00196] Table 2 summarizes the main physicochemical properties of the bare HSA nanoparticles (without further stabilization procedures) and the control properties obtained after cross-linking with glutaraldehyde. Encapsulation of bevacizumab in albumin nanoparticles produced stable nanoparticles with a high antibody content. Interestingly, the encapsulation efficiency calculated as the active monoclonal antibody loaded onto nanoparticles was close to 90% with a bevacizumab load of approximately 13%. When the Petition 870200019339, dated 10 / 02 / 2020, page 56 / 100 53 / 78 nanoparticles loaded with bevacizumab were cross-linked with glutaraldehyde; the resulting nanoparticles were slightly smaller than those produced without the cross-linking chemical. However, treatment of nanoparticles with glutaraldehyde inactivated the monoclonal antibody, and very low levels of the antibody were quantified by ELISA analysis. Table 2. Physicochemical characteristics of cross-linked and untreated albumin nanoparticles and with glutaraldehyde. The nanoparticles were prepared in a bevacizumab / albumin ratio of 0.15 and 10 min. of incubation before nanoparticle formation. Data expressed as mean + / - SD (n = 3). PDI: polydispersity index; BEVA: bevacizumab; EE: encapsulation efficiency; NP: nanoparticles; GLU: glutaraldehyde. Size (nm) PDI Zeta Potential (mV) Yield (%) BEVA Loading (ug / mg NP) EE (%) NP NA NA NA NA - - B-NP 310 ± 3 0.14 ± 0.02 -14 ± 1 85 ± 3 132 ± 5 89 ± 0 NP- GLU 163 ± 2 0.17 ± 0.01 -36 ± 0 6 6 ± 5 - - B-NP- GLU 270 ± 3 0.11 ± 0.03 -39 ± 1 68 ± 2 0.1 ± 0.3 0.1 ± 1.3 Example 3: Characterization of nanoparticles loaded with bevacizumab HE HAS

[00197] Figure 2 shows the morphology of bevacizumab-loaded nanoparticles (B-NPs), which have a spherical shape and an irregular surface. Petition 870200019339, dated 10 / 02 / 2020, page 57 / 100 54 / 78 FT-IR determinations

[00198] IR allows the evaluation of the appearance of conformational changes in the secondary structure of the protein. Figure 3 shows the FT-IR spectra of nanoparticles loaded with bevacizumab, compared to those of the monoclonal antibody alone and the protein and the physical mixture thereof.

[00199] Infrared protein spectra exhibit a number of amide bands, which represent different vibrations of the peptide moieties. These signals, amide band I ranging from 1600 to 1700 cm⁻¹ (mainly C=O stretching) and amide band II at 1550 cm⁻¹ (CN stretching coupled to the NH folding mode), have been used as evidence of the presence of this chemical and are directly related to the protein's secondary structure. However, amide band I is more sensitive to changes in the protein's secondary structure than amide band II. Thus, changes in the frequencies and intensities of these signals are evidence of interaction with the protein.

[00200] In this case, and due to the fact that the nanoparticles are formed by two different proteins (albumin and bevacizumab), a small variation in the frequency of the signal corresponding to the amide I peak was observed (1,642 for HSA and 1,645 cm-1 for B-NP).

[00201] As a control, glutaraldehyde-crosslinked nanoparticles were also studied. In this case, a slight shift in the frequencies of amide I (1,642 to 1,645 cm-1) was also found as a result of the interaction between glutaraldehyde and albumin. Petition 870200019339, dated 10 / 02 / 2020, page 58 / 100 55 / 78 X-ray studies

[00202] Figure 4 shows the X-ray spectrum of human serum albumin, bevacizumab, and bevacizumab-laden nanoparticles. In all cases, these spectra show an amorphous structure. Thermal analysis

[00203] Thermal analyses were performed to determine the reaction between the functional groups of HSA and bevacizumab. Figure 5 shows the thermograms of native albumin (HSA) and bevacizumab (BEVA) (A), bevacizumab nanoparticles (B-NP) and the physical mixture (PM) of albumin and bevacizumab (B), native albumin (HSA) and glutaraldehyde (GLU) (C), cross-linked glutaraldehyde nanoparticles (NP-GLU) and the physical mixture (PM) of albumin and glutaraldehyde (D).

[00204] The thermograms show that native albumin exhibits an exothermic effect around 30°C, which corresponds to a reversible transition, and a second thermal effect due to an endothermic glass transition.

[00205] The absence of the exothermic signal corresponding to albumin in NPs can be attributed to a combination of albumin with protein (BEVA) and a crosslinking agent (glutaraldehyde). Thus, bevacizumab and albumin would form a complex. Elementary Analysis

[00206] Table 3 shows the elemental analysis of human serum albumin, bevacizumab, glutaraldehyde-crosslinked albumin nanoparticles, and bevacizumab-loaded albumin nanoparticles. Bevacizumab exhibits a significantly lower carbon and nitrogen content. Petition 870200019339, dated 10 / 02 / 2020, page 59 / 100 56 / 78 that of human serum albumin. Conversely, the oxygen content in the monoclonal antibody is about 2 times higher than in albumin. Similarly, nanoparticles loaded with bevacizumab (B-NP) showed a lower percentage of nitrogen and a higher oxygen content than native albumin. Table 3. Elemental analysis of human serum albumin (HSA), bevacizumab (BEVA), glutaraldehyde-crosslinked albumin nanoparticles (GLU-NPs), and bevacizumab-loaded nanoparticles (B-NPs). %C %H %N %0O HSA 48.34 6. 96 17.80 26. 91 BEVA 36. 70 6. 64 4.25 52.41 NP-GLU 48.09 6.87 15.19 29.85 B-NP 48. 77 6.86 14.90 29.48 Example 4: Stability of nanoparticles loaded with bevacizumab

[00207] The stability of the nanoparticles was evaluated in ultrapure water. Samples were dispersed in purified water and stored at room temperature for 3 days. At different time intervals, stability was evaluated by measuring the size, polydispersity index, and zeta potential of the nanoparticles.

[00208] After dispersion in water (pH adjusted to 7.4), the nanoparticles loaded with bevacizumab remained stable for at least 24 hours (Figure 6). Their behavior was similar to that observed for empty nanoparticles crosslinked with glutaraldehyde (Figure 6). Similarly, the polydispersity index Petition 870200019339, dated 10 / 02 / 2020, page 60 / 100 The 57 / 78 (PDI) of B-NP was not affected during the experiment. Thus, at t = 0, the PDI was 0.19 ± 0.01 and, 24 hours later, this parameter was calculated at 0.16 ± 0.03 (data not shown). Example 5: In vitro release of bevacizumab from nanoparticles

[00209] Figure 7 shows the in vitro release profile of bevacizumab from albumin nanoparticles in PBS. This profile was characterized by an initial disruption effect of approximately 23% of the loaded antibody during the first 5 minutes, followed by a controlled slow release over the next 24 hours. At the end of the experiment, approximately 40% of the loaded bevacizumab had been released. The disruption release could be related to the antibody adsorbed on the surface of the nanoparticles. Example 6 - Preparation and characterization of albumin nanoparticles coated with bevacizumab

[00210] The encapsulation of bevacizumab in human serum albumin nanoparticles decorated with different compounds was prepared by a 4-step procedure. In particular, non-ionic HPMC-P and PEG35 were selected to explore their capabilities to decorate bevacizumab-loaded nanoparticles. The Eudragit S-100 ionic coating was also used.

[00211] The first stage was dedicated to the production of nanoparticles in an aqueous environment. Then, the surface of the nanoparticles was decorated by simple incubation in an aqueous environment. The third stage was used to purify the resulting nanoparticles, which... Petition 870200019339, dated 10 / 02 / 2020, page 61 / 100 58 / 78 were finally dried.

[00212] Step one - 100 mg of HSA and a variable amount of bevacizumab (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 with 1 M HCl. The mixture was incubated at room temperature for 10 minutes. The nanoparticles were obtained by continuous addition of 16 mL of ethanol used as a desolvent under continuous stirring (500 rpm) at room temperature.

[00213] Second stage - For coating the newly formed bevacizumab-laden nanoparticles, one of the following compounds was added: PEG 35,000, hydroxymethylpropyl cellulose phthalate or Eudragit S-100.

[00214] As a control, bevacizumab-loaded nanoparticles were stabilized by cross-linking with 12.5 pg of glutaraldehyde in ethanol (300 pL) for minutes, as described above.

[00215] Third stage - The resulting nanoparticles were purified. Two different procedures were used: ultracentrifugation and ultrafiltration. In the first, the nanoparticles were purified twice by centrifugation at 21,000 xg for 20 min. at 4°C (Sigma 3K30, Osterodeam Harz, Germany) and redispersion of the microsphere in the original volume in water. In the latter, the nanoparticles were purified by ultrafiltration through a polysulfone membrane cartridge with a pore size of 50 kDa (Medica SPA, Italy).

[00216] Fourth stage - Finally, the nanoparticles were freeze-dried in a Genesis apparatus. Petition 870200019339, dated 10 / 02 / 2020, page 62 / 100 59 / 78 12EL (Virtis, New York, USA). When ultracentrifugation was used as a purification method, the microsphere from the last centrifugation was dispersed in a 5% aqueous sucrose solution. When ultrafiltration was used, the sediment was also redispersed in a 5% aqueous sucrose solution before lyophilization.

[00217] Table 4 summarizes the main physicochemical properties of these nanoparticles. Overall, the amount of bevacizumab loaded was always similar and close to 14%. However, incubation of bevacizumab-loaded nanoparticles with different excipients for coating purposes produced nanoparticles with modified physicochemical properties. Thus, PEG35-coated nanoparticles encapsulating bevacizumab (B-NP-PEG35) exhibited average sizes and negative zeta potentials similar to bare bevacizumab-loaded nanoparticles (B-NP). Conversely, when B-NP was incubated with HPMC-P, the average size of the resulting nanoparticles increased significantly compared to B-NP. When incubation was performed with the ionic Eudragit® S-100, the resulting nanoparticles exhibited a reduced size and an increased negative zeta potential, compared to B-NP.Through SEM, the albumin nanoparticles showed a spherical shape and a smooth surface.

[00218] Table 4 - Physicochemical characteristics of bevacizumab encapsulated in coated albumin nanoparticles. Data expressed as mean ± SD (n = 3). PDI: polydispersity index. Coating agents: S-100 (Eudragit® S100 ionic), HPMC-P (phthalate of Petition 870200019339, dated 10 / 02 / 2020, page 63 / 100 60 / 78 hydroxypropylmethylcellulose), PEG35 (polyethylene glycol 35,000). CA / protein ratio: coating agent / albumin ratio. B-NP-GLU: bevacizumab-loaded albumin nanoparticles cross-linked with glutaraldehyde. B-NP: bevacizumab loaded onto bare albumin nanoparticles. Protein / CA Ratio Incubation Time Size (nm) PDI Zeta Potential (mV) Yield (%) Loading BEVA (pg / mg NP) B-NP-GLU - 180 ± 3 0.11±0.01 -36 ± 1 75 ± 2 0.1 ± 1 B-NP - 310 ± 3 0.14±0.02 -14 ± 1 85 ± 3 132 ± 5 B-NP-HPMCP 0.1; 10 min. 369 ± 1 0.15±0.01 -13 ± 1 76 ± 4 142 ± 4 B-NP-PEG35 0.5; 35 min. 301 ± 2 0.13±0.03 -17 ± 1 63 ± 7 145 ± 6 B-NP-SIOO 0.25; 10 min. 252 ± 4 0.07±0.01 -27 ± 1 86 ± 3 148 ± 5

[00219] The morphological study (Figure 8) of bevacizumab-loaded nanoparticles coated with PEG35 (B-NP-PEG35) shows that they are spherical with an irregular surface and homogeneous dispersion. Example 7: In vitro release of bevacizumab from coated nanoparticles

[00220] Figure 9 shows the in vitro release profile of bevacizumab from albumin nanoparticles coated with two non-ionic polymers (PEG35 and HPMC-P) and with the ionic Eudragit® S100 in PBS at pH 7.4. For PEG35-coated nanoparticles (B-NP-PEG35), the profile was similar to that observed for bare nanoparticles (BNP), with the difference that, at the end of the experiment, the amount of bevacizumab released was greater than for B-NP. In any case, these pegylated nanoparticles Petition 870200019339, dated 10 / 02 / 2020, page 64 / 100 61 / 78 offered a biphasic release pattern characterized by an initial disruption effect in the first 5 minutes of approximately 22%, followed by a more sustained and slower release rate for at least 24 hours. The disruption release was almost 22% and may be related to antibody adsorbed on the surface of the nanoparticles. The biphasic section, ignoring the first 5 minutes of disruption release, was fitted using the Korsmeyer-Peppas equation for a diffusion profile (n = 0.54; R2= 0.994). During the diffusion stage, bevacizumab release was up to 48%, reaching a plateau after the first two hours.

[00221] For HPMC-P coated nanoparticles (B-NP-HPMC-P), again, the amount of bevacizumab released during the first 60 minutes was about 40%. Then, a continuous release rate of the remaining antibody was observed. However, in this case, the release rate of bevacizumab was faster than for BNP or B-NP-PEG35. Thus, after 8 h of incubation, almost 100% of the bevacizumab content was released from HPMC-P coated nanoparticles.

[00222] Unlike nanoparticles coated with non-ionic polymers, nanoparticles coated with ionic Eudragit® S-100 (B-NP-S-100) exhibited an immediate release profile. Example 8 - Integrity of bevacizumab after encapsulation in albumin nanoparticles

[00223] To corroborate the results obtained with the ELISA kit used to quantify bevacizumab loaded in albumin nanoparticles, the integrity of the antibody Petition 870200019339, dated 10 / 02 / 2020, page 65 / 100 62 / 78 (bevacizumab) encapsulated in different nanoparticles was analyzed by automated microfluidic electrophoresis using the Experion™ Automated Electrophoresis System (Bio Rad, USA). Samples were evaluated under non-reducing and reducing conditions using 2-mercaptoethanol. The data obtained were processed using the Experion System software.

[00224] The nanoparticles were weighed and broken down with 1 mL of 0.005 N NaOH. The concentration of the different solutions was approximately 400 ng of protein / μL (the linear dynamic range of the test is 5-2,000 ng / μL). Free protein samples (albumin and bevacizumab) were used as controls. All these samples were evaluated as obtained or after treatment with β-mercaptoethanol and heat. Subsequently, the samples were treated following the protocol of the Experion System Pro260 analysis kit (Bio-Rad Lab., USA). Once the samples and controls were loaded onto the chip, they were analyzed by the Experion™ Automated Electrophoresis System (Bio-Rad, USA).

[00225] The results were obtained as densitometric bands on a virtual gel. Each band corresponded to a different sample. The Experion software identifies the different size peaks and expresses them in kilodaltons (kDa) in the system's control band.

[00226] The results of the studies are shown in Figure 10. In lane 5, bevacizumab appears as a strong band around 150 kDa. A similar band was observed in lanes 2 (B-NP) and 3 (B-NP-PEG35). Similarly, the bands corresponding to albumin (lane 4) also appear clearly in lanes 1-3. Petition 870200019339, dated 10 / 02 / 2020, page 66 / 100 63 / 78 Example 9 - Biodistribution study of ocularly administered nanoparticles in Wistar rats Radiolabeling and biodistribution study of NP in Wistar rats for in vivo SPECT-CT imaging.

[00227] Radiolabeling of the nanoparticles was performed with 99mTc by reduction of 99mTc-pertechnetate with tin chloride, following a method described elsewhere. Briefly, 20 pL of a tin chloride dihydrate solution in water for injection and a final tin concentration of 0.02 mg / mL was added to 9 mg of lyophilized nanoparticles, followed by the addition of 60 pL of 99mTcO4 eluate to the reduced tin. A quantity of 4 pL of the radiolabeled nanoparticle suspension (5 MBq) was mixed with 0.6 mg of unlabeled nanoparticle formulation and this mixture was carefully administered to the right eye of Wistar rats anesthetized with isoflurane. Ophthalmic administration in Wistar rats for in vivo SPECT-CT imaging.

[00228] The animals were kept under anesthesia for one hour to prevent active removal of the eye suspension, after which they were awakened and SPECT-CT images were obtained at six different times between 5 and 17 hours and 30 min after nanoparticle administration.

[00229] For imaging studies, animals were anesthetized immediately before each study with isoflurane and placed in the prone position in a Symbia T2 Truepoint SPECT-CT system (Siemens). Images were acquired using a 128x128 matrix, 7 images / s; CT was set to 110 mAs and 130 kV, 130 images of 3 mm thickness. Image fusion was performed using the Petition 870200019339, dated 10 / 02 / 2020, page 67 / 100 64 / 78 software Syngo MI Applications TrueD. The images were processed and quantified using the embedded software system. Quantitative values ​​were obtained through the automatic drawing of an isocontour in the three planes over the selected areas to obtain the Volumes of Interest (VOIs), from which the average count values ​​were obtained.

[00230] Figures 11 and 12 show the biodistribution of B-NP and B-NP-PEG35 after ocular administration as eye drops. The radioactivity associated with the nanoparticles remains in the eye for at least 4 h in the case of B-NP and for 8 h in the case of B-NP-PEG35, although it slowly disappears from the point of administration and enters the gastrointestinal tract. The transit of radiolabeled nanoparticles through the animal's pharynx can be seen in the leftmost image of Figure 11. The intensity of the SPECT images in Figure 11 has been scaled to the highest intensity point in each individual image to better appreciate the position of the radioactivity in the animal's body.

[00231] The evolution of radioactivity over time corrected for semi-quantitative decay is plotted in Figure 12 for B-NP. The results with B-NP-PEG35 were very similar; however, the nanoparticles take longer to be excreted because they remain in the eye for a longer time. Example 10 - Biodistribution of nanoparticles after intravenous administration in male Wistar rats

[00232] Bevacizumab-loaded HSA nanoparticles (B-NP) and HSA nanoparticles loaded with Petition 870200019339, dated 10 / 02 / 2020, page 68 / 100 65 / 78 bevacizumab and PEG35-coated (B-NP-PEG35) tablets were administered for in vivo biodistribution imaging experiments in Wistar rats. 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 after administration.

[00233] One hour after intravenous administration, radioactivity associated with the injection of B-NP and BNP-PEG35 is observed in the liver and kidneys, as can be seen in Figure 13. It is also worth noting that there is less hepatic uptake of B-NP-PEG35. B-NP and B-NP-PEG35 do not accumulate in any organ. Example 11 - Effect of albumin nanoparticles loaded with bevacizumab on corneal neovascularization

[00234] Male Wistar rats weighing approximately 200 g were obtained from Harlan to test the efficacy of bevacizumab-loaded nanoparticles in a rat model of corneal neovascularization. The studies were approved by the Ethical Committee for Animal Experimentation of the Institution (protocol number 172-14), in accordance with European legislation on animal experimentation.

[00235] The animals were kept under sedation 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 cycloplegic eye drops (Coliricusi Tropicamide, 10 mg / mL, Alcon) was administered to each eye of the rats. After 5 minutes, the corneas of the rats were burned by applying a silver nitrate stick. Petition 870200019339, dated 10 / 02 / 2020, p. 69 / 100 66 / 78 (Argepenal, Braun) was applied to the surface of the eyes for 5 seconds. Finally, the eyes were washed with a sterile 0.9% w / v NaCl solution.

[00236] Twelve hours later, the animals were anesthetized with isoflurane (Isovet, Spain) and divided into different groups. The following treatments were applied as eye drops to the animals: (i) 10 μL aqueous solution of 4 mg / mL bevacizumab (Avastin®) every 12 hours for 7 days, (ii) 10 μL of an aqueous solution of 4 mg / mL aflibercept (Eylea®) every 12 hours for 7 days, (iii) 10 μL of a 0.1% aqueous solution of dexamethasone phosphate (Coliriculi dexamethasone®) every 12 hours for 7 days, (iv) bevacizumab-loaded albumin nanoparticles (B-NP; 10 μL suspension containing 40 μg of bevacizumab) daily for one week, and (v) PEG35-coated bevacizumab-loaded albumin nanoparticles. (B-NP-PEG35; 10 μL, suspension containing 40 μg of bevacizumab) every day for 1 week.One group of animals received physiological saline solution (PBS) as a control, and another group of animals received human serum albumin dissolved in PBS (HSA) in a quantity similar to that administered with B-NP-PEG35.

[00237] Figure 14 corresponds to the schematic timeline showing corneal cauterization occurring at 0 h (day 0) and the first treatment at 24 h (day 1).

[00238] For the calculations, digital images of the corneas were taken and analyzed using ImageJ software (public domain, http: / / rsb.info.nih.gov / ij / ). The images were analyzed in binary mode, transforming the Petition 870200019339, dated 10 / 02 / 2020, page 70 / 100 67 / 78 images in black and white format. From these images, the total corneal area was determined, as well as the corneal surface area occupied by the silver nitrate burn (lesion) and the area affected by the generation of new vessels (corneal neovascularization) by pixel counting. From these parameters, the invasive area (IA) and the CNV (corneal neovascularization normalized by the lesion surface) were determined as follows: IA = [(area affected by the genesis of new vessels) / (total corneal area)] x 100 CNV = IA / (eye surface area affected by the burn)

[00239] Table 5 summarizes the effectiveness of the different bevacizumab treatments as a result of the reduction in the eye surface area affected by lesion-induced neovascularization (Figure 15). In all cases, the lesion induced in the eyes of the animals was similar and no statistical differences were found in the lesion areas between the four groups (p > 0.05; Figure 16).

[00240] The group of animals treated with bevacizumab solution (BEVA) showed a lower surface area of ​​the eye affected by neovascularization, compared to animals that received PBS (negative control). On the other hand, in animals treated with bevacizumab-loaded nanoparticles (B-NP), the surface area of ​​the eye affected by corneal neovascularization was 2.7 times smaller than in animals treated with Avastin® (BEVA) (Figures 17 and 18). When animals were treated with bevacizumab loaded onto a pegylated nanoparticle, the decrease in the surface area affected by neovascularization was Petition 870200019339, dated 10 / 02 / 2020, page 71 / 100 68 / 78 was approximately 1.4 times lower than in animals treated with Avastin®. It is important to note that animals treated with nanoparticles received 50% less bevacizumab than animals treated with Avastin®. Another important observation was that, under our experimental conditions, neither dexamethasone nor Eylea® demonstrated a positive effect on neovascularization (Table 9, Figure 15).

[00241] Table 5 - Effect of bevacizumab formulations on corneal neovascularization induced by burning with a silver nitrate stick. BEVA: bevacizumab solution (Avastin®, 4 mg / mL, twice daily for 7 days); B-NP: bevacizumab-loaded albumin nanoparticles (4 mg / mL, once daily for 7 days); B-NP-PEG35: bevacizumab-loaded, PEG35-coated albumin nanoparticles (4 mg / mL, once daily for 7 days); HSA: human serum albumin solution; Dexamethasone: 0.1% solution, twice daily for 7 days; Eylea: Aflibercept solution (Eylea®, 4 mg / mL, twice daily for 7 days); Control: PBS. IA: area of ​​invasion. CNV: corneal neovascularization normalized by the lesion surface. Data expressed as mean + / - SD of n = 9. Surface area of ​​the eye affected by lesion (%) IA (%) CNV Control (-) 17.3 ± 4.0a 31.3 ± 4.6a 1.89 ± 0.49a BEVA 15.1 ± 2.6a 24.4 ± 4.7c 1.69 ± 0.53a B-NP 15.1 ± 2.9a 9.4 ± 1.6e 0.74 ± 0.24d B-NP-PEG35 16.0 ± 4.1a 17.7 ± 2.9d 1.18 ± 0.38b HSA 15.7 ± 3.9a 36.0 ± 5.9a 2.41 ± 0.60a Dexamethasone 17.7 ± 1.1a 34.4 ± 6.7a 1.94 ± 0.35a Petition 870200019339, dated 10 / 02 / 2020, page 72 / 100 69 / 78 Eylea 15.5 ± 2.2a 34.1 ± 6.5a 2.27 ± 0.68a bp <0.05 ANOVA followed by Tukey's test significantly different from control (-) cp <0.01 ANOVA followed by Tukey's test significantly different from control (-) dp <0.005 ANOVA followed by Tukey's test significantly different from control (-) ep <0.001 ANOVA followed by Tukey's test significantly different from control (-) Histology

[00242] For histological study of the eyes; with After corneal neovascularization, at the end of treatment, 2 eyes from each group were enucleated. The ocular surface was washed with saline solution and the anterior pole was separated from the posterior pole. The corneas were mounted in a plane, fixed with 4% paraformaldehyde for 24 hours, and then several washes were performed on each sample with PBS. The corneas were kept in 70% methanol for subsequent sectioning and analysis.

[00243] For corneal analysis, corneas were embedded in paraffin, and 4-micrometer sections were cut from the center of the cornea and the area of ​​neovascularization. These were then stained with hematoxylin-eosin and analyzed by light microscopy. Evaluation of the sections included the intensity of neovascularization, intensity of inflammation, fibrosis, edema, and average corneal thickness. The study was conducted by an examiner blinded to the treatment groups. Images were acquired using a Nikon Eclipse microscope. Petition 870200019339, dated 10 / 02 / 2020, page 73 / 100 70 / 78 Ci was equipped with a Nikon DS-Ri 1 digital camera. The images were analyzed using Nikon's NIS-element calibrated digital image analysis system.

[00244] To assess the intensity of neovascularization, the following classification was used: 0 = absence of neovascularization; 1 = minimal or almost negative vascularization; 2 = mild vascularization; 3 = limited or focal vascularization in the subepithelial and prestromal areas (moderate neovascularization); 4 = very frequent or intense; 5 = diffuse and intense vascularization. Similarly, the intensity of inflammation was classified as follows: 0 = absence of inflammation; 1 = minimal or almost negative inflammation; 2 = low focal count of mixed inflammatory cell types, such as lymphocytes, neutrophils, and eosinophils; 3 = moderate inflammation; 4 = very frequent or intense; 5 = intense, diffuse, and mixed inflammatory cell types.The scaling system for fibroblast activity was: 0 = absence of fibroblast activity; 1 = minimal or near-negative fibroblast activity; 2 = focal fibroblast activity; 3 = moderate fibroblast activity; 4 = very frequent; 5 = diffuse and intense fibroblast activity. Finally, edema was classified as follows: 0 = absence of edema; 1 = minimal or near-negative edema; 2 = mild edema; 3 = moderate edema; 4 = very frequent or intense; 5 = diffuse and intense edema.

[00245] Figure 19 shows the histological studies of the eyes of the animals involved in the study. The lesions in the corneas treated with B-NP (figure 19B) and B-NP Petition 870200019339, dated 10 / 02 / 2020, page 74 / 100 71 / 78 PEG35 (Figure 19C) are in the recovery phase and, although they were previously affecting vision temporarily, they are no longer doing so. Therefore, the damage is reversible. Conversely, corneal lesions treated with saline solution are very severe, affecting vision and appear irreversible (Figure 19F).

[00246] Table 6 - Histopathological evaluations of samples obtained from animal eyes. Control: animals treated with physiological saline; BEVA: animals treated with Avastin®; B-NP: animals treated with bevacizumab-loaded nanoparticles; B-NP-PEG35: animals treated with pegylated bevacizumab-loaded nanoparticles. Sample Fibrosis Inflammation Average thickness (J, lm) Vascularization Edema Control 4 4 774.7 4 3 BEVA 1 4 570.5 2 1 B-NP 1 2 287.7 1 0 B-NPPEG35 1 2 430.9 4 1

[00247] Table 6 summarizes the histopathological evaluations in the different groups. Corneas treated with serum showed a thickness 1.4 times greater than those treated with bevacizumab and 2.7 times greater than those treated with B-NP. On the other hand, corneas treated with Avastin® (BEVA) showed a thickness 2 times greater than corneas treated with B-NP and 1.3 times greater than those treated with BNP-PEG35. Similarly, corneas treated with BNP presented the best symptoms with a low degree of fibrosis, low vascularization, and absence of edema. Petition 870200019339, dated 10 / 02 / 2020, page 75 / 100 72 / 78 Example 12 - Biodistribution of nanoparticles after intravenous administration in tumor-bearing mice

[00248] For tumor imaging experiments in mice, human hepatocellular carcinoma (HepG2) cells were cultured under standard conditions, harvested one week after plating, and suspended in PBS. Tumors were induced in nude mice after subcutaneous injection of 5x10⁵ HepG2 cells at two different sites: the right limb and the upper back. Tumor growth was followed for 12–15 days until clearly visible, and 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). One and four hours after intravenous administration, the animals were sacrificed, and both tumors and a portion of the contralateral (tumor-free) leg muscle were excised.The samples were counted on a gamma counter calibrated for 99mTc, corrected for sample weight and drop, and the tumor / non-tumor ratio was calculated using contralateral leg muscle as a backdrop.

[00249] In tumor-bearing mice, the radioactivity associated with intravenously injected NP-PEG35 is concentrated in the tumors (compared to normal tissue), as can be seen in Figure 20. This phenomenon appears to be time-dependent, since 4 hours after administration of the nanoparticles the amount present in the tumors decreases, although it already remains at high levels (tumor / non-tumor ratio > 6). Petition 870200019339, dated 10 / 02 / 2020, page 76 / 100 73 / 78 Example 13 - Effect of albumin nanoparticles loaded with bevacizumab in a murine colorectal cancer model

[00250] The studies were approved by the Ethical Committee for Animal Experimentation of the Institution (protocol number 107-16), in accordance with European legislation on animal experimentation. For the experiments, forty-two naked male athymic rats, weighing approximately 20 grams and 3 weeks old, were acquired from Harlan Sprague Dawley, Inc. The rats were kept in a controlled environment, in accordance with institutional guidelines. Food and water were provided ad libitum.

[00251] Human cancer cells (HT-29) were cultured under standard conditions, harvested one week after plating, and suspended in PBS. For tumor induction, mice were anesthetized with isoflurane by inhalation, and 100 pL containing 2-3 x 10⁶ HT-29 tumor cells (bevacizumab-sensitive human colon cancer cell line) were injected subcutaneously into the right lateral flank of each animal. Tumor growth was monitored for 12-15 days until clearly visible (diameter 0.4-0.6 cm), and then treatment was initiated.

[00252] Mice were randomized into six groups of 7 animals each: (i) aqueous bevacizumab solution (Avastin), (ii) bevacizumab-loaded albumin nanoparticles (B-NP), (iii) PEG35,000-coated bevacizumab-loaded albumin nanoparticles (B-NP-PEG35), (iv) physiological saline (PBS), (v) PEG35,000-coated empty nanoparticles (NP-PEG35) Petition 870200019339, dated 10 / 02 / 2020, page 77 / 100 74 / 78 in a quantity similar to that administered with B-NP-PEG35 and (vi) an aqueous solution of HSA containing a similar amount of albumin as the group that received B-NP. In all cases, 150–200 μL of the preparations containing 5 mg bevacizumab / kg body weight were administered twice weekly intravenously. The control group received an intravenous injection of 0.9% saline solution at the same times.

[00253] Blood samples were collected on day 0 (before the first administration), day 15, day 22, and day 26 after the first administration. Serum bevacizumab concentration was measured by a specific enzyme immunoassay (Shikari Q-Beva).

[00254] Tumor volumes and weights were recorded 1-2 times / week. Tumors (V) were measured in two dimensions, width (W) and length (L) with a caliper and calculated using the following equation (equation 5) V (mm3) = length x (cm)2 x 0.5 [equation 5]

[00255] Figure 21 shows tumor volume versus time. A tumor with identical volume was cultured in each group and no statistical differences in tumor volume were found between the six groups at the beginning of the experiment (p> 0.05).

[00256] On day 12, the group that received B-NP-PEG35 showed a significantly smaller tumor volume (p <0.05) than the other groups. On day 14, the groups that received BEVA and B-NP-PEG35 showed a significantly smaller tumor volume (p <0.05) than the other groups. On day 22, the groups that did not receive Petition 870200019339, dated 10 / 02 / 2020, page 78 / 100 75 / 78 treated animals presented a larger tumor volume than animals treated with bevacizumab-loaded nanoparticles (B-NP), bevacizumab (BEVA), and bevacizumab-loaded pegylated nanoparticles (B-NP-PEG35). It is worth noting that, at the end of the experiment, half of the animals in the group that did not receive bevacizumab developed ulcers in the tumors.

[00257] Figure 22 shows the serum levels of bevacizumab. Samples were collected on days: 0 (before administration), 15, 22, and 26 after the first administration. It is worth noting that there is an increase in serum bevacizumab levels on day 22, which corresponds to the day after a weekly administration. It can be seen in Figure 22 that serum bevacizumab levels in mice receiving the free drug are up to 6 times higher than those receiving the nanoparticles (B-NP and B-NP-PEG35).

[00258] The benefits of administering bevacizumab encapsulated in nanoparticles are lower serum levels of bevacizumab. After intravenous administration of free bevacizumab, there is a high concentration of the drug in the bloodstream, which can cause side effects. This concentration slowly decreases to reach a plateau. However, after administration of a new dose, the concentration of bevacizumab in the bloodstream increases (see Figure 22), thus increasing the likelihood of side effects.

[00259] On the other hand, after intravenous administration of bevacizumab nanoparticles, serum drug levels increase slowly to reach a plateau. This concentration is six times lower than that obtained Petition 870200019339, dated 10 / 02 / 2020, page 79 / 100 76 / 78 with free bevacizumab, which remains constant. Furthermore, whenever a new dose is administered, the peak concentration of bevacizumab in the blood is much lower. This reduces the likelihood of side effects.

[00260] Polyethylene glycol also provides a steric barrier to the surface of the nanoparticles, preventing opsonization, which is the main mechanism for the loss of the injected dose (ID) within a few hours after intravenous injection. Pet Imaging

[00261] At the end of the experiment, three mice loaded with bevacizumab and bevacizumab in treatment with pegylated nanoparticles and saline were selected to undergo 18FFDG-PET imaging. For this, the mice were fasted overnight but were allowed to drink water ad libitum. The following day, the mice were anesthetized with 2% isoflurane in 100% O2 gas and kept immobile during 18F-FDG. Forty minutes before scanning, 18F-FDG (10 MBq ± 2 in 80-100 μL) was injected via the tail vein. PET imaging was performed on a Philips Mosaic small animal computed tomography scanner (Cleveland, OH) with a resolution of 2 mm, an axial field of view (FOV) of 11.9 cm, and a transaxial FOV of 12.8 cm. Anesthetized mice were placed horizontally on the PET scanner bed for a 15-minute static acquisition (sinogram). Images were reconstructed using the 3D Ramla algorithm (a true 3D reconstruction) with 2 iterations and a relaxation parameter of 0.024 on a 128 x 128 matrix with a voxel size of 1 mm, applying time corrections. Petition 870200019339, dated 10 / 02 / 2020, page 80 / 100 77 / 78 inactive, drop, random, and scatter. For the evaluation of 18F-FDG tumor uptake, all studies were exported and analyzed using PMOD software (PMOD Technologies Ltd., Adliswil, Switzerland). Regions of interest (ROIs) were drawn on 1 mm thick coronal PET images of small animals in consecutive slices, including the entire tumor. Finally, the maximum standardized uptake value (SUV) was calculated for each tumor using the formula: SUV = [concentration of tissue activity (Bq / cm3) / injected dose (Bq)] x body weight (g)

[00262] Table 7 summarizes the PET imaging results in the different groups. Regarding SUVmax (maximum tumor uptake), the lowest value corresponded to the group treated with B-NP-PEG35, while the highest corresponded to the group that received saline solution.

[00263] In terms of volume, the lowest values ​​belonged to the groups treated with B-NP-PEG35 and HSA. However, the animals selected from the HSA group were not representative, as they were the only ones that did not present ulcers. The highest value again belonged to the group that did not receive any treatment (physiological saline). The B-NP-PEG35 group presented a volume 3 times smaller than the physiological saline group and 1.5 times smaller than the BEVA group.

[00264] Finally, TLG (total glycolysis of the lesion) showed the lowest value for the group treated with B-NPPEG35, which was 1.5 times lower than the group treated with BEVA and 3.5 times lower than the group that received saline solution. Table 7 - PET imaging results. PS: animals treated with saline solution; B-NP-PEG35: animals Petition 870200019339, dated 10 / 02 / 2020, page 81 / 100 78 / 78 treated with pegylated nanoparticles loaded with bevacizumab; BEVA: animals treated with Avastin®. SUV max Volume TLG PS 1.35 0.91 0.77 B-NP- PEG35 0.97 0.33 0.22 BEVA 1.15 0.48 0.34 Example 14 - Preparation of human serum nanoparticles loaded with ranibizumab

[00265] Ranibizumab-loaded HSA nanoparticles were prepared following the same procedure described in Example 1 for bevacizumab-loaded albumin nanoparticles.

[00266] For ranibizumab-loaded nanoparticles, the ideal antibody / albumin ratio was 0.15. The ranibizumab nanoparticles exhibited a particle size of approximately 210 nm with a PDI less than 0.3 and a zeta potential of -15 mV. Petition 870200019339, dated 10 / 02 / 2020, page 82 / 100

Claims

1 / 4 CLAIMS 1. Nanoparticle characterized by comprising a solid core, said solid core comprising a non-crosslinked albumin matrix and a monoclonal antibody, wherein the monoclonal antibody is distributed throughout the albumin matrix, wherein said solid core is coated with a non-ionic polymer.

2. Nanoparticle, according to claim 1, characterized in that the weight ratio of monoclonal antibody / albumin varies between 0.01 and 0.

5.

3. Nanoparticle, according to claim 1 or 2, characterized in that the ratio of the non-ionic polymer / albumin varies between 0.02 and 0.5 (weight / weight).

4. Nanoparticle, according to any one of claims 1 to 3, characterized in that the albumin is human serum albumin or bovine serum albumin.

5. Nanoparticle, according to any one of claims 1 to 4, characterized in that the monoclonal antibody is selected from bevacizumab, ranibizumab, trastuzumab, cetuximab and rituximab.

6. Nanoparticle, according to any one of claims 1 to 5, characterized in that the nonionic 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 trade name Eudragit; or a polyalkylene glycol. Petition 870260066553, dated 06 / 07 / 2026, page 14 / 34 2 / 4 7. Nanoparticle, according to any one of claims 1 to 6, characterized in that the monoclonal antibody is homogeneously distributed throughout the albumin matrix.

8. Nanoparticle, according to any one of claims 1 to 7, characterized in that the monoclonal antibody is selected from an anti-VEGF or anti-VEGF R2 antibody.

9. Pharmaceutical composition characterized by comprising: - a plurality of nanoparticles, as defined in any one of claims 1 to 8; and - a pharmaceutically acceptable excipient, carrier or vehicle.

10. Pharmaceutical composition, according to claim 9, characterized in that the nanoparticles are in the form of a dry powder.

11. Pharmaceutical composition, according to claim 9 or 10, characterized in that the pharmaceutically acceptable excipient, carrier or vehicle is suitable for oral, topical or parenteral administration.

12. Pharmaceutical composition, according to any one of claims 9 to 11, characterized in that the albumin is human serum albumin or bovine serum albumin.

13. Pharmaceutical composition, according to any one of claims 9 to 12, characterized in that the monoclonal antibody is selected from bevacizumab, ranibizumab, trastuzumab, cetuximab and rituximab.

14. Pharmaceutical composition, according to any one of claims 9 to 13, characterized by the fact that the non-ionic polymer is a water-soluble cellulose selected from hydroxyethylcellulose, hydroxyn-propylcellulose, hydroxy-n-butylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate and ethylhydroxyethylcellulose; starch; dextran; polyvinylpyrrolidone, a polyester selected from compounds under the trade name Eudragit; or a polyalkylene glycol.

15. Pharmaceutical composition, according to any one of claims 9 to 14, characterized in that the monoclonal antibody is homogeneously distributed throughout the albumin matrix.

14. Use of a nanoparticle, as defined in claim 1, characterized in that it is used in the manufacture of a medicament for the treatment of eye diseases or cancer.

15. Use, according to claim 14, characterized in that the eye disease is selected from macular degeneration, neovascularization or angiogenesis of the cornea, neovascularization or angiogenesis of the iris, neovascularization or angiogenesis of the retina, diabetic proliferative retinopathy, non-diabetic proliferative retinopathy, glaucoma, infectious conjunctivitis, allergic conjunctivitis, ulcerative keratitis, non-ulcerative keratitis, episcleritis, scleritis, diabetic retinopathy, uveitis, endophthalmitis, infectious eye conditions and inflammatory eye conditions.

16. Use, according to claim 14 or 15, characterized in that said antibody is an anti-VEGF antibody and the cancer is breast cancer, lung cancer, pancreatic cancer, multiple myeloma, renal cell carcinoma, prostate cancer, melanoma, cancer of Petition 870260066553, dated 06 / 07 / 2026, page.16 / 34 4 / 4 colon, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, liver cancer, renal and gastric cancer, bladder cancer or squamous cell carcinoma; wherein the antibody is trastuzumab and the cancer is breast cancer, lung cancer, pancreatic cancer, multiple myeloma, renal cell carcinoma, prostate cancer, melanoma, colon cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, liver cancer, renal and gastric cancer, bladder cancer or squamous cell carcinoma; wherein the antibody is cetuximab and the cancer is lung cancer, renal cell carcinoma, colorectal cancer, cervical cancer, ovarian cancer or squamous cell carcinoma; or the antibody is rituximab and the cancer is lymphoma or leukemia. Petition 870260066553, dated 06 / 07 / 2026, page 17 / 34.

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