WO2018198030A1

WO2018198030A1 - Method for modulating phagocytic uptake of an active ingredient or a precursor thereof by macrophages

Info

Publication number: WO2018198030A1
Application number: PCT/IB2018/052849
Authority: WO
Inventors: Alessandro DALPIAZ; Barbara PAVAN; Marco FOGAGNOLO; Guglielmo PAGANETTO; Eliana Grazia LEO; Valentina IANNUCCELLI; Eleonora MARETTI
Original assignee: Universita degli Studi di Ferrara; Universita Degli Studi di Modena e Reggio Emilia
Current assignee: Universita degli Studi di Ferrara; Universita Degli Studi di Modena e Reggio Emilia
Priority date: 2017-04-26
Filing date: 2018-04-24
Publication date: 2018-11-01
Anticipated expiration: 2019-10-26
Also published as: IT201700045255A1

Abstract

The present invention relates to a method for modulating phagocytic uptake of a water-insoluble active ingredient or a water-insoluble precursor thereof, by macrophages, involving the use of nanoparticles of the active ingredient or of the precursor thereof coated with at least one bile acid or a salt thereof, in order to selectively direct said active ingredient, once it is in circulation, towards the target site of interest. The invention further relates to the use of said nanoparticles in the treatment or diagnosis of a disease affecting the macrophages or of a solid tumor.

Description

METHOD FOR MODULATING PHAGOCYTIC UPTAKE OF AN ACTIVE INGREDIENT OR A PRECURSOR THEREOF BY MACROPHAGES

The present invention relates to a method for modulating phagocytic uptake of a water-insoluble active ingredient or a water-insoluble precursor thereof by macrophages, which involves the use of nanoparticles of the active ingredient or of the precursor thereof coated with at least one bile acid or a salt thereof, in order to selectively direct said active ingredient, once it is in circulation, towards the target site of interest. The invention further relates to the use of said nanoparticles in the treatment or diagnosis of a disease affecting the macrophages or a solid tumor.

The selectivity of an active ingredient once in circulation and the efficiency with which it reaches the target site of interest are two aspects of fundamental importance for said active ingredient to be effective. In particular, with regard to the nanoparticles, both for the systems for directing an active ingredient based on "passive targeting" and for those based on "active targeting" it is essential to avoid the uptake of the active ingredient by the macrophages of the reticuloendothelial system (RES), dedicated to removing foreign particles from the system circulation .

Strategies have been developed for modifying- protecting nanoparticles of a polymeric, lipidic or phospholipidic nature containing active ingredients to prevent their phagocytosis by macrophages, such as ' PEGylation ' , i.e. the functionalisation with polyethylene glycol (PEG) . Such modification allows steric stabilisation of a particular agent or vector of an agent of interest, protecting it from the immediate phagocytosis and destruction by the macrophages residing in the RES, consequently increasing the stability and hence the therapeutic effectiveness of the incapsulated agent. Such strategy, however, requires the need to anchor by chemical bonds the PEG chains to polymeric or lipidic or phospholipidic matrices which subsequently will need to be formulated as nanoparticles in which the active ingredient is to be incapsulated, with related high costs of formulation and purification of the preparations. Moreover, the polymeric, lipidic or phospholipidic components and the PEG components cannot be controlled once they are introduced into the body in nanoparticle form and in the case of accumulation in areas of the body during prolonged therapies they can constitute elements of severe toxicity.

WO 2010/080754 describes nanoparticles comprising at least one water-insoluble compound and at least one bile acid, wherein the use of bile acids to prepare such nanoparticles makes it possible to obtain the reduction by 5-10 times of the average dimension of the nanoparticles and of the related polydispersity index ("PDI") of the particles produced. In particular, in the case of pharmaceutically active ingredients, the possibility of controlling the dimensions of the nanoparticles makes it possible to obtain improved pharmaceutical properties of the product itself (e.g., controlled dissolution rate, stability and improved effectiveness and targeting ability) .

The Applicant addressed the problem of selectively directing an active ingredient in circulation towards the target site of interest and of increasing the efficiency with which it reaches such site.

The Applicant has found that it is possible to solve such technical problem by coating nanoparticles of a water-insoluble active ingredient or a water- insoluble precursor thereof with at least one bile acid or a salt thereof. In particular, the Applicant has identified in bile acids components able to modulate the phagocytic uptake by macrophages, i.e. to modulate the phagocytic activity of macrophages. Consequently, thanks to the coating of the active ingredient of interest with a bile acid that reduces the phagocytic activity of the macrophages it is possible to direct the active ingredient into the site to be treated, maintaining said active ingredient in circulation for a longer time. If the target site is the macrophage itself, for example to treat a disease caused by a pathogen that infects the macrophages, it is possible to direct the active ingredient into the macrophage thus increasing the phagocytic activity that is already normally carried out by macrophages. Such system does not represent a chemical-type protection system with respect to the phagocytic activity of the macrophages, but rather it acts directly on the macrophages modulating their phagocytic activity on the basis of the active ingredient and of the target site of interest .

Therefore, a first aspect of the present invention is a method for modulating phagocytic uptake of an active ingredient or a precursor thereof by macrophages, said active ingredient or a precursor thereof being water-insoluble, which comprises coating said active ingredient or a precursor thereof in the form of nanoparticles with at least one bile acid or a salt thereof, preferably a salt thereof.

In a second aspect, the present invention relates to a method for conferring to nanoparticles of an active ingredient or a precursor thereof the ability to modulate phagocytic uptake by macrophages of said active ingredient or a precursor thereof, said active ingredient or a precursor thereof being water- insoluble, wherein said method comprises coating said nanoparticles with at least one bile acid or a salt thereof, preferably a salt thereof.

In a third aspect, the present invention relates to the use of at least one bile acid or a salt thereof, preferably a salt thereof, for conferring to nanoparticles of an active ingredient or a precursor thereof the ability to modulate the phagocytic uptake by macrophages of said active ingredient or a precursor thereof, said active ingredient or a precursor thereof being insoluble in water.

In the present description and in the appended claims, the term "precursor" means a prodrug, i.e. a molecule, inactive in itself from the pharmacological point of view, which, once it is introduced into the body, is metabolised so as to form the pharmacologically active molecule (active ingredient) .

In the present description and in the appended claims, the term "conjugate or derivative of an active ingredient with a bile acid" means that said active ingredient has been functionalised with a bile acid, for example through a covalent or ionic bond.

In the present description and in the appended claims, the term "average diameter" of the nanoparticles means, unless otherwise indicated, the d50 (median value) diameter i.e. the value of the diameter below which is found 50% by weight of the population of the nanoparticles (see "A Guidebook to Particle Size Analysis" published by Horiba Instruments Inc. - 2016, available from https : //www.horiba. com/ fileadmin/uploads/Scientific/ eMag/PSA/Guidebook/pdf/PSA_Guidebook.pdf) . It can be determined by Dynamic Light Scattering (DLS), in accordance with the ISO 13320:2009 standard.

Advantageously, coating a water-insoluble active ingredient or a water-insoluble precursor thereof with at least one bile acid (or a salt thereof) makes it possible to modulate the phagocytic activity of the macrophages, on the basis of the type of bile acid used in the coating. The Applicant has in fact found that a first class of bile acids, the sulfonated ones, which includes for example taurocholic acid, increases the phagocytic activity of the macrophages and hence the uptake of the active ingredient of interest by the macrophages. This is particularly advantageous in the case of pathogens that infect the macrophages and that therefore are located in the macrophages themselves. By contrast, a second class of bile acids, which includes those characterised by the free carboxyl group, which includes for example ursodeoxycholic acid, cause the reduction in the phagocytic activity of the macrophages and consequently a decrease in terms of uptake of the active ingredient of interest. In this case, the active ingredient remains in circulation and hence is destined to selectively reach the specific target on which it acts pharmacologically or, in the case of diagnostic agents, the disease to be diagnosed or monitor.

An additional advantage of the present invention is given by the fact that the nanoparticles of the present invention represent a system for the delivery of an active ingredient or of a precursor thereof characterised by the absence of toxicity and by high biocompatibility .

Furthermore, the proposed invention makes it possible to formulate the active ingredient itself as nanoparticles coated with bile acid (or a salt thereof) by means of a single, simple and economical operation in the absence of any matrix of a polymeric, lipidic or phospholipidic nature. The absence of this type of matrices allows to avoid the need to modify/protect the matrices themselves by functionalisation, for example with PEG. This then allows to avoid all the steps involving chemical reactions and thus to considerably reduce the formulative steps, thus sharply lowering the costs and the side effects caused by the matrices following administration. Moreover, the proposed invention allows to obtain nanoparticles with a relatively high content of free active ingredient ready for dissolution and for the consequent therapeutic action, once the target site is reached, in the absence of any phenomena of undesired accumulation of polymeric and lipidic matrices. The formulative simplicity of the nanoparticles of the present invention should also enable to carry out simplified procedures in any marketing authorisation requests.

Brief description of the figures

The present invention shall now be described, by way of non-limiting illustration, according to preferred embodiments thereof, with particular reference to the accompanying figures, in which:

Figure 1 shows a SEM (scanning electron microscope) photograph of nanoparticles of UDCA-AZT coated with UDCA;

Figure 2 shows a SEM (scanning electron microscope) photograph of nanoparticles of UDCA-AZT coated with tauro;

Figure 3 shows a SEM photograph in STEM mode of nanoparticles of UDCA-AZT coated with UDCA;

Figure 4 shows a SEM photograph in STEM mode of nanoparticles of UDCA-AZT coated with taurocholate (tauro) ;

Figure 5 shows the cellular uptake of UDCA-AZT after 15 minutes of incubation in free form, both in the absence (UA 100 uM) and in the presence of the bile acid salts taurocholate (UA + tauro) and ursodeoxycholate (UA + UDCA) , or in nanoparticle form obtained in the presence of taurocholate (nano tauro) or ursodeoxycholate (nano UDCA) ;

Figure 6 shows the number of healthy cells observed after 15 minutes of incubation with the nanoparticle systems coated with taurocholate or ursodeoxycholate .

Further features and advantages of the present invention will be apparent from the detailed description that follows.

With reference to the present invention, the term active ingredient means both a pharmacologically active ingredient and a diagnostic agent, both being water- insoluble .

The pharmaceutically active ingredient may be selected among analgesics, anti-inflammatory agents, anthelmintics, antiarrhythmic agents, antibiotics, anticoagulants, antidepressants, antidiabetic agents, antiepileptics , antihistamines, antihypertensive agents, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, for example paclitaxel, docetaxel, cabazitaxel, zidovudine (AZT) , immunosuppressants, antithyroid agents, antiviral agents, for example zidovudine (AZT) , anxiolytic sedatives, astringents, alpha and beta blockers, adrenoceptors, corticosteroids, antitussives, diagnostic agents, imaging diagnostic agents, diuretics, dopaminergics, haemostatics, immunologic agents, lipid regulation agents, muscle relaxants, parasympathomimetics, calcitonin parathyroid, prostaglandins, radiopharmaceuticals, sex hormones, anti-allergy agents, stimulants, sympathomimetics, thyroid agents, vasodilators, bronchodilators

(xanthines), xanthenes, components of essential oils (for example geraniol, eugenol, carvacrol), active ingredients extracted from plants or their synthetic equivalents, for example substances comprising polyphenols, sulfides, monoterpenes , saponins, phytosterols , carotenoids, capsaicin or pharmaceutically acceptable forms thereof. Other non- limiting examples of pharmaceutically active ingredients are: immunosuppressant agents such as cyclosporins including cyclosporin A, tacrolimus, and mycophenolate mofetil; immunoactive agents, antiviral and antifungal agents, antineoplastic agents, analgesic and anti-inflammatory agents, antibiotics, antiepileptics , anaesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, anticonvulsant agents, antagonists, neuronal blocking agents, anticholinergics and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergic and antiarrhythmic agents, antihypertensive drugs, antineoplastic agents, hormones, antihyperlipemics ; antimicrobials, for example, antibacterials such as sulfadiazine, antimycotics such as itraconazole; non steroidal anti inflammatory drugs, for example, indomethacin; antihypercholesterolemic agents, for example probucol; and steroidal compounds.

With reference to the diseases that can affect macrophages and that can be diagnosed and/or treated by means of the nanoparticles according to the invention, such diseases can be caused by pathogen agents, for example viral pathogen agents, bacterial pathogen agents, mycetes and/or parasites belonging to the protozoa, known to the person skilled in the art. Examples of viral pathogen agents that infect macrophages are the HIV virus, the HTLV-III virus, the Dengue virus (DENV) and Zika virus. An example of bacterial pathogen agents that infect macrophages are the mycobacteria that cause tuberculosis. Examples of diseases caused by parasites belonging to protozoa are animal leishmaniasis, toxoplasmosis and African trypanosomiasis .

According to a preferred aspect of the present invention, the diagnostic agent is preferably an imaging agent or a contrast medium, for example folic acid for tumor cells or 2-fluoro-2-deoxy-D-glucose marked to monitor the reduction of the metabolism of the tumor cells following chemotherapy with cytostatic drugs. Another example consists of the non-invasive detection of macrophages carried out with magnetic resonance using iron oxide nanoparticles (USPIO) . After intravenous or intra-articular administration, the USPIO are specifically ingested by the activated macrophages and, because of their magnetic properties, cause signal variations in tissues that exhibit macrophage infiltrations.

According to a preferred aspect of the present invention, the water-insoluble precursor of the active ingredient is a conjugate or a derivative, which serves as a prodrug of the active ingredient. Through the conjugation or functionalisation of an active ingredient, it is in fact possible to make water- insoluble an active ingredient that is per se water- soluble. Preferably, a water-soluble active ingredient is conjugated or functionalised with a bile acid, more preferably a sulfonated bile acid, for example taurocholic acid, or a bile acid characterised by the free carboxyl group, for example ursodeoxycholic acid (UDCA) . According to a preferred embodiment of the present invention, the sulfonated bile acid with which the active ingredient is conjugated or functionalised is taurocholic acid. According to another preferred embodiment of the present invention, the bile acid characterised by the free carboxyl group with which the active ingredient is conjugated or functionalised is ursodeoxycholic acid (UDCA) .

An example of a precursor according to the invention is the UDCA-AZT derivative having the following formula:

Preferably, the water-insoluble active ingredient or a water-insoluble precursor thereof has a water solubility lower than 0.01 mg/ml, more preferably lower than 0.005 mg/ml, even more preferably 0.003 mg/ml, measured at room temperature (approximately 25°C) and physiological pH between 6.5 and 7.4.

The nanoparticles of a water-insoluble active ingredient or a water-insoluble precursor thereof according to the invention are coated with at least one bile acid or a salt thereof, preferably a salt thereof. According to a preferred aspect of the invention, said at least one bile acid is selected among sulfonated bile acids, preferred taurocholic acid. According to another preferred aspect of the invention, said at least one bile acid is selected among those characterised by the free carboxyl group, preferably ursodeoxycholic acid.

Preferably, the salt of the bile acid is a salt of an alkaline metal, more preferably a sodium salt.

According to a preferred aspect of the present invention, the nanoparticles have an average diameter comprised between 110 and 290 nm, preferably between 150 and 250 nm.

According to a preferred aspect of the present invention, the nanoparticles consist of the water- insoluble active ingredient or a water-insoluble precursor thereof, as described above.

According to another preferred aspect of the present invention, the nanoparticles consist of the water-insoluble active ingredient or a water-insoluble precursor thereof, as described above, and a coating consisting of at least one bile acid or a salt thereof, preferably a salt thereof, as described above.

The nanoparticles of the invention can be prepared according to the following process which comprises the steps of:

a) dissolving a water-insoluble active ingredient or a water-insoluble precursor thereof in an organic solvent, preferably an alcohol, for example methanol, ethanol, isopropanol, acetone, acetonitrile ;

b) adding the solution obtained in step a) to an aqueous solution of at least one bile acid, preferably selected between taurocholic acid or ursodeoxycholic acid, or a salt thereof;

c) maintaining the suspension obtained in step b) under stirring until evaporation of the non-aqueous solution.

As stated above in the present description, the

Applicant has found that the nanoparticles as defined above play a role in the modulation of the phagocytic activity of the macrophages, i.e. of the phagocytic uptake of an active ingredient.

Therefore, another aspect of the present invention consists of nanoparticles of an active ingredient or a precursor thereof, wherein said active ingredient or precursor thereof is water-insoluble, said nanoparticles being coated with at least one bile acid or a salt thereof, for use in the treatment or diagnosis of a disease caused by pathogen agents that infect the macrophages or of a solid tumor.

Based on the possibility of increasing or reducing the phagocytic activity of the macrophages, it is possible to produce respectively nanoparticles that carry the active ingredient of interest into the macrophages (increasing the phagocytic activity of the macrophages), or nanoparticles that reduce the phagocytic activity of the macrophages, thereby allowing the active ingredient to remain in circulation, and to reach the target against which its pharmacological activity is to be carried out, for example a solid tumor tissue, in the case of anti-tumor drugs as an active ingredient. Such modulation activity is directly tied to the type of bile acid used in the coating. Consequently, the nanoparticles of the present invention can be used to carry an active ingredient into the macrophages and/or to carry an active ingredient to an infected tissue, preferably a solid tumor tissue.

According to a preferred aspect, when the nanoparticles according to the invention are used to carry the active ingredient in the macrophages, the at least one bile acid is a sulfonated bile acid, preferably taurocholic acid.

According to another preferred aspect of the present invention, when the nanoparticle according to the invention is used to maintain the active ingredient in circulation, for example if it is to be carried to an infected tissue, preferably a tumor tissue, the at least one bile acid is a bile acid characterised by the free carboxyl group, preferably ursodeoxycholic acid. With reference to this latter aspect, the nanoparticle allows to extend the half-life of the active ingredient which otherwise would tend to be ingested by the macrophages. As stated above, in fact, macrophages have a natural tendency to ingest all particles recognised as "foreign bodies" and even an active ingredient inside a nanoparticle, once in circulation, can be recognised as a foreign body and hence be ingested by the macrophages. Preferably, the modulation of the phagocytic activity of the macrophages determines the reduction of such phagocytic activity and consequently the extension of the half-life of the active ingredient. According to another preferred aspect of the invention, the modulation of the phagocytic activity of the macrophages determines the increase of such phagocytic activity and consequently the uptake of the active ingredient by the macrophages.

Another aspect of the present invention consists of nanoparticles of a conjugate or a derivative of an active ingredient, preferably water-soluble, with a bile acid, preferably a sulfonated bile acid, more preferably taurocholic acid, or a bile acid characterised by the free carboxyl group, more preferably ursodeoxycholic acid, coated with at least a bile acid or a salt thereof, preferably a salt thereof. Preferably, said particles are coated with at least one sulfonated bile acid, more preferably taurocholic acid, or a salt thereof. Preferably, said particles are coated with at least one bile acid characterised by the free carboxyl group, more preferably ursodeoxycholic acid, or a salt thereof, preferably a salt thereof.

Another aspect of the present invention consists of the aforesaid nanoparticles for use as a medicament.

Another aspect of the present invention consists of the aforesaid nanoparticles for use in the treatment or diagnosis of a disease caused by pathogen agents that infect the macrophages or of a solid tumor.

The present invention will now be further illustrated through some embodiment examples as described below.

Examples

EXAMPLE 1.

Nanoparticles according to the present invention were prepared as described below. Through the functionalisation of the active ingredient zidovudine (AZT) and from ursodeoxycholic acid (UDCA) , the derivative UDCA-AZT was obtained. The derivative UDCA- AZT (20 mg) was fully solubilised in methanol. The solution obtained was added in an aqueous solution of sodium ursodeoxycholate, under magnetic agitation. The suspension obtained was kept under stirring under a fume hood for 12 hours to allow the removal of the methanol. The samples thus obtained were freeze-dried to obtain a powder.

EXAMPLE 2 Example 1 was repeated replacing the aqueous solution of UDCA with an aqueous solution of sodium taurocholate (tauro) .

EXAMPLE 3

The samples of lyophilised powder obtained according to examples 1 and 2 were placed on an aluminium stub for analysis with a SEM scanning electron microscope (SEM, Nova NanoSEM 450, Fei, Eindhoven, The Netherlands) . The images obtained are shown in Figures 1 and 2. The particles under SEM analysis had a spherical appearance with smooth surface and dimensions comprised between 150 and 250 nm. The dimensional analysis carried out with PCS (photon correlation spectroscopy) (Nanosizer ZS, Malvern, Germany) confirms the data observed by SEM and indicates the presence of a homogenous population of particles for both preparations, as shown below in Table 1, which indicates average diameter, PDI and surface charge (zeta potential) of the two samples right after they were prepared and after reconstitution of the freeze-dried powder, obtained by Nanosizer ZS (Malvern, Germany) .

Sample Average PDI Z-potential diameter

Fresh AZT-UDCA/Tauro 190 + 10 0.063 + 0.02 - 55.6 + 6.53

Freeze-dried AZT- 217+ 15 0.066 + 0.03 - 59.6 + 9.11 UDCA/Tauro

fresh AZT-UDCA/UDCA 215 + 20 0.116 + 0.07 - 51.8 + 7.46

Freeze-dried AZT- 248 + 20 0.183 + 0.09 - 61.4 + 6.91 UDCA/UDCA The suspension of fresh nanoparticles was subjected to morphological analysis using the scanning electron microscope SEM in STEM (i.e. transmission) mode (see Figures 3 and 4) . In both cases the nanoparticles appear surrounded by a halo attributed to the coating with the bile salt.

EXAMPLE 4

To evaluate the content of UDCA-AZT in the nanoparticles, they were dissolved in methanol in the ratio of 2.6 ml/mg and after filtration (0.45 μπι) , the solution was quantified via HPLC (see below) . The content of UDCA-AZT was found to be 16.7% both in the nanoparticles obtained in the presence of taurocholate, and in those in the presence of ursodeoxycholate.

EXAMPLE 5

Assessment of the phagocytic activity

To assess the degree of phagocytosis of the proposed nanoparticles, they were incubated with murine macrophages (line J774A.1) and the intracellular content of UDCA-AZT of the nanoparticles was analysed via HPLC. The data were compared with the intracellular content of UDCA-AZT derived from the incubation of the free molecule, both in the absence and in the presence of the salts of the free bile acids employed for the formulation of the nanoparticles. In particular, the cells J774A.1 were seeded in 12-well cell culture plates at a density of 5 x 10⁵ cells per well, and upon reaching the semi-confluent growth stage the cells were treated for 15 min with a growth medium containing 100 uM free UDCA-AZT (in the presence and in the absence of free taurocholate (Tauro) or free ursodeoxycholate (UDCA) ) or incapsulated in nanoparticles . At the end of the treatment, the cells were washed and lysed. The cell lysates were dried under nitrogen flow, resuspended in methanol and centrifuged to remove cell detritus. The levels of AZT and UDCA-AZT were measured in the supernatant (10 ul) through HPLC analysis. All values obtained from the experiments with J774A.1 cells are the average of three independent experiments.

Cell count.

The cell count was carried out as follows. The cells J774A.1 were counted using the Scepter 2.0 cell counter (Merck Millipore, Milan, Italy) . Briefly, the cells were scraped to be able to detach them from the wells and they were then resuspended in the culture medium. Subsequently, cell suspensions were transferred into 1.5 ml micro-centrifuge test tubes and the number of cells was quantified with the counter fitted with 60 μπι sensor, based on the manufacturer's recommendation. All values obtained from the experiments with J774A.1 cells are the average of three independent experiments.

HPLC Analysis

The chromatographic apparatus consisted of a Shimadzu modular system (Kyoto, Japan) consisting of four-way pump (LC-10 AD VD model) and a variable wavelength UV detector (SPD 10A VP model) and an injection valve fitted with 20 μΐ loading loop (Rheodyne model 7225, Torrance, CA, USA) . Chromatographic separation was carried out at ambient temperature using a Hypersil BDS C-18 column (5 μπι, 150 mm x 4.6 mm) fitted with pre-column made of the same material (Alltech Italia, Milan) . The UV detector was set to 260 nm. The mobile phase consisted of a H20/MeOH mixture in the ratio of 20:80 (V/V) with a flow of 1 ml per minute. The retention time of UDCA-AZT was found to be 4.8 minutes. Chromatographic precision was obtained from 6 repeated analyses of the same sample at the concentration of 10 μΜ in methanol and it was found to be expressed by a relative standard deviation of 0.95%. Calibration was carried out in the range of concentrations in methanol between 0.2 μΜ and 20 μΜ, the linearity of which was observed (n = 8, r = 0.995, P< 0.001) .

Toxicity test

The cells J774A.1 were seeded in 96-well plates at a density of 8000 cells per well to reach an optimal growth within 48-72 hours. Subsequently, the cells were incubated for 15 min in 200 uL of culture medium in the presence and absence of 100 uM UDCA-AZT. At the end of the "time-course", the incubation medium was removed and replaced with 200 i of fresh culture medium, then to each well were added 20 uL of stock solution (5 mg/ml) of 3- ( 4 , 5-Dimethylthiazol-2-yl ) -2 , 5- diphenyltetrazolium bromide (MTT) and the entire plate was incubated at 37 °C for 4 hours. Moreover, a negative control of 20 μΐ of the MTT stock solution added to 200 μΐ of medium alone was included in the plate. Then, 100 μΐ of DMSO were added to each well and the entire plate was incubated again at 37 °C for 2 hours in an orbital incubator/shaker. Lastly, the absorbance of each well was measured at 570 nm with a microplate reader.

Statistical analysis

A one-way variance analysis was carried out, followed by Bonferroni or Dunnet analysis

Results

Figure 5 shows the different modes of uptake in the murine macrophages of UDCA-AZT, both in free form and in nanoparticle form. In particular, after 15 minutes of incubation it is found that 238 ± 25 ng/106 cells of free UDCA-AZT entered the macrophages. It was tested whether the presence of the free bile salts (in the same quantity present in the incubated nanoparticle systems) could influence the uptake of free UDCA-AZT: in the presence of free taurocholate (UA + tauro) , after 15 minutes of incubation, 263 ± 36 ng/106 cells of UDCA-AZT entered the macrophages, while the quantity recorded in the presence of free ursodeoxycholate (UA + UDCA) 9 was found to be 441 ± 51 ng/106 cells. These values were not found to be significantly different from the result obtained in the absence of free bile salts (p > 0.05) . The incubation for 15 minutes of the nanoparticles obtained in the presence of taurocholate (nano tauro) led to an entry of UDCA-AZT into the macrophages of 16231 ± 2458 ng/106 cells, i.e. a quantity approximately 70 times greater than that obtained with free UDCA-AZT (p < 0.001) . In the same conditions, the incubation of the nanoparticles obtained in the presence of ursodeoxycholate (nano UDCA) led to the entry of UDCA-AZT of 3448 ± 637 ng/106 cells, a quantity approximately 14 times greater than that obtained with free UDCA-AZT. However, this difference in uptake between free UDCA-AZT and nanoparticles coated with ursodeoxycholate was not found significant (p > 0.05) . The difference in uptake of UDCA-AZT obtained with the two investigated nanoparticle systems was instead found to be significant (p < 0.001) .

Figure 6 shows, in percentage terms, the number of healthy cells observed following incubation with the nanoparticle systems. The results show that the toxic impact of these systems is not significant. With reference to the number of healthy cells (expressed as % relative to the control) observed after 15 minutes of incubation with the nanoparticle systems coated with taurocholate (nano tauro) or ursodeoxycholate (nano UDCA) , it can be observed that there were no significant differences with respect to the control.

Claims

1. Method for modulating the phagocytic uptake of an active ingredient or a precursor thereof by macrophages, said active ingredient or a precursor thereof being water-insoluble, which comprises coating said active ingredient or a precursor thereof in the form of nanoparticles with at least one bile acid or a salt thereof.

2. Method according to claim 1, wherein the at least one bile acid is selected from the sulfonated bile acids, preferably taurocholic acid, or bile acids characterized by the free carboxyl group, preferably ursodeoxycholic acid.

3. Method according to any one of the preceding claims, wherein the water-insoluble precursor is obtained by conjugation or functionalization of the active ingredient with a bile acid, preferably a sulfonated bile acid or a bile acid characterized by the free carboxyl group.

4. Method according to claim 3, wherein the sulfonated bile acid is taurocholic acid.

5. Method according to claim 3, wherein the bile acid characterized by the free carboxyl group is ursodeoxycholic acid.

6. Method according to any one of the claims from

3 to 5, wherein the active ingredient is zidovudine

(AZT) .

7. Method according to any one of the preceding claims, wherein said nanoparticles have an average diameter comprised between 110 and 290 nm, preferably between 150 and 250 nm.

8. Method according to any one of the preceding claims, wherein the water-insoluble active ingredient or a water-insoluble precursor thereof has a water solubility lower than 0.01 mg/ml, more preferably lower than 0.005 mg/ml, even more preferably 0.003 mg/ml, measured at room temperature (about 25°C) and physiological pH between 6.5 and 7.4.

9. Method according to any one of the preceding claims, wherein the modulation of the phagocytic uptake causes the reduction in such phagocytic activity and accordingly the extension of the half-life of the active ingredient.

10. Method according to any one of the preceding claims, wherein the modulation of the phagocytic uptake causes the increase in such phagocytic activity and accordingly the uptake of the active ingredient by the macrophages.

11. Nanoparticles of a conjugate or a derivative of an active ingredient, preferably water-soluble, with a bile acid, preferably a sulfonated bile acid, more preferably taurocholic acid, or a bile acid characterized by the free carboxyl group, more preferably ursodeoxycholic acid, coated with at least one bile acid or a salt thereof.

12. Nanoparticles according to claim 11, wherein said nanoparticles are coated with at least one sulfonated bile acid, preferably taurocholic acid, or a salt thereof.

13. Nanoparticles according to claim 11, wherein said nanoparticles are coated with at least one bile acid characterized by the free carboxyl group, preferably ursodeoxycholic acid or a salt thereof.

14. Nanoparticles according to any one of the claims from 11 to 13, wherein the active ingredient is zidovudine (AZT) .

15. Nanoparticles as defined in any one of the claims from 11 to 14 for use as medicament.

16. Nanoparticles as defined in any one of the claims from 11 to 14 for use in the treatment or diagnosis of a disease caused by pathogen agents which infect the macrophages or of a solid tumor.