INORGANIC NANOPARTICLE-BASED VACCINE COMPOSITIONS FOR CANCER TREATMENT

Abstract

The present invention is related to biotechnology, particularly to the field of human health. It provides new vaccine compositions that comprise as active principle a system that contains the recombinant human EGF, or peptides thereof, and a carrier protein or peptide, bound to a nucleus constituted by inorganic nanoparticles, with nanometric or submicrometric scale dimensions. These vaccine compositions are useful for the chronic treatment of cancer and have as advantages that their administration does not result in the appearance of adverse effects at the injection site and that they do not accumulate in the body.

Claims

1. A vaccine composition to induce an immune response against the epidermal growth factor (EGF), comprising as active principle a system that contains the recombinant human EGF (rhEGF), or peptides thereof, and a carrier protein, bound to a nucleus formed by inorganic nanoparticles.

2. The vaccine composition of claim 1, wherein the inorganic nucleus is formed by salts, oxides or hydroxides selected from the group comprising of: calcium, iron, zinc, magnesium, zirconium, cerium, beryllium, silicon, or the mixture of two or more of them.

3. The vaccine composition of claim 1, wherein the nucleus is of calcium phosphate.

4. The vaccine composition of claim 3, wherein the calcium phosphate is hydroxyapatite (HAp).

5. The vaccine composition of claim 4, wherein the HAp is of amorphous type.

6. The vaccine composition of claim 4, wherein the HAp has low cristallinity.

7. The vaccine composition of claim 4, wherein the hydroxyapatite is partially coated by an organic ligand.

8. The vaccine composition of claim 7, wherein the organic ligand is sodium citrate.

9. The vaccine composition of claim 1, wherein the carrier protein is selected from the group comprising: cholera toxin B subunit, tetanus toxoid, KLH, and P64k of Neisseria meningitidis.

10. The vaccine composition of claim 1, wherein the active principle is on the surface of a HAp nanoparticle.

11. The vaccine composition of claim 10, characterized by wherein the active principle is bound to the HAp nanoparticle by means of one of the following methods: Covalent bond of the chemical conjugate of the rhEGF or peptides thereof and the carrier protein or peptide with the HAp nanoparticle. Covalent bond of the rhEGF or peptides thereof and the carrier protein or peptide with the HAp nanoparticle in an independent way. Successive covalent bond of the rhEGF or peptides thereof and the carrier protein or peptide with the HAp nanoparticle. Multiple conjugation of the rhEGF or peptides thereof and the carrier protein or peptide on the surface of the HAp. Encapsulation or physical binding of the rhEGF and the carrier protein or peptide on the surface of the HAp.

12. The vaccine composition of claim 1 in combination with other adjuvants that are selected from the group consisting of: incomplete Freund's adjuvants, squalene-based adjuvants, synthetic origin adjuvants, mineral origin adjuvants, vegetable origin adjuvants, animal origin adjuvants, particulated proteic adjuvants and liposomes.

13. A method of treating cancer comprising administering the vaccine composition of claim 1.

14. A method of treating a disease for a subject in need thereof comprising administering a therapeutically effective amount of the vaccine composition of claim 1.

15. The method of claim 14, wherein a previous immune response induction stage is achieved with another vaccine composition against the EGF.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0091] FIG. 1. X-Ray Diffraction Pattern: A) Amorphous HAp particles, obtained in the present invention. B) Standard HAp pattern—JCPDS: PDF Ref. 09-0432.

[0092] FIG. 2. FTIR Spectrum: A) Amorphous HAp particles obtained in the present invention. B) Sodium citrate.

[0093] FIG. 3. Size of the amorphous HAp particles obtained in the present invention, determined by electronic transmission microscopy. A) Image of the particles recorded at 25000× magnification. B) Image of the particles recorded at 500000× magnification. C) Size distribution of the particles.

[0094] FIG. 4.Thermogram of the amorphous HAp nanoparticles obtained in the present invention.

[0095] FIG. 5. Size distribution of the HAp-rhEGF-rP64k system covalently bound, determined by the method of dynamic light scattering (DLS).

[0096] FIG. 6. Characterization of the HAp-rhEGF-rP64k system by means of: A) SDS-PAGE: 1—Molecular mass pattern 2—Positive control of the rhEGF-rP64k conjugate elaborated according to U.S. Pat. No. 8,778,879, invention 3—HAp-rhEGF-rP64k system. B) Western Blot profile: 1—Positive control of rhEGF-rP64k conjugate, 2—HAp-rhEGF-rP64k system.

[0097] FIG. 7. Anti-EGF antibody response of the C57BL/6 mice immunized with the HAp-rhEGF-rP64k system covalently linked and the control group (Montanide-rhEGF-rP64k).

[0098] FIG. 8. Ratio of antibody subclasses (IgG2b+IgG2c)/IgG1, contained in sera of C57BL/6 mice immunized with the HAp-rhEGF-rP64k systems and the control group (Montanide-rhEGF-rP64k).

[0099] FIG. 9. Photograph of the effects of the HAp-rhEGF-rP64k and the Montanide-rhEGF-rP64k systems at the injection site in BALB/c mice. A) Effect of the Montanide-rhEGF-rP64k system. B) Effect of the HAp-rhEGF-rP64k system.

[0100] FIG. 10. Anti-EGF antibody response in C57BL/6 mice immunized with the HAp-rhEGF-rP64k system combined with VSSP and with rhEGF-rP64k encapsulated in liposomes.

[0101] FIG. 11. Ratio of antibody subclasses (IgG2b+IgG2c)/IgG1, contained in sera of C57BL/6 mice immunized with the HAp-rhEGF-rP64k system combined with VSSP and with rhEGF-rP64k encapsulated in liposomes.

[0102] FIG. 12. Maintenance of the anti-EGF antibody response of the HAp-rhEGF-rP64k system, with previous induction of the immune response with one or two doses of the Montanide-rhEGF-rP64k system.

[0103] FIG. 13. Size distribution of the HAp-PI system constructed on the particle, determined by DLS.

[0104] FIG. 14. Characterization of the HAp-PI system by means of: A) SDS-PAGE: 1—Molecular mass pattern, 2—Positive control of rP64k, 3—Positive control of rhEGF, 4—HAp-PI system and B) Western Blot Profile: 1—Positive control of rP64k, 2—HAp-PI system.

[0105] FIG. 15. Size distribution of the HAp-CM system, determined by DLS.

[0106] FIG. 16. Characterization of the HAp-CM system by means of: A) SDS-PAGE: 1—Molecular mass pattern, 2—Positive control of rhEGF-rP64k conjugate elaborated according to U.S. Pat. No. 8,778,879, invention 3—HAp-CM system and B) Western Blot Profile 1—Positive control of rhEGF-rP64k conjugate, 2—HAp-CM system.

[0107] FIG. 17. Anti-EGF antibody response in C57BL/6 mice immunized with HAp-rhEGF-rP64k, HAp-PI and HAp-CM systems.

[0108] FIG. 18. Ratio of antibody subclasses (IgG2b+IgG2c)/IgG1, contained in sera of C57BL/6 mice immunized with the HAp-rhEGF-rP64k, HAp-PI and HAp-CM systems and the Montanide-rhEGF-rP64k control.

[0109] FIG. 19. Photographic images of the histological sections of muscle tissue extracted at the inoculation site and adjacent muscle tissue of C57BL/6 mice, 10× magnification. .star-solid. Represents the fibroblastic repair tissue. The mice were treated with: A-C: Montanide-rhEGF-rP64k, D: HAp-rhEGF-rP64k, E: HAp-CM, F: HAp-PI.

[0110] FIG. 20. Number of lesions observed in the tissues at the sites of injection in C57BL/6 mice immunized with the HAp-rhEGF-rP64k, HAp-PI and HAp-CM systems and the Montanide-rhEGF-rP64k control.

EXAMPLES

Example No. 1. Synthesis, Characterization and Activation of HAp Nanoparticles

[0111] Following a modified variant of the procedure described by Koroleva M., et al., Russian Journal of inorganic chemistry (2016), 61(6): 674-680), nanoparticles of amorphous HAp coated with sodium citrate were obtained. A solution was prepared under controlled environmental conditions, formed by CaCl.sub.2 0.05 mol/L and sodium citrate at a sodium citrate/calcium molar ratio of 4:1 (A solution). Next, a sterile B solution was added, formed by NaH.sub.2PO.sub.4 0.06 mol/L at a flow rate of 1 mL/min, maintaining a Ca/P molar ratio of 1.67. Subsequently, the reaction was adjusted to pH 10 with a dissolution of sodium hydroxide and it was kept stirring for three hours at room temperature. Once the reaction was concluded, it was washed with purified water and separated by filtration using Amicon polyethersulf one membrane of 10 kDa. The obtained solid was vacuum dried at room temperature. Next, they were sterilized with pure steam at 120° C. for 30 min and vacuum dried.

[0112] By means of the X-Rays Diffraction assay it was demonstrated that HAp amorphous nanoparticles, with crystallinity degree of 9.3% and crystallite size of 16.5 nm were generated (FIG. 1). In this assay copper radiation was used (Cu Kα) in a tube operated at 45 kV and 40 mA. The range was from 10° to 90° with steps of 0.013° at nine seconds intervals.

[0113] The Fourier transformed infrared spectra (FTIR) of the nanoparticles (A) and the sodium citrate used as control (B) are shown in FIG. 2. The bands observed at 1090, 1030, 962, 604, 561 and 472 cm.sup.−1 in the spectrum of the nanoparticles confirmed the presence of the phosphate groups corresponding to the HAp. Also, at 3400 cm.sup.−1 a wide band attributed to residual water and the OH.sup.− groups was observed. The bands of the carboxylate groups of the citrate were also observed at 1610 and 1413 cm.sup.−1 in both spectra, which confirms the presence of this ligand on the surface.

[0114] The nanoparticles, determined by Electronic transmission Microscopy, presented a spherical morphology (FIGS. 3A and 3B), with a size average of 62±13 nm (FIG. 3C).

[0115] In the thermography a loss of 6.2% of the mass at temperatures higher than 200° C. (FIG. 4) related to the presence of sodium citrate on the surface of the nanoparticles was found. The range of temperatures analyzed was between 25° C. and 1000° C. with a heating rate of 20 K/min, using an argon flow of 60 mL/min.

[0116] The nanoparticles of amorphous HAp were treated with a sterile dissolution of EDC maintaining a mass ratio EDC/HAp of 2:1 for one hour, under controlled environmental conditions. Then the suspension was centrifuged at 6708 gravities and the excess EDC was removed.

Example 2. Obtainment of the HAp-rhEGF-rP64k System by Means of the Covalent Bond Between the Amorphous HAp Nanoparticles and the rhEGF-rP64k Conjugate

[0117] The nanoparticles of amorphous HAp obtained and activated with the procedure described in Example 1 were mixed under controlled environmental conditions, with a sterile solution of PBS, pH 7±0.3, containing the rhEGF-rP64k chemical conjugate obtained with the methodology described in U.S. Pat. No. 8,778,879 invention at a rhEGF-rP64k/HAp mass ratio of 1:2.5. The suspension formed was kept under shaking at 140 cycles per minute for 2 h at room temperature.

[0118] The HAp-rhEGF-rP64k system obtained was dispersed with EDTA, the conditions of the medium were adjusted to a pH of 7±0.3 and protein concentration of 1±0.2 mg/mL, with sterile solutions of PBS and sodium hydroxide.

[0119] The DLS assay showed the presence of nanoparticles with mean hydrodynamic diameter of 97.5 nm and a polydispersity index of 0.36. The obtained system was polydisperse with particle diameter ranging from 9 to 366 nm, measured by intensity (FIG. 5).

[0120] The nanoparticulate system, obtained by means of the previously explained procedure, was characterized by means of SDS-PAGE and Western Blot (FIG. 6). The electrophoresis showed the rhEGF-rP64k conjugate had a pattern of bands similar to the one of the positive control (that is, predominant bands with molecular mass equal to or greater than 66 kDa), which indicates the presence of conjugate in the system. In the Western Blot assay, a band similar to the control of the free rhEGF-rP64k conjugate was observed, demonstrating the presence of the rhEGF in conjugates with molecular mass greater than 200 kDa. With this analysis it was proved that the rhEGF-rP64k conjugate bound to the nanoparticles was able to maintain its structural and functional integrity, as confirmed by the fact that it was still recognized by the anti-EGF antibodies.

Example 3. The HAp-rhEGF-rP64k System Generates a Humoral Response Against the EGF Without Visible Adverse Effects at the Injection Sites.

[0121] C57BL/6 (n=5) mice were immunized with 63 μg of proteins, contained in the system described in Example 2. A batch of the product obtained with the methodology described in U.S. Pat. No. 8,778,879 invention (Montanide-rhEGF-rP64k) was used as positive control of the assay. An immunization protocol consisting of four doses was applied (days: 0, 14, 28 and 42).

[0122] Two days before beginning the immunization protocol and on days 35 and 56, the total IgG antibody titers against the EGF were determined in both groups of mice by means of ELISA. The ratio (IgG2b+IgG2c)/IgG1 specific to the EGF was also determined in the immune sera on day 56. The statistical analysis was performed using the test of comparison of means of Kruskal-Wallis, different letters showed statistically significant differences (p<0.05).

[0123] The HAp-rhEGF-rP64k system generated anti-EGF antibodies, that were detectable in the immune serum at a dilution of 1/10000 on day 56 (FIG. 7). This result demonstrates that the binding of the rhEGF-rP64k conjugate to the particle does not affect the conjugate integrity, and that the HAp potentiates an anti-EGF humoral response in spite of its being significantly lower than the one of the group control. On the other hand, the ratio (IgG2b+IgG2c)/IgG1 showed there were no statistically significant differences in the responses obtained with the HAp-rhEGF-rP64k system and the control (Montanide-rhEGF-rP64k) (FIG. 8). In both systems, the prevailing responses were of humoral type (Th2), which makes them highly appropriate for the depletion of the EGF, as part of the treatment of epithelial tissue origin cancer.

[0124] The effect of the HAp-rhEGF-rP64k system at the injection site was determined. To this, purpose five BALB/c mice were treated with the anti-EGF system described in Example 2 (HaP-rhEGF-rP64k) and five with the Montanide-rhEGF-rP64k control. The vaccination protocol was similar to the one previously described. After concluding the experiment, photographic images of the injection sites of the mice from the two treated groups were taken. As can be seen in FIG. 9A, the mice of the control group showed accumulations of mineral oil at the injection sites, caused by Montanide administration. In contrast, in the mice treated with the HAp-rhEGF-rP64k system produced according to the procedure described in Example 2(FIG. 9B), no damage was found at the injection sites. These results indicate that the new system developed in the present invention substantially reduces injuries at the injection site, and thus shows great potential for chronic administration, particularly for the treatment of cancers that require periodic inoculation for several years.

Example 4. The Combination of the HAp-rhEGF-rP64k System with Particulate Adjuvants Generates an Anti-EGF IgG Antibody Response and Induces a Th1-type Response Pattern

[0125] C57BL/6 mice divided into three groups of five animals each were used. The animals were immunized in the following way:

[0126] Group 1: 63 μg of proteins of the vaccine composition described in U.S. Pat. No. 8,778,879 (Montanide-rhEGF-rhP64k) (Positive control).

[0127] Group 2: 63 μg of proteins of the HAp-rhEGF-rP64k system with 100 μg of proteins of the nanoparticulate adjuvant VSSP.

[0128] Group 3: 31.5 μg of proteins of the HAp-rhEGF-rP64k system and 31.5 μg of the rhEGF-rP64k conjugate, encapsulated in liposomal vesicles (DRV's) obtained by the methodology of dehydration-rehydration (Kirby and Gregoriadis, Biotechnology, (1984) 2: 979-984).

[0129] The immunizations were carried out on days 0, 14, 28 and 42. Blood extractions were performed two days before the beginning of the protocol and on days 35 and 56 and the total IgG antibody titers against the EGF in the serum obtained was determined by means of ELISA. The ratio (IgG2b +IgG2c)/IgG1 specific to the EGF in the sera was also determined on day 56. The statistical analysis was performed by means of the test of comparison of means of Kruskal-Wallis, different letters indicate statistically significant differences (p<0.05). The generation of anti-EGF antibodies was evidenced in the three groups studied. The combinations of the HAp-rhEGF-rP64k system with the VSSP and the DRV's liposomes produced antibodies that were detectable in the immune serum up to a dilution of 1/10000 and were same during the two extractions (FIG. 10). The existence of statistically significant differences of the combination with respect to the control was demonstrated.

[0130] The analysis of the ratio (IgG2b+IgG2c)/IgG1 specific to the EGF on day 56, evidenced both adjuvant combinations exhibited similar responses, and they were both statistically superior as compared to the control (FIG. 11). These results demonstrated that the combination of HAp-based formulations with other particulate adjuvants potentiates a specific immune response to the EGF that induces a superior Th1 response pattern, which is the most favorable one for targeted cancer treatments.

Example 5. The HAp-rhEGF-rP64k System Maintains the Anti-EGF IgG Antibody Response Previously Induced with Montanide-rhEGF-rP64k

[0131] Three groups of C57BU6 mice were used (n=5), which were immunized on days: 0, 14, 28, 42 and 70, with the following immunization schedules:

[0132] Group 1. (Control) the mice were immunized with 63 μg of proteins contained in the system described in U.S. Pat. No. 8,778,879 patent (Montanide-rhEGF-rhP64k).

[0133] Group 2. The mice were immunized on day 0 with 63 μg of proteins contained in the Montanide-rhEGF-rhP64k system and with same quantity of proteins contained in the HAp-rhEGF-rP64k system on the rest of the immunizations performed.

[0134] Group 3. The mice were immunized on days 0 and 14 with 63 μg of proteins contained in the Montanide-rhEGF-rhP64k system and with same quantity of proteins contained in the HAp-rhEGF-rP64k system on the rest of the immunizations performed.

[0135] Two days before to the first immunization, the pre-immune serum was extracted and on days 35, 56 and 84 days the extractions of the immune sera were performed and the total IgG antibody titers against the EGF were quantified by means of ELISA.

[0136] Anti-EGF antibody titers were detectable at the three times studied in the three groups, without statistical significant differences across them, at the 1/50000 dilution on day 35 and at the 1/100000 dilution on days 56 and 84 (FIG. 12). The inoculation of the HAp-rhEGF-rP64k system was able to maintain the anti-EGF IgG antibody response induced by the Montanide-rhEGF-rP64k systems, during the time period studied. This result supports the feasibility of substituting Montanide by HAp as adjuvant of the rhEGF-rP64k conjugate in the maintenance phase of the anti-EGF immune response and its suitability in the chronic treatment of epithelial origin cancer.

Example 6. Construction of the HAp-PI system by Means of the Covalent Binding of the rhEGF and rP64k Proteins on the Surface of the Amorphous HAp Nanoparticles

[0137] The HAp nanoparticles previously coated with sodium citrate and activated with EDC, obtained according to the methodology described in Example 1, were mixed under controlled environmental conditions with a sterile PBS solution, pH 7±0.3, containing rhEGF and rP64k at a molar ratio of 6 rhEGF per rP64k and proteins/HAp mass ratio of 1: 2.5. The suspension formed was kept under shaking at 140 cycles per minute for 2 h at room temperature. By means of this procedure, a rhEGF-HAp-rP64k system was obtained characterized in that the proteins are bound to the HAp nanoparticle but not to each other. This system was dispersed with EDTA, adjusting the pH to 7±0.3 and the protein concentration to 1±0.2 mg/mL.

[0138] FIG. 13 shows the particle size profile measured by DLS. The mean diameter was of 111.2 nm, with a polydispersity index of 0.347. The system obtained was similar to the one generated in Example 2 in terms of size, polydispersity and DLS profiles. The above-mentioned aspects indicate when the rhEGF and the rP64k are bound to the HAp nanoparticles, but not to each other, they had a dispersion similar to the one of the rhEGF-rP64k conjugate.

[0139] As can be seen in FIG. 14A, which shows results from the SDS-PAGE assay, in the lane corresponding to the HAp-PI system, a main band appears at the height of 66 kDa, which is characteristic of the rP64k protein, and another band at similar height than that of the rhEGF control (6 kDa). These results demonstrated that both recombinant proteins were bound to the HAp nanoparticle and that when reduced they maintained their characteristic molecular mass because they were not bound to each other.

[0140] As evidenced in FIG. 14B there is a band approximately at the height of 66 kDa in lanes 1 and 2, which confirms the presence of the rP64k protein in the HAp-PI system and also that the protein maintains its integrity when it is bound to the HAp nanoparticles.

Example 7. Construction of the HAp-CM System on the Surface of the Amorphous HAp Nanoparticles by Means of the Multiple Conjugation of the rhEGF and rP64k Proteins

[0141] The amorphous HAp nanoparticles obtained in Example 1 were treated under controlled environmental conditions for 1 h with a sterile EDC solution, maintaining a mass ratio of 2.7 EDC per HAp. Next, a sterile PBS solution, pH 7±0.3, was added containing the rhEGF and rP64k at a molar ratio of 6 rhEGF per rP64k and proteins/HAp mass ratio of 1:2.5. The suspension formed was kept under shaking at 140 cycles per minute for 2 h at room temperature.

[0142] A system formed by HAp nanoparticles coated with both recombinant proteins bound to each other and to the nanoparticles was obtained as a result of this procedure. This system was dispersed with EDTA and the pH was adjusted to 7±0.3. Subsequently, it was diluted with PBS solution until a protein concentration of 1±0.2 mg/mL was reached.

[0143] By means of the multiple conjugation previously described, a complex HAp-rhEGF-rP64k system with mean size of 90.2 nm, measured by DLS, and polydispersity index of 0.337 was obtained (FIG. 15).

[0144] The characterizations performed by means of the SDS-PAGE electrophoresis and Western Blot showed the conjugation of the proteins. A similar product to the rhEGF-rP64k positive control, that recognizes the anti-EGF antibodies, was obtained (FIG. 16).

Example 8. The New Systems Constructed on the HAp Nanoparticles Generate Anti-EGF IgG Antibody Responses and Produce a Smaller Number of Epidermal Lesions at the Injection Site

[0145] Three groups of five C57BL/6 mice each were immunized, with 63 μg of proteins, contained in the following systems:

[0146] Group 1: HAp-rhEGF-rhP64k system, obtained according to the methodology described in Example 2.

[0147] Group 2: HAp-PI system, obtained according to the methodology described in Example 6.

[0148] Group 3: HAp-CM system, obtained according to the methodology described in Example 7.

[0149] The immunizations were performed on days 0, 14, 28 and 42. Blood extractions were carried out two days before the beginning of the protocol and on days 35 and 56. The total IgG antibody titers against the EGF in the obtained sera were determined by means of ELISA. The ratio (IgG2b+IgG2c)/IgG1 specific to the EGF in the sera was also determined on day 56. The statistical analysis was performed by means of the test of comparison of means of Kruskal-Wallis, different letters indicate statistically significant differences (p<0.05).

[0150] The HAp-rhEGF-rP64k and HAp-CM systems generated anti-EGF antibodies that were detectable in the immune serum up to a dilution of 1/10000 and in the HAp-PI system up to a dilution of 1/8000 on days 35 and 56 (FIG. 17). The results obtained by SDS-PAGE and Western Blot were confirmed, (Examples 6 and 7) and it was demonstrated that all the systems constructed on the HAp nanoparticles were immunogenic, although in the HAp-PI system the rhEGF is not in the context of a covalent bond with the rP64k. This system constitutes an evidence that it is feasible to produce systems that generate an anti-EGF response immune without the need of chemically binding the rhEGF to a carrier protein.

[0151] The analysis of the ratio (IgG2b IgG2c)/IgG1 revealed there were no statistically significant differences in the response produced by them. The prevailing responses were of humoral type (Th2), which makes them appropriate for the depletion of the EGF, as part of the treatment of epithelial tissue origin cancer. (FIG. 18).

[0152] At 120 days after the beginning of the experiment, the C57BL/6 mice were sacrificed and tissue samples from the injection sites were extracted for the anatomopathological analysis, previously staining them with hematoxylin-eosin. The tissue sections were fixed in neutral buffered formalin and they were processed by means of the paraffin embedding method. As can be seen in FIG. 19A-C, which shows the results from the control group (Montanide-rhEGF-rP64k), there is a loss of the normal structure of the muscle tissue at the inoculation site. The A zone is characterized by inflammatory infiltrate and dilated blood vessels with discontinuity in the adjacent muscle tissue. In FIGS. 19B and C, longitudinal (B) and traversal (C) fibers of adjacent muscle tissue at the inoculation site with invasion of a reaction of fibroblastic repair tissue can be observed. However, in FIGS. 19D-F, which show results from the mice treated with the new systems object of the present invention, a normal structure at the inoculation site and adjacent muscle tissue can be observed. D: HAp-rhEGF-rP64k. E: HAp-CM. F: HAp-PI.

[0153] In each mouse of the three previous groups, the presence or absence of the following lesions was evaluated:

[0154] Discontinuity of the epidermis and lymphocytic infiltration of the dermis.

[0155] Inflammatory infiltrate.

[0156] Invasion of the muscle tissue.

[0157] Vacuolar degeneration of epithelial cells.

[0158] Dilated blood vessels.

[0159] A point was added whenever a lesion was found, the values corresponding to each mouse in the groups were added up and the result was graphed.

[0160] The study evidenced that the groups treated with the systems developed in the present invention exhibited a very small number of lesions, contrary to that observed in the control group inoculated with the Montanide-rhEGF-rP64k system (FIG. 20). This result confirms the visual observations of Example 3.

[0161] These results corroborate the low toxicity of the anti-EGF systems based on HAp nanoparticles and the adverse effects in the injection sites caused by the mineral oil contained in the Montanide-rhEGF-rP64k system.