Injectable suspensions or emulsions of coated magnetite nanoparticles for treatment of solid tumors

Abstract

The present invention relates to an injectable suspension or emulsion of organic layer coated magnetite nanoparticles, which can be used for the treatment of solid tumors.

Description

TECHNICAL FIELD

[0001] The present invention relates to an injectable suspension or emulsion of organic layer-coated magnetite nanoparticles, which can be used in a method for treating solid tumors. BACKGROUND

[0002] Hyperthermia is a therapy proposed in oncology to treat tumors. In short, the principle of this therapy is to heat the tumor cells to about 41 °C to 45 °C, causing them to undergo apoptosis; this treatment affects cancer cells in particular, as they are more sensitive to temperature increases than healthy tissue cells, which have an effective cooling system due to their vascularization. In addition, the vascular system of the tumor is not sufficient to guarantee the normal supply of oxygen and nutrients under conditions of thermal stress. Hyperthermia can be induced by radio waves, microwaves, ultrasound and other forms of energy.

[0003] Despite the simplicity of the principle, which is conceptually simple, to date it has not been possible to implement it widely in therapeutic methods or protocols, due to the presence of limitations, side effects and practical difficulties. The main obstacle to the application of this method is the need for a highly selective overheating of the tissue, which raises the temperature of the tumor cells to the effective range without affecting the surrounding healthy tissue.

[0004] Due to these difficulties, the method has only been applied in limited cases and only for local treatment. The first case in which hyperthermia is used is when the tumor to be treated is close to the body surface; in this case, the overheating is generated by a machine outside the body directly on the tumor. The second application of hyperthermia is to insert a fine needle probe into the tumor for a short period of time, usually about 10 to 30 minutes; the tip of the probe emits high-frequency radiation, which generates very high temperatures and destroys the cells of the surrounding area.

[0005] For more extensive areas of the body, i.e. body regions or the whole body, simple hyperthermia cannot be applied, the method being used only to support other main treatments, such as chemotherapy or radiotherapy; in these cases, the tissue is slightly heated to kill the tumor cells weakened by the main treatment.

[0006] To overcome these difficulties, it has been proposed to use magnetic particles, usually nanoparticles, to reach the tumor area and heat them by radiofrequency irradiation.

[0007] A large number of studies have focused on producing and characterizing magnetic particles with controlled and reproducible properties; see, for example, the review "Iron oxide based MR contrast agents: from chemistry to cell labeling", S. Laurent et al., Current Medicinal Chemistry, 2009, 16, 4712-4727; the paper "Hydrothermal preparation and characterization of ultrafine magnetite powders", Q. Yitai et al., Materials Research Bulletin, Vol. 29(9), 1994, 953-957; the paper "Magnetic properties of bacterial magnetosomes as potential diagnostic and therapeutic tools", R. Hergt et al., Journal of Magnetism and Magnetic Materials 293 (2005) 80-86; and the patent application WO 2011 / 073922 A1.

[0008] These studies show the possibility of using magnetic (nano) particles in diagnostic and therapeutic applications, but the reported characterizations are carried out only on the particles as such, or at most in vitro, and do not show how to implement a real therapeutic hyperthermia treatment in vivo; in particular, no practical details are provided on how to selectively deliver the particles to the tumor area.

[0009] The patent application WO 2015 / 104664 A1 discloses a method for reaching the tumor area with magnetite nanoparticles. The method comprises: preparing a construct comprising a plurality of magnetite nanoparticles in a biocompatible polymeric shell; extracting immune system cells from the patient's body; incorporating the cells into the construct; reintroducing the cells loaded with the construct into the body, so that they reach the tumor site. This method is in principle effective, but is not advantageous for standardization and therefore is difficult to apply widely, since it requires the extraction of cells from a specific patient and then their injection back into the same patient each time.

[0010] Therefore, a first object of the present application is to provide a suspension or emulsion of organic layer-coated magnetite nanoparticles that can be used directly for hyperthermia, as well as a method for using the same in the treatment of solid tumors. SUMMARY

[0011] According to the present application, there is provided a suspension or emulsion of organic layer-coated magnetite nanoparticles selected from:

[0012] A) aqueous suspensions to which surfactants, as well as one or more buffering systems and stabilizers, are added; and

[0013] B) water-based lipid suspensions or emulsions to which one or more components selected from the group consisting of lecithin, medium-chain triglycerides, long-chain triglycerides, glycerol, linoleic acid, cholesterol and sodium oleate are added. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The average tumor volume as a function of time is shown for mice that received an allograft of IGROVl-luc cells (human ovarian cancer cell line) and were then treated according to the present application, compared to mice that were not treated according to the present application;

[0015] Figure 2 The average tumor volume as a function of time is shown for mice that received an allograft of DETROIT-562 cells (subcutaneous pharyngeal cancer cell line) and were then treated according to the present application, compared to mice that were not treated according to the present application. DETAILED DESCRIPTION

[0016] In the remainder of the present specification, the following terms and abbreviations are used, with the following meanings:

[0017] "Room temperature" means a temperature in the range of 18°C to 30°C;

[0018] "NPs" stands for magnetite nanoparticles;

[0019] For the sake of simplicity, the suspension or emulsion of the present application will be referred to as "injectable composition" when no further specific indication is required;

[0020] "Medium-chain triglycerides" means triglycerides obtained from C6-C12 fatty acids, while "long-chain triglycerides" means triglycerides obtained from C14-C60 fatty acids.

[0021] In its first aspect, the present application relates to a suspension or emulsion of organic layer-coated magnetite (Fe3O4) NPs as described above.

[0022] Before the organic layer is applied, the size of the NPs is between 1 nm and 100 nm, preferably between 10 nm and 60 nm, more preferably between 20 nm and 30 nm. These NPs can be prepared according to methods known in the art, for example the method described in patent application WO 2011 / 073922 A1. In brief, the method comprises: i) etching metallic iron with an organic acid in a polyol solvent (e.g. glycerol) at a temperature of 130 °C to 200 °C, forming Fe 2+ ions; ii) bubbling an oxidizing gas (air) into the solution at a temperature lower than 100 °C, oxidizing Fe 2+ ions to Fe 3+ ions, and filtering off the residual metallic iron; iii) treating the solution with water in a water / Fe 3+ ion molar ratio of 1.5 to 5, in the presence of a mineral acid. In this method, the size of the NPs is controlled by the water / Fe 3+ ion molar ratio, operating within the above molar ratio range ensures obtaining particles in the desired size range.

[0023] The NPs thus obtained are subsequently coated with an organic compound. For the coating, the surface of the magnetite NPs is first functionalized with a catechol derivative bearing a lipophilic group on the benzene ring; the two hydroxyl groups present in the catechol molecule form a polar head which binds to the NP surface by electrostatic interaction, while the lipophilicity makes the assembly compatible with the polymer which forms a shell around the NPs. The polymer which can be used for the shell of the NPs coated with catechol is chitosan, sodium alginate, polymethyl methacrylate and polysaccharides, etc. The preferred polymer for the preparation of the shell is a block copolymer of poly(lactic acid / glycolic acid) and polyethylene glycol carboxylate; for a detailed description of this copolymer and the method for coating the NPs with it, reference is made to patent application WO 2015 / 104664 A1.

[0024] The coated NPs thus obtained generally have an average particle size in the range 35 nm to 60 nm, and an average polydispersity index of 0.10-0.15, indicating a narrow particle size distribution.

[0025] Subsequently, the coated NPs obtained as described above are suspended in a suspending medium, to prepare one of the suspensions or emulsions A) or B) of the application.

[0026] These injectable compositions must of course have a series of properties to be suitable for the intended use; in particular:

[0027] - they must be sterile and pyrogen-free, to avoid problems in the event of leakage from the tumor mass to the surrounding healthy tissue;

[0028] - for substantially the same reason, the injectable composition must have an osmotic concentration that is isotonic with the body fluids; the preparation of isotonic compositions is within the reach of the person skilled in the art, for example by using NaCl at a concentration of 0.9% by weight;

[0029] - the pH of the injectable composition must be in the range 8.0 ± 0.2, i.e. between 7.8 and 8.2; the inventors have found that at different pH values the polymeric shell surrounding the NPs is not stable;

[0030] - finally, in order to have an effective thermo-therapeutic effect, but without affecting healthy tissues at a medium distance from the injection site, it is necessary to control the amount of NPs delivered; the inventors have found that the above-mentioned effects are achieved when the concentration of NPs in the injectable composition is in the range 10 mM to 30 mM, preferably between 15 mM and 30 mM. Concentrations higher than this range cause excessive temperature increases, which can damage healthy cells and tissues in the vicinity of the treated area; on the other hand, concentrations lower than this range require the injection of excessively high volumes of suspension in order to achieve an effective amount of NPs, and the injection of excessively high volumes of suspension can cause accidental rupture of the tumour cells, with consequent leakage of the substance and possible delivery through the whole body.

[0031] In a first embodiment, the injectable composition of the present application is a water suspension A) containing NPs and a surfactant at a concentration in the above-mentioned range. In this base composition, at least one further ingredient is added, selected from the group consisting of a buffer system and a stabiliser.

[0032] The surfactant has the function of promoting the stability of the suspension, avoiding the precipitation of the NPs. The surfactant used for the purposes of the present application is a fatty acid sorbitan ester, sold under the trade name SPAN® (including 20 (sorbitan monolaurate), 40 (sorbitan monopalmitate), 60 (sorbitan monostearate), 65 (tristearin sorbitan) of the family, and other products sold under the trade name SPAN® 30, 80 and 85; polymeric sorbitan fatty acid esters, sold under the trade name TWEEN® (with grades 20, 21, 40, 60, 65, 80, 85); sorbitol and sorbitol esters; cyclodextrins, in particular β-cyclodextrins; known as CAVITOL® sulfobutyl ether cyclodextrin derivatives family; linoleic acid; and polyethylene glycol (PEG), in particular the products sold under the trade name Macrogol (for example Macrogol 3350, Macrogol 4000 and Macrogol 6000). When present, the concentration of this ingredient in the suspension A) is comprised between 1% and 25%, according to the different excipients selected.

[0033] The role of the buffer system is to maintain the pH of the suspension in the range 8.0 ± 0.2, which is suitable for in vivo injection. Another role of the buffer system, achieved by stabilizing the pH, is to avoid the degradation of the magnetite NPs and of their surface organic coating. The buffer systems that help to maintain the pH of the suspension in this range include, for example, phosphate buffers (formed by the salts Na2HP04 / NaH2P04), citrate buffers (obtained by mixing citric acid trisodium citrate (C6H807 H20 / Na3C6H507 H20)), histidine buffers, or some "universal buffers", so called because their most effective pH value can be adjusted in a wide range according to the proportions of the ingredients, for example the saline-sodium citrate (SSC) buffer, prepared from sodium chloride and trisodium citrate and adjusted in pH with hydrochloric acid, or the McIlvaine buffer, prepared from a solution of Na2HP04and citric acid in the appropriate proportions. The methods of preparation of these buffer solutions are widely known in the art and can be prepared by an ordinary chemist.

[0034] Finally, the stabilizers in the injectable composition A) are stabilizers with different roles or contributions, for example:

[0035] - antioxidants, for example ascorbic acid at a concentration comprised between 0.001 and 0.005% w / w;

[0036] - complexing agents, for example EDTA at a concentration comprised between 0.025 and 0.075% w / w; cyclodextrins (at a concentration comprised between 1 and 15% w / w) or others; or PEG (at a concentration comprised between 0.1 and 10% w / w);

[0037] - preservatives, for example ethanol (at a concentration comprised between 10 and 15% w / w), benzyl alcohol (at a concentration comprised between 0.10 and 0.15% w / w) and sodium benzoate (at a concentration comprised between 0.10 and 0.15% w / w).

[0038] Examples of water-based suspensions suitable for the purposes of the present application are the following:

[0039] ​i) a suspension comprising 3 mg of magnetite NPs; 2 g of disodium ethylenediaminetetraacetic acid (EDTA); 5 g of polysorbate 80 (polyoxyethylene (20) sorbitan monooleate, trade name Span® 80); 9 mg of NaCl; a phosphate buffer in an amount such that the pH of the suspension is 8.0 ± 0.2; and water to bring the total volume of the suspension to 1000 mL; 80); 9 mg of NaCl; a phosphate buffer in an amount such that the pH of the suspension is 8.0 ± 0.2; and water to bring the total volume of the suspension to 1000 mL;

[0040] ii) a suspension comprising 3 mg of magnetite; 9 mg of NaCl; 2 mg of polyethylene glycol with an average molecular weight of 300 Da (trade name PEG Macrogol 300); a phosphate buffer in an amount such that the pH of the suspension is 8.0 ± 0.2; and water to bring the total volume of the suspension to 1000 mL;

[0041] iii) a suspension comprising 3 mg of magnetite; 9 mg of NaCl; 1 mg of polysorbate 80 (polyoxyethylene (20) sorbitan monooleate, trade name Span® 80); a phosphate buffer in an amount such that the pH of the suspension is 8.0 ± 0.2; and water to bring the total volume of the suspension to 1000 mL.

[0042] In a second embodiment, the injectable composition of the present application is a lipid suspension or emulsion B) in which the main component of the liquid phase is oleic acid, linoleic acid, stearic acid, polyethylene glycol or mixtures thereof, to which one or more components selected from the group consisting of medium-chain triglycerides, long-chain triglycerides, glycerol and sodium oleate can be added in the range of 0.1% w / w to 10% w / w.

[0043] An example of a lipid composition of the present application is:

[0044] iv) a suspension comprising 3 mg of magnetite and 1 g of linoleic acid.

[0045] In a preferred embodiment, the injectable composition of the present application can be provided in kit form, comprising:

[0046] - a first container containing the liquid phase of each of the compositions A) and B), i.e. water in the case of composition A) and oleic acid in the case of composition B);

[0047] - one or more containers containing a predetermined amount of lyophilized powder of surface-coated magnetite NPs, and at least one component selected from the group consisting of surfactants, bulking agents, salts or compounds forming a buffer system and stabilizers.

[0048] The surfactants, the components of the buffer system and the stabilizers used to prepare the kit of the present application are the same as described above in composition A). The bulking agents can be selected from the group consisting of mannitol, lactose, sucrose and glycine. ​

[0049] The injectable composition of the application, provided in kit form, has the advantage of completely avoiding the ageing effects of the liquid phase composition, such as solid precipitation or chemical interactions that can occur between the components. At the time of use, the contents of the various containers of the kit are simply mixed, usually by gentle stirring, to reconstitute the liquid phase composition.

[0050] The composition of the application is preferably stored at a temperature lower than room temperature, more preferably in the temperature range of 2-8°C.

[0051] The composition of the application can be used to treat solid tumours, in particular head and neck cancers, by injecting one of the above compositions directly into the cancerous mass, followed by radiofrequency irradiation of the body region in which the injection site is located.

[0052] Head and neck cancers are the most suitable types of cancer for treatment using the method of the application, because the tumour masses located in these regions are relatively close to the body surface and are easily accessible using a needle for injection.

[0053] The injectable composition of the application is administered by direct injection through the skin with a needle into the tumour mass; the injection volume of the above composition is between 20 μL and 1 mL, preferably between 50 μL and 200 μL for tumours having a volume of between 100 mm 3 and 500 mm 3 If the tumour volume is outside this range, it is recommended to perform multiple injections at different locations of the tumour mass, with each injection volume still being within the above range.

[0054] The subsequent radiofrequency (RF) irradiation is performed at a frequency typically in the range of 10-400, with a magnetic field strength H0 of between 20 kA / m and 25 kA / m, for an irradiation time of 20 to 60 minutes, preferably 30 ± 5 minutes.

[0055] With regard to the treatment regimen, the inventors found that the best results in terms of inhibition of tumour growth were obtained with a regimen of 7 injection / irradiation cycles, with one irradiation after injection in each cycle, and a time interval of 1 to 7 days between the cycles.

[0056] The features and advantages of the application will also become more apparent from the following examples.

[0057] Instruments, methods and experimental conditions

[0058] The tests described below were conducted in accordance with the principles of good laboratory practice on athymic female nude mice, 5 to 7 weeks old (provided by Envigo RMS S.r.l., San Pietro al Natisone, Udine, Italy), in compliance with the current Italian regulations (Decree No. 26 of 4 March 2014), which enforce the Directive 2010 / 63 / EU on the protection of animals used for biomedical research. The animals were marked with non-erasable ink and randomly grouped and assigned according to the tumour volume and clinical observations, and were sacrificed at the end of the study. All the animal procedures (housing, health monitoring, restraint, administration,...), as well as the ethical review, were performed in compliance with the current Italian regulations (Decree No. 26 of 4 March 2014), which enforce the Directive 2010 / 63 / EU on the protection of animals used for biomedical research.

[0059] The RF irradiation was performed using a NovaStar 5W instrument by Ameritherm, Scottsville, NY, USA.

[0060] The MS analysis was performed using an Applied Biosystems / Sciex triple quadrupole mass spectrometer equipped with an Agilent 1100 HPLC pump and a CTC PAL autosampler.

[0061] Example 1

[0062] This example relates to the preparation of a suspension of coated magnetite NPs.

[0063] Magnetite NPs were prepared according to the polyol synthesis route described in patent application WO 2011 / 073922 Al; once prepared, the NPs were first functionalized with the organic ligand N-(3,4-dihydroxyphenylethyl)-dodecanamide (DDA) and then dispersed in tetrahydrofuran (THF). Subsequently, the organic coated magnetite nanoparticles were encapsulated in a matrix of polylactic-co-glycolic acid-polyethylene glycol copolymer (PLGA-b-PEG-COOH) mentioned in WO 2011 / 073922 Al. According to the nanoprecipitation technique, using a ratio of 1 : 10 of THF: water, the process formed a stable phosphate buffer suspension. The resulting suspension was dialyzed in a tangential flow membrane to remove the organic solvents, impurities and unreacted reagents and then concentrated to 0.3% (w / w) in Fe3O4(150 mL); finally, to the suspension were added 2 mg / ml of EDTA and 5 mg / ml of 80.

[0064] Subsequently, the coated NPs in suspension were fully characterized.

[0065] The average particle size and average polydispersity index of the coated NPs were determined by dynamic light scattering (DLS) to be 50.8 ± 0.6 nm and 0.11 ± 0.01, respectively, indicating a narrow particle size distribution. Transmission electron microscopy (TEM) images of the coated NPs showed that a small number of inorganic particles were uniformly dispersed in a clustered manner within the polymer matrix, with an average diameter of 50 nm. The zeta potential showed that the surface carried a negative charge of -43.0 ± 0.2 mV, which can be attributed to the presence of multiple carboxylic acid groups on the outer surface. The iron content was determined by inductively coupled plasma atomic emission spectrometry to be 2.7 mg / mL.

[0066] The resulting suspension was dispensed into syringes for subsequent biological testing.

[0067] Example 2

[0068] This embodiment relates to ovarian cancer growth inhibition achieved using the method of the present invention.

[0069] On day 0, 200 μl of a thymic substance containing 5 × 10⁻⁶ thymic molecules was subcutaneously injected into the left side of 32 female nude mice. 6 Culture medium for IGROV1-luc cells (human ovarian cancer cell line). IGROV1-luc cells can be purchased from companies such as Creative Biolabs, BPS Biosciences, or Affimetrix.

[0070] Daily examinations were conducted on the animals to assess tumor development, mortality, and clinical signs. Toxicity was evaluated based on physiological or distressing behaviors (e.g., weight loss ≥20%), paralysis, loss of muscle tone, tremors, any orifice bleeding, dyspnea, decreased activity, neurological symptoms, and kyphosis. No unexpected deaths occurred during the testing period.

[0071] Assess tumor progression using calipers. Assess tumor volume and body weight twice weekly. Calculate tumor volume using Formula 1 below:

[0072] Tumor volume (mm) 3 = Length (mm) × Width 2 (mm 2 )× 0.5 (1)

[0073] The tumor growth inhibition rate (%TGI) was calculated according to Formula 2:

[0074] % TGI = 100 – (Mtt / Mtc × 100) (2)

[0075] in:

[0076] Mtt = Average tumor volume in the treatment group;

[0077] Mtc = mean tumor volume of the control group.

[0078] When the mean tumor volume reached 482 mm3 3 (day 8), the mice were randomly grouped and assigned to the various experimental groups, with the goal of 8 mice per group.

[0079] On day 9, mice of groups 2 to 4 received an intratumoral single injection of 50 μl of the suspension of example 1, respectively. Mice of group 1 (control group) did not receive an injection of NPs suspension.

[0080] Mice of groups 3 and 4 started to receive radiotherapy one hour after the intratumoral injection of NPs suspension. Mice were anesthetized by inhalation of isoflurane in a mouse cage with active scavenging. The parameters of the radiotherapy instrument were: frequency = 360 kHz, field strength (H0) = 21 kA / m.

[0081] Mice of group 2 did not receive radiotherapy, mice of group 3 received one 40-minute session of radiotherapy, and mice of group 4 received one 40-minute session of radiotherapy per day for five consecutive days, with a 24-hour interval between each session.

[0082] The RF radiotherapy conditions are summarized in Table 1 below:

[0083] Table 1

[0084] Treatment group Duration of radiotherapy (min) Radiotherapy schedule 1 (control) / / 2 / / 3 40 Once 4 40 5 consecutive days, 24 hours apart

[0085] All mice were sacrificed when the respective humane endpoint limit was reached: no significant internal lesions were observed at the macroscopic necropsy.

[0086] Tables 2-5 below report the volumes (in cm3) of mice of groups 1 to 4, respectively, as a function of the number of days tested for all mice of the four groups. 3

[0087] Table 2

[0088]

[0089] Table 3

[0090]

[0091] Table 4

[0092]

[0093] Table 5

[0094]

[0095] ​Table 6 reports the average tumor volume (in cm3) for each group, as a function of the number of days of test, together with the standard error (SE) and the standard deviation (SD). 3

[0096] Table 6

[0097]

[0098] The average tumor volume (in mm3) growth data ± SE as a function of the number of days of test are also shown in graphical form in 3 Figure 1 The numbers in the graphs represent the groups (1 to 4) to which the data refer; the data are presented at the level of significance.

[0099] Table 7 reports the values of the tumor growth inhibition rate (% T.G.I.) calculated from the data in Table 6.

[0100] Table 7

[0101] Day Group 1 Group 2 Group 3 Group 4 8 0.0 0.6 0.7 1.3 10 0.0 2.9 11.5 14.2 12 0.0 4.6 15.2 20.1 14 0.0 3.4 11.3 35.1 16 0.0 3.0 8.0 39.4 18 0.0 2.0 9.3 43.0

[0102] From the 15th day, all the mice in the study showed a slight weight loss and a slight erythema in the area surrounding the tumor. Although the trend of the weight loss curve slowed down around the 15th day, the local erythema did not show any signs of recovery until the end of the study.

[0103] The tumor burden of the control mice (Group 1) showed an exponential growth.

[0104] The mice of Group 2 did not show significant differences compared to the mice of Group 1.

[0105] The mice of Group 3, which received only a single radiotherapy treatment, also did not show significant differences compared to the mice of Group 1.

[0106] The mice of Group 4, on the other hand, showed a significant increase in the anti-tumor effect, which reached 43.0% by the 18th day.

[0107] No toxic reactions were observed in all the mice in the study, which confirms that the injection of coated NPs according to the present application does not produce harmful side effects.

[0108] Example 3

[0109] This example relates to the inhibition of the growth of subcutaneous pharyngeal carcinoma achieved using the method of the present application.

[0110] On day 0, 49 athymic female nude mice were injected subcutaneously on the left side with 200 μl of culture medium containing 2 x 1010 6 cells of the DETROIT-562 cell line (subcutaneous pharyngeal carcinoma).

[0111] ​​The animals were monitored following the same general procedure detailed in Example 2 for the first few days of cancer growth. No unexpected deaths occurred during the test period.

[0112] When the average tumor volume reached 113 mm3 3 (day 7), the mice were randomly grouped and assigned to 7 experimental groups, with a target of 7 mice per group.

[0113] From day 8 onwards, groups 2 to 7 received multiple intratumoral injections of different solutions / suspensions according to two different schedules:

[0114] Once a week for a total of four times (q7dx4);

[0115] Once every four days for a total of seven times (q4dx7).

[0116] Specifically, groups 2 and 3 received multiple injections of 50 μL of a buffered solution (phosphate buffered saline, PBS); groups 4 to 7 received multiple injections of 50 μL of the suspension in Example 1, but only groups 6 and 7 mice were subsequently anesthetized and subjected to RF irradiation, under the same conditions as described in Example 2.

[0117] Group 6 mice received q7dx4 intratumoral treatment with NPs; after each treatment, they were subjected to three consecutive sessions of radiotherapy, 24 hours apart, each of 40 minutes.

[0118] Group 7 mice received q4dx7 intratumoral treatment with NPs; after each treatment, they were subjected to one session of radiotherapy.

[0119] Group 1 (control group) mice did not receive any injection.

[0120] The injection / irradiation conditions are summarized in Table 8 below.

[0121] Table 8

[0122]

[0123] Table 9 summarizes the average tumor volume (in cm 3 ) for each group, with the standard error (SE) and standard deviation (SD) given as a function of the number of days of the test.

[0124] The average tumor volume (mm 3 ) growth data ± SE as a function of the number of days of the test are also shown in graphical form in Figure 2 . In the graph, the data curves for groups 1 to 5 almost overlap, while the data curves for groups 6 and 7 are marked with the corresponding number in the graph; the data are presented with the level of significance.

[0125]

[0126] Table 10 reports the values of tumor growth inhibition (%T.G.I.) calculated from the data in Table 9.

[0127] Table 10

[0128] Day Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 7 0.0 -0.6 0.2 -2.3 0.0 -1.1 -0.1 11 0.0 1.5 0.6 -1.8 4.5 20.4 16.6 14 0.0 0.7 -3.6 3.7 3.8 28.8 23.5 17 0.0 1.5 -2.0 -1.5 3.5 31.2 28.8 21 0.0 3.7 -3.2 4.7 5.1 36.6 40.0 24 0.0 1.2 -3.4 4.4 4.2 37.2 42.8 28 0.0 0.5 -1.8 1.5 -0.9 31.0 41.0 31 0.0 0.4 1.8 -1.2 -2.0 29.5 38.5 35 0.0 2.3 1.3 -0.3 0.1 24.3 31.0

[0129] No significant anti-tumor effect was observed for the treatments with PBS (groups 2 and 3) and for the treatments with coated NPs followed by no radiotherapy (groups 4 and 5) compared to the control group (group 1).

[0130] On the contrary, multiple radiotherapy sessions after intratumoral injection of coated NPs were able to enhance the anti-tumor effect. A progressive delay in tumor growth was observed up to day 24, when a significant highest tumor growth inhibition was observed for groups 6 and 7, of 37.2% (p<0.01) and 42.8% (p<0.01), respectively. Mann-Whitney Test showed that the therapeutic effect of groups 6 and 7 was significant compared to group 1 (control group). No other significant anti-tumor effect was observed for the other groups.

[0131] No weight loss and side effects were observed.

[0132] Results comment

[0133] The data obtained in the series of tests of Example 2 and Example 3 show that the method of the present application, comprising intratumoral injection of a composition containing magnetite NPs followed by RF irradiation, is able to significantly slow down the progression of cancer growth.

[0134] One advantage of this method is that the tissues surrounding the cancer mass to be treated are substantially not affected by the treatment, so that the treatment can be repeated as many times as needed; this is different from the known thermotherapy methods that use RF irradiation only, which, due to the use of a higher frequency range, cause changes in the entire area of the body and therefore cannot be repeated over time.

Claims

1. An injectable composition for transdermal administration to solid tumors via direct injection into a tumor mass, said injectable composition comprising a suspension or emulsion of organically coated magnetite nanoparticles, selected from: A) An aqueous suspension containing surfactants and one or more buffer systems and stabilizers; and B) A water-based lipid suspension or emulsion, wherein the water-based lipid suspension or emulsion contains one or more components selected from lecithin, medium-chain triglycerides, long-chain triglycerides, glycerol, linoleic acid, cholesterol, and sodium oleate. The injectable composition has the following characteristics: Sterile and pyrogen-free; Isotonic with body fluids; The pH of the injectable composition is 7.8 to 8.2; The concentration of magnetite nanoparticles in the injectable composition ranges from 10 mM to 30 mM.

2. The injectable composition according to claim 1, wherein, The size of the magnetite nanoparticles is between 1 nm and 100 nm, preferably between 10 nm and 60 nm, and more preferably between 20 nm and 30 nm.

3. The injectable composition according to claim 1 or 2, wherein, The surface of the magnetite nanoparticles is first functionalized with a catechol derivative to form a component surrounded by a polymer shell, wherein the polymer is selected from chitosan, sodium alginate, polymethyl methacrylate, polysaccharides, block copolymers of poly(lactic acid / glycolic acid) and polyethylene glycol carboxylate, and combinations thereof.

4. The injectable composition according to any one of the preceding claims, wherein, The surfactant in suspension A) is selected from fatty acid dehydrated sorbitan esters, polysorbate fatty acid esters, sorbitol, sorbitan esters, cyclodextrin, sulfonyl ether cyclodextrin derivatives, linoleic acid, polyethylene glycol, and mixtures thereof, and when present, is added to suspension A) at a concentration of 1% to 25% by weight.

5. The injectable composition according to any one of the preceding claims, wherein, The buffer system in suspension A) is selected from: phosphate buffer formed by the salt pair Na2HPO4 / NaH2PO4; citrate buffer formed by trisodium citrate (C6H8O7·H2O / Na3C6H5O7·H2O); histidine buffer; physiological saline-sodium citrate buffer formed by sodium chloride, trisodium citrate and hydrochloric acid; and McIlvaine buffer formed by Na2HPO4 and citric acid solution.

6. The injectable composition according to any one of the preceding claims, wherein, The stabilizer in suspension A) is at least one of the following: -An antioxidant, wherein the concentration of the antioxidant is from 0.001% to 0.005% by weight; - A complexing agent selected from EDTA at a concentration of 0.025% to 0.075% by weight, cyclodextrin at a concentration of 1% to 15% by weight, PEG at a concentration of 0.1% to 10% by weight, and mixtures thereof; - A preservative, wherein the preservative is selected from ethanol at a concentration of 10% to 15% by weight, benzyl alcohol at a concentration of 0.10% to 0.15% by weight, sodium benzoate at a concentration of 0.10% to 0.15% by weight, and mixtures thereof.

7. The injectable composition according to any one of claims 1 to 3, wherein, In suspension or emulsion B), the component is added to the composition at a concentration of 0.1% to 10% by weight.

8. An injectable composition according to any one of the preceding claims, in kit form, comprising: - A first container containing a liquid phase of either composition A) or B), i.e., water in the case of composition A) and one or more of oleic acid, linoleic acid, stearic acid and polyethylene glycol in the case of composition B); - One or more containers containing a predetermined amount of surface-coated lyophilized magnetite nanoparticle powder, and at least one component selected from surfactants, fillers, salts or compounds that form buffer systems, and stabilizers.

9. The injectable composition in kit form according to claim 8, wherein, The filler is selected from mannitol, lactose, sucrose, glycine, and mixtures thereof.

10. The injectable composition according to any one of the preceding claims, for a volume of 100 mm² 3 Up to 500mm 3 For tumors between the two, administer a dose of 20 μL to 1 mL.

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