EA047692B1 - PHARMACEUTICAL COMPOSITION, ITS PRODUCTION AND APPLICATIONS - Google Patents

Description

Field of technology to which the invention relates

The invention relates to a pharmaceutical composition comprising a combination of (i) at least one biocompatible nanoparticle and (ii) at least one carrier comprising at least one compound of interest, typically at least one pharmaceutical compound to be administered to a subject in need of such at least one compound of interest, wherein the combination of at least one biocompatible nanoparticle and at least one carrier comprising at least one compound of interest enhances the effectiveness of the compound(s) of interest. The largest dimension of the biocompatible nanoparticle is typically from about 4 to about 500 nm, and the absolute value of the surface charge is at least 10 mV (|10 mV|). The carrier does not contain any surface sterically stabilizing agent.

The invention also relates to such a composition for use in administering a compound(s) of interest to a subject in need thereof, wherein at least one nanoparticle, on the one hand, and at least one carrier containing the compound of interest, on the other hand, are to be administered to said subject sequentially, typically at an interval of more than 5 minutes to about 72 hours from each other.

The combined and, as a rule, sequential administration to a subject of at least one biocompatible nanoparticle and at least one carrier containing a compound(s) of interest preserves the pharmaceutical (i.e., therapeutic, prophylactic or diagnostic) effectiveness of said compound(s) of interest with its reduced toxicity in said subject or increases its pharmaceutical effectiveness with equivalent or reduced toxicity compared to the pharmaceutical effectiveness and toxicity caused by said compound(s) when administered in a standard pharmaceutical dose, as a rule, in the absence of any biocompatible nanoparticle and/or carrier.

The pharmaceutical composition of the invention typically allows for a reduction of at least 10% in the pharmaceutical dose(s) of the compound(s) administered compared to a standard pharmaceutical dose(s) of said compound(s), typically in the absence of any biocompatible nanoparticle and/or carrier, while maintaining the same pharmaceutical efficacy with equivalent toxicity, preferably with reduced toxicity, to the subject or with an increase in pharmaceutical efficacy with equivalent or reduced toxicity to the subject.

State of the art

The use of nanotechnology to deliver therapeutic and diagnostic agents in a safer and more effective manner to patients has led to increased interest in this field over the past decades. Drug delivery systems have emerged, typically carriers such as liposomes, emulsions or micelles, designed to maximize the therapeutic efficacy of drugs by controlling their biodistribution profile. These systems offer the opportunity to encapsulate a poorly soluble drug, protect the drug from degradation or elimination, and/or alter the blood circulation and distribution of the drug.

The observed rapid blood clearance of first-generation drug delivery systems (DDS) (due to their uptake by the mononuclear phagocytic system (MPS)) prompted the development of second-generation DDSs possessing a surface modified with sterically stabilizing agents selected to impart stealth properties to the DDS when attached to its surface. These agents are typically flexible and/or hydrophilic polymers such as polyethylene glycol (PEG) polymers and can typically carry a surface charge that is weakly negative or positive. Steric stabilization prevents non-specific surface binding of DDSs to blood components and reduces its rapid uptake and clearance in vivo by MPS cells, resulting in prolonged circulation time of DDSs in the bloodstream [Jain K.R. and Stylianopoulos T. Delivering nanomedicine to solid tumors. Nature Reviews. Clinical Oncology, 2010, 7, 653-664]. Liposomal long-circulating nanoparticulate drug delivery systems (NDDS) are the most commonly studied type of NDDS; however, synthetic amphiphilic polymers have also been used to sterically stabilize other types of NDDS to alter their biodistribution [Torchilin V.P. Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nature Reviews. Drug Discovery, 2014, 13, 813-827].

Despite this increase in blood circulation time (i.e., improved transport in the blood), which was considered important for the delivery of the therapeutic compound to its target site, it was found that a flexible and/or hydrophilic polymer coating, typically a PEG coating, negatively affected the intracellular delivery of the pharmaceutical compound (i.e., the release of the compound at its target site), ultimately resulting in a loss of activity for the delivery system. A way to overcome this limitation is to use cleavable PEG systems. However, the increasing complexity of the design of such carriers can create difficulties in the reproducibility of the carrier surface properties, resulting in unacceptable variability in the characteristics from pair to pair

- 1,047,692 tii per batch. Moreover, the prolongation of the action time of these subtle DDS has been associated with a large number of side effects. For example, DOXIL, a pegylated liposomal formulation containing doxorubicin, has been found to cause serious side effects such as hand-foot syndrome or mucositis.

The hydrophilic coating of liposomes has been investigated as a possible contributor to their accumulation in the eccrine sweat gland on the palmar surface of the hand and the sole of the foot [Pegylated liposomal doxorubicin-related palmar-plantar erythrodysesthesia (hand-foot syndrome). D. Lorusso et al. Annals of Oncology. 2007; 18, 1159-1164].

WO 2005/063305 relates to an assembly comprising a gas-filled microbubble (typically at least 0.5 μm in size) and a component (less than about 100 nm in size) attached to said microbubble. The resulting assembly is intended for use as a pharmaceutically active component in diagnostic and/or therapeutically active formulations. The two components, i.e. the gas-filled microbubble and the component attached to the microbubble, are administered simultaneously, typically for image enhancement in the field of ultrasound contrast imaging, including targeted ultrasound imaging, ultrasound-mediated drug delivery and other imaging techniques.

As is evident from the prior art and despite the long-standing medical need, safe and effective delivery of pharmaceutical compounds (including therapeutic, prophylactic and diagnostic compounds) to their target site(s) remains a problem. There is a clear need to improve the efficacy and safety of the compound, or in other words, to transport and release the pharmaceutical compound so that said compound reaches its target site in a subject in the required and sufficient amount to obtain the desired diagnostic, therapeutic or prophylactic effect.

Detailed description

The present invention now allows to optimize the effectiveness of a compound of interest (herein also simply referred to as a compound) regardless of its intended use in the context of therapy, prophylaxis or diagnostics. The composition described herein, which is a combination of (i) at least one biocompatible nanoparticle and (ii) at least one carrier containing at least one compound of interest, optimizes the pharmacokinetic parameters of at least one compound of interest and, as a consequence, now makes it possible to develop pharmaceutical compounds that could not be developed otherwise, for example, due to their unacceptable toxicity. As a rule, the biocompatible nanoparticle is not used as such as a pharmaceutical compound, i.e. as a therapeutic, prophylactic or diagnostic compound.

A typical composition of the invention (usually referred to herein as a pharmaceutical composition) is a composition comprising a combination of (i) at least a biocompatible nanoparticle and (ii) at least a carrier comprising at least one compound (the compound of interest), wherein the largest or greatest dimension of the biocompatible nanoparticle is typically from about 4 to about 500 nm and the absolute value of the surface charge of the biocompatible nanoparticle is at least 10 mV and wherein the carrier does not contain any surface sterically stabilizing agent, i.e. does not contain a flexible and/or hydrophilic polymer, preferably does not contain a hydrophilic polymer bearing a weakly negative or positive charge to the surface of the carrier, such as PEG.

Typically, the ratio between (at least one) biocompatible nanoparticles and (at least one) carriers containing at least one compound of interest is from 0.1/1 to 1000/1 or from 0.5/1 to 1000/1, preferably from 0.5/1 to 500/1, even more preferably from 0.5/1 to 300/1.

The terms approximately and about, when associated with a value such as, for example, the size of a nanoparticle or a time interval, indicate that a deviation from the stated value, which would be recognized by a skilled person as a small deviation, does not significantly affect the properties of the object to which it is associated, and that the stated object remains within the scope of the claimed invention.

A preferred subject of the invention is a pharmaceutical composition comprising a combination of (i) at least one biocompatible nanoparticle and (ii) at least one carrier comprising at least one compound of interest, typically at least one pharmaceutical compound, wherein the largest or greatest dimension of the biocompatible nanoparticle is from about 4 to about 500 nm and the absolute value of the surface charge of the biocompatible nanoparticle is at least 10 mV (|10 mV|) and wherein the carrier does not contain any surface sterically stabilizing agent, for use in administering at least one compound of interest to a subject in need thereof, wherein the at least one nanoparticle, on the one hand, and at least

- 2 047692 one carrier containing a compound of interest, on the other hand, should be administered separately to a subject in need of said at least one compound of interest, typically at an interval of more than 5 minutes to about 72 hours from each other, and where the biocompatible nanoparticle is not used as such as a pharmaceutical compound.

The combined and, as a rule, sequential administration to a subject of at least one biocompatible nanoparticle and at least one carrier containing the compound(s) of interest, by means of the composition of the invention, usually provides (maintains) the same pharmaceutical (i.e. therapeutic, prophylactic or diagnostic) effectiveness of the compound(s) with its reduced toxicity to the subject, or increases the pharmaceutical effectiveness of the compound(s) with an equivalent or reduced toxicity to the subject (preferably with reduced toxicity) compared to the pharmaceutical effectiveness and toxicity caused by a standard pharmaceutical dose of said compound(s), as a rule, in the absence of any biocompatible nanoparticle and/or carrier.

The pharmaceutical composition of the invention typically allows for a reduction of at least 10%, preferably at least 15%, in the administered pharmaceutical (i.e. therapeutic, prophylactic or diagnostic) dose(s) of the compound(s) compared to a standard pharmaceutical dose(s) of said compound(s), typically in the absence of any biocompatible nanoparticle and/or carrier, (i) while maintaining the same pharmaceutical efficacy with equivalent toxicity, preferably with reduced toxicity, to the subject or (ii) while increasing the pharmaceutical efficacy with equivalent or reduced toxicity to the subject.

Biocompatible nanoparticle.

Since the shape of a particle can influence its biocompatibility, particles having a qualitatively uniform shape are preferred. Thus, for pharmacokinetic reasons, nanoparticles that are essentially spherical/round or ovoid in shape are preferred. Such a shape also facilitates the interaction of the nanoparticles with cells or their uptake by them. A spherical/round shape is particularly preferred.

In the essence of the invention, the term nanoparticle refers to a product, in particular a synthetic product, with a size in the nanometer range, typically from about 1 to about 500 nm, preferably from about 4 to about 500 nm, from about 4 to about 400 nm, from about 30 to about 300 nm, from about 20 to about 300 nm, from about 10 to about 300 nm, for example from about 4 to about 100 nm, for example about 10, 15 or 20 nm and about 100 nm, or from about 100 to about 500 nm, typically from about 100 to about 300 nm.

The terms "nanoparticle size", "largest nanoparticle size" and "largest nanoparticle size" in this specification generally refer to the largest or biggest nanoparticle size or the diameter of a nanoparticle when it is spherical/round or ovoid in shape. Transmission electron microscopy (TEM) or cryo-TEM can be used to measure the size of a nanoparticle. In addition, dynamic light scattering (DLS) can be used to measure the hydrodynamic diameter of nanoparticles in solution. These two methods can also be used one after another to compare the hydrodynamic diameter of a nanoparticle measured by DLS with the size of the said nanoparticle measured by TEM or cryo-TEM to confirm the said size.

The preferred method is DLS (see International Standard ISO22412 Particle size analysis - Dynamic light scattering, International Organization for Standardization (ISO) 2008).

For use in the context of the invention, the absolute electrostatic surface charge (also referred to herein as charge or surface charge) of the biocompatible nanoparticle should be greater than |10 mV| (absolute value). The surface charge of the nanoparticle is typically determined by measuring the zeta potential in an aqueous medium for a nanoparticle concentration of 0.2 to 10 g/L, for a pH of 6 to 8, and typically for electrolyte concentrations in an aqueous medium of 0.001 and 0.2 M, such as 0.01 or 0.15 M.

Typically, the biocompatible nanoparticle of the present invention has an electronic surface charge of at least |10 mV|, i.e. below -10 mV or above 10 mV, for example below -12 mV or -15 mV to -20 mV or above 12 mV or 15 mV to 20 mV, typically below -15 mV or above 15 mV. Preferably, the biocompatible nanoparticle of the present invention has an absolute value of the electronic surface charge (absolute value of the surface charge) of more than 10 mV, wherein said charge is even more preferably a negative charge.

The combined properties, size and surface charge, of the nanoparticles allow for a short circulation time of the nanoparticles in the blood and extravasation into the liver. Therefore, by sequentially administering the biocompatible nanoparticles of the invention and a carrier containing the compound(s) of interest, no co-circulation or limited co-circulation

- 3 047692 the combination of two compounds (i.e. the biocompatible nanoparticle and the carrier containing the compound(s)) is not achieved. Thus, the combined properties of the biocompatible nanoparticles, size and surface charge, allow the safe use of the compound of interest while providing (maintaining) the same pharmaceutical (i.e. therapeutic, prophylactic or diagnostic) efficacy of the compound(s) with its reduced toxicity to the subject or, in other words, with an increase in the pharmaceutical efficacy of the compound(s) with its equivalent or reduced toxicity to the subject (preferably reduced toxicity) compared to the pharmaceutical efficacy and toxicity caused by a standard pharmaceutical dose of the said compound(s), typically in the absence of any biocompatible nanoparticle and/or carrier.

Provided that it is charged, the nanoparticle used in the context of the invention may be organic or inorganic. A mixture of organic and inorganic nanoparticles may also be used.

When it is organic, the nanoparticle may be a lipid-based nanoparticle (glycerolipid, phospholipid, sterol lipid, etc.), such as a solid lipid nanoparticle, a protein-based nanoparticle, also referred to herein as a protein nanoparticle (e.g., albumin), a polymer-based nanoparticle (polymer nanoparticle), a copolymer-based nanoparticle (copolymer nanoparticle), a carbon-based nanoparticle, a virus-like nanoparticle (e.g., a viral vector).

The organic nanoparticle may also be a nanosphere (solid nanoparticle) or a nanocapsule (hollow nanoparticle), such as a liposome, gel, hydrogel, micelle, dendrimer, etc. A mixture of the organic nanoparticles described herein may also be used.

The polymer or copolymer may be of natural or synthetic origin.

Examples of synthetic (artificial) and natural polymers or copolymers used in the context of the invention for producing organic nanoparticles can be selected from polylactic acid (PLA), poly(lactide-co-glycolic) acid (PLGA), polyethylene glycol (PEG), polyglactin, polylactide, polyoxyethylene fatty acid esters, polypropylene glycol, polysorbate, polyvinyl alcohol, polyacrylamide, polymethyl methacrylate, polyalkyl cyanoacrylate, polylactate co-glycolate, poly(amidoamine), poly(ethyleneimine), alginate, cellulose and cellulose derivatives of polymers, collagen, hyaluronic acid, polyglutamic acid (PGA), actin, polysaccharide and gelatin.

When it is inorganic and when its largest dimension is generally below about 10 nm, for example below about 8 nm, below about 7 nm, generally from about 7 to about 4 nm, for example below about 6 nm, below about 5 nm or below about 4 nm, the nanoparticle can be made of any inorganic material. The inorganic material can, for example, contain a metallic element from the 3rd, 4th, 5th, 6th periods of the periodic table, including lanthanides. When the largest dimension of the nanoparticle is generally below about 10 nm, the nanoparticles can assemble into larger structures. The assembly of the nanoparticles into a larger structure can usually be caused by the interaction between the nanoparticles and the biocompatible polymer(s), protein(s), etc. A larger structure can also be obtained by entrapping the nanoparticles in a carrier, typically a solid carrier such as a gelatin structure (also referred to herein as a gelatin nanoparticle) or a hollow carrier such as a liposome. These larger structures can also be designed by one skilled in the art to release the nanoparticles after administration in vivo.

When it is inorganic and when the largest dimension of said nanoparticle is generally at least 10 nm, generally from 10 to 500 nm, the nanoparticle may comprise at least one of or may consist of (i) one or more divalent metallic elements selected, for example, from Mg, Ca, Ba and Sr, (ii) one or more trivalent metallic elements selected, for example, from Fe and Al, and (iii) one or more tetravalent metallic elements, including Si.

In a particular embodiment, the inorganic material of the nanoparticle is selected from (i) one or more divalent metallic elements selected from, for example, Mg, Ca, Ba and Sr, (ii) one or more trivalent metallic elements selected from, for example, Fe and Al, and (iii) one or more tetravalent metallic elements, including Si.

In a further specific embodiment, the inorganic material of the nanoparticle is selected from calcium carbonate ( _CaCO3 ), magnesium carbonate ( _MgCO3 ), magnesium hydroxide (Mg^H):), iron hydroxide (Fe(OH)2), iron oxyhydroxide (FeOOH _{), iron oxide (Fe3O4 or Fe2O3), aluminum oxide (Al3O4), aluminum hydroxide (Al(OH)3 ) , aluminum oxyhydroxide} (AlOOH) and silicon oxide (SiO2).

The nanoparticles used in the compositions described herein must be biocompatible, i.e. compatible with living tissue. In cases where this is required by their composition, the nanoparticles must accordingly be coated with a biocompatible material in order to be suitable for use. In a particular embodiment of the invention, the nanoparticles described herein

- 4 047692 nanoparticle is thus coated with a biocompatible coating.

The biocompatible material may be an agent that allows interaction with a biological target. Such an agent typically brings a positive or negative charge to the surface of the nanoparticle when the absolute charge of the nanoparticle is at least 10 mV.

The agent forming a positive charge on the surface of the nanoparticles can be selected, for example, from aminopropyltriethoxysilane or polylysine. The agent forming a negative charge on the surface of the nanoparticles can be selected, for example, from phosphate (for example, polyphosphate, metaphosphate, pyrophosphate, etc.), carboxylate (for example, citrate or dicarboxylic acid, in particular succinic acid) and sulfate.

In a particular embodiment, since the absolute charge of the nanoparticle is at least 10 mV (|10 mV|), the nanoparticle can be coated with a biocompatible material containing an agent having a steric group, such an agent which is also referred to herein as a surface sterically stabilizing agent.

Such an agent having a steric group can be selected from, for example, polyethylene glycol (PEG); polyethylene oxide; polyvinyl alcohol; polyacrylate; polyacrylamide (poly(K-isopropylacrylamide)); polyurea; biopolymer; polysaccharide such as dextran, xylan and cellulose; collagen; zwitterionic compound such as polysulfobetaine; etc.

A biocompatible coating can be preferably a continuous coating (a complete monolayer). This implies the presence of a very high density of biocompatible molecules that create a corresponding charge on the entire surface of the nanoparticle.

The biocompatible coating may further comprise a labeling agent, typically an agent that allows the color to be visualized using standard imaging equipment.

The combined administration of at least one biocompatible nanoparticle together with at least one carrier containing at least one compound of interest maintains the pharmaceutical (i.e., therapeutic, prophylactic or diagnostic), generally therapeutic, efficacy for the compound(s) of interest with reduced toxicity or increases the pharmaceutical efficacy of the compound(s) of interest with equivalent or reduced toxicity to a subject, typically when administered to a subject in need of the compound(s) of interest at an interval of from more than 5 minutes to about 72 hours from each other, compared to the pharmaceutical efficacy and toxicity caused by standard pharmaceutical, usually therapeutic, dose(s) of said compound(s), typically in the absence of any biocompatible nanoparticle and/or carrier.

In a particular embodiment, the combined administration of at least one biocompatible nanoparticle and at least one carrier comprising at least one compound of interest allows for a decrease in the administered therapeutic dose of the compound(s) by at least 10%, preferably at least 15%, typically when administered to a subject in need of at least one compound of interest, at an interval of from more than 5 minutes to about 72 hours from each other, compared to a standard therapeutic dose(s) of said compound(s), typically in the absence of any biocompatible nanoparticle and/or carrier, while maintaining the same therapeutic efficacy with equivalent toxicity or reduced toxicity (preferably reduced toxicity) of the compound(s) to the subject; or while increasing the therapeutic efficacy with equivalent or reduced toxicity of the compound(s) to the subject.

In a particular embodiment, at least one nanoparticle is administered with multiple carriers, typically at least two carriers, each of said carriers containing at least one compound of interest. The compounds of interest present in the first carrier may be identical to or different from those present in the second or another separate carrier.

The nanoparticle is preferably eliminated from the subject to which it has been administered, typically within 1 hour to 6 weeks, such as 1 month (4 weeks), within 1 hour to 1 month, such as 1 hour to 3 weeks, or 1 hour to 2 weeks, or 1 hour to 1 week, after its administration to a subject in need of the compound of interest.

The material of which a nanoparticle is composed (including its biocompatible coating, if present) is important in determining the biopersistence (i.e., the ability to persist in the body of a subject) of the nanoparticle. A nanoparticle can be classified as biodegradable (when it is composed, for example, of a biodegradable polymer such as PLGA or PLA) and/or soluble (e.g., iron oxide), or non-biodegradable and insoluble. Biodegradable and soluble nanoparticles are cleared from a subject more quickly than non-biodegradable and/or insoluble nanoparticles.

Compound of interest.

Various molecules or agents can be used in accordance with the present invention as at least one compound of interest, typically as at least one pharmaceutical compound of interest. This compound can

- 5 047692 may be a therapeutic, prophylactic or diagnostic compound as described above. It may be an organic compound or an inorganic compound.

Examples of the compound used as the compound of interest are usually selected from a small molecule, a cytotoxic compound, and a transition metal coordination complex.

In the context of the present invention, a small molecule is a low molecular weight (<900 Da) organic compound with a size of about 10 ^-9 m. Most drugs are small molecules.

In a particular embodiment, the compound of interest used in the context of the present invention is a targeted small molecule. A targeted small molecule typically inhibits enzymatic domains on mutated, overexpressed or other critical proteins (potential targets in the context of cancer treatment) in malignant cells. Targeted small molecules include those molecules that target cell division (e.g., an aurora kinase inhibitor or a cyclin-dependent kinase inhibitor) or another biological mechanism such as protein turnover or chromatin modification (e.g., a histone deacetylase inhibitor). Examples of targeted small molecules include imatinib, rapamycin, gefitinib, erlotinib, sorafenib, sunitinib, nilotinib, dasatinib, lapatinib, bortezomib, atorvastatin, etc.

In another specific embodiment, the compound of interest used in the context of the present invention is a cytotoxic compound, such as a chemotherapeutic agent. The cytotoxic compound may, for example, be selected from a DNA modifying agent such as an anthracycline (e.g. doxorubicin, daunorubicin, etc.); an alkylating agent (e.g. melphalan or temozolomide); and a drug that precisely interferes with certain physiological mechanisms such as microtubule polymerization (e.g. taxol) or metabolite synthesis (e.g. methotrexate). In a specific embodiment, the cytotoxic compound is an activatable cytotoxic compound. Photofrin is an example of such an activatable cytotoxic compound, which is commonly used in the context of photodynamic therapy. Photofrin is activated by a laser source to obtain its therapeutic effect.

In another specific embodiment, the compound of interest used in the context of the present invention is a transition metal coordination complex. Transition metal coordination complexes have potential advantages over more common organic drug agents, including a wide range of coordination numbers and geometries, accessible redox states, tunability of thermodynamics and ligand substitution kinetics, and broad structural diversity. Metal-based substances interact with molecular targets of cells to affect biochemical functions, resulting in the destruction of malignant cells. Transition metal coordination complexes are typically cytotoxic agents (e.g., platinum coordination complexes: cisplatin, carboplatin, oxaloplatin, or ruthenium or gold coordination complexes) that target DNA structures.

Carrier.

At least one compound of interest is encapsulated or impregnated into a carrier or grafted (linked) to such a carrier according to methods known to a person skilled in the art. Schematic representations of carriers containing at least one compound of interest are shown in Fig. 1.

The carrier may be an organic carrier. The organic carrier is typically selected from a lipid carrier (e.g., glycerolipid, phospholipid, sterol, etc.); a polymer carrier; a copolymer carrier; a carbon-containing carrier; and a virus-like carrier (e.g., a viral vector).

The polymer or copolymer that makes up the carrier may be of natural or synthetic origin.

Examples of synthetic (artificial) and natural polymers or copolymers used in the context of the invention for producing a carrier can be selected from polylactic acid (PLA), poly(lactide-co-glycolic) acid (PLGA), poly(glutamic acid) (PGA), poly(caprolactone) (PCL), poly(amino acid), polyglactin, polylactide, polyoxyethylene fatty acid esters, polysorbate, polyvinyl alcohol, polyacrylamide, polymethyl methacrylate, polyalkyl cyanoacrylate, polylactate-co-glycolate, poly(amidoamine), poly(ethyleneimine), alginate, cellulose and cellulose derivatives of polymers, collagen, hyaluronic acid, actin, polysaccharide and gelatin.

The carrier may be an inorganic carrier. The inorganic carrier is typically a nanoparticle.

The nanoparticle is usually selected from a metal nanoparticle, a metal oxide nanoparticle, and a mixture thereof.

- 6 047692

The carrier may be a solid carrier such as a nanosphere (solid nanoparticle) or a hollow carrier such as a nanocapsule (hollow nanoparticle).

Preferred carriers are, for example, selected from a liposome, a micelle, a polymeric (or polymer) carrier, a hydrogel, a dendrimer, a gel, a copolymer carrier, a protein carrier, and an inorganic carrier, as defined herein.

The surface of the carrier of the present invention typically and preferably does not contain (or in other words is devoid of or does not exhibit) any surface sterically stabilizing agent, i.e. any hydrophilic and/or flexible polymer. For example, the carrier of the present invention does not contain or does not exhibit a polymer selected from dextran, polysialic acid (PSA), hyaluronic acid, chitosan, heparin, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyacrylamide, poly(ethylene glycol) (PEG) and a PEG-based copolymer such as poloxamer, poloxamine or polysorbate. Preferably, the carrier of the invention does not contain any hydrophilic polymer that imparts a weakly negative or positive surface charge to the surface of the carrier, such as poly(ethylene glycol) (PEG) or a PEG-based copolymer, polyvinyl alcohol (PVA) or polyvinylpyrrolidone (PVP).

The pharmaceutical composition of the present invention (cf. Fig. 2b) can be advantageously substituted for existing carriers (or drug delivery systems) containing or exhibiting a surface sterically stabilizing agent (Fig. 2a), typically such as a hydrophilic and flexible polymer, more particularly a hydrophilic polymer which imparts a weakly negative or positive surface charge to the surface of the carrier (e.g. a polyethylene glycol polymer), such negatively or positively charged surface being considered neutral by those skilled in the art.

The pharmaceutical composition of the present invention maintains the pharmaceutical (i.e. therapeutic, prophylactic or diagnostic) efficacy of the compound of interest with reduced toxicity in said subject or increases its pharmaceutical efficacy with equivalent or reduced toxicity compared to the pharmaceutical efficacy and toxicity caused by said compound when administered in a standard pharmaceutical dose, typically in the absence of any nanoparticle and/or carrier.

The pharmaceutical composition of the invention typically allows for a reduction of at least 10% in the administered pharmaceutical dose of the compound compared to a standard pharmaceutical dose of said compound, typically in the absence of any nanoparticle and/or carrier, while maintaining the same pharmaceutical efficacy with equivalent toxicity, preferably with reduced toxicity to the subject, or with an increase in pharmaceutical efficacy with equivalent or reduced toxicity to the subject.

The carrier allows the compound of interest to be released, preferably in a controlled manner. The carrier can generally be designed to release the compound(s) of interest at a predetermined or controlled rate or in response to an external stimulus.

In a particular embodiment, the carrier allows for the release of the compound(s) of interest, typically by time-controlled release, by diffusion of the compound of interest from the carrier, by erosion and/or degradation of the carrier.

In another specific embodiment, the carrier allows the release of the compound(s) of interest by intracellular or extracellular activation, i.e. in response to an intracellular or extracellular stimulus, such as a change in pH or the action of an enzyme.

In another specific embodiment, the carrier allows the compound(s) of interest to be released in response to an external stimulus. Examples of an external stimulus are electromagnetic radiation (e.g., ionizing radiation such as X-rays, gamma radiation, or non-ionizing radiation such as UV, visible light or infrared radiation), ultrasound and a magnetic field. The pharmaceutical compound is, for example, released from the carrier when said carrier is exposed to an external stimulus selected from electromagnetic radiation, ultrasound and a magnetic field.

The carrier, which does not contain any surface sterically stabilizing agent, may be, for example, a liposome with a membrane phase transition temperature between 37 and 45°C, containing dipalmitoylphosphatidylcholine (DPPC) 62 mol.%, hydrogenated soy phosphatidylcholine (HSPC) 22 mol.% and cholesterol (Chol) 16 mol.% or dipalmitoylphosphatidylcholine (DPPC) 90 mol.% and monopalmitoylphosphatidylcholine (MPPC) 10 mol.%.

The carrier, which does not contain any surface sterically stabilizing agent, may also be, for example, a liposome containing a synthetic phospholipid, such as a 1,3-diamidophospholipid, which is sensitive to shear stress.

A carrier that does not contain any surface sterically stabilizing agent may also be, for example, a liposome containing a peptide that changes its conformation (alpha-helix to beta-pleated sheet) when exposed to pH or temperature.

- 7 047692

A carrier which does not contain any surface sterically stabilizing agent may also be, for example, an amphoteric liposome containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a molar ratio of 3:1 and an equal amount of weak cationic and weak anionic amphiphiles, both derived from cholesterol, a-(3'-O-cholesteroloxycarbonyl)-N-(N-ethylmorpholine)succinamide (MoChol) and cholesteryl hemisuccinate (CHEMS).

The pharmaceutical composition of the invention (defined by a combination of at least one biocompatible nanoparticle and at least one carrier containing at least one compound of interest) can be used in many fields, in particular in human or veterinary medicine. This composition is usually used in an animal, preferably in a mammal, even more preferably in a human, regardless of its age or sex.

The pharmaceutical composition of the invention can be used for the prevention or treatment of a disease or disorder selected from a cardiovascular disease, a central nervous system (CNS) disease, a gastrointestinal disease, a genetic disorder, a hematological disorder, a hormonal disorder, an immune disorder, an infectious disease, a metabolic disorder, a musculoskeletal disorder, cancer, a respiratory disease and intoxication, etc. In a preferred embodiment, the pharmaceutical composition is intended for the prevention or treatment of a disease or disorder selected from a cardiovascular disease, a CNS disease, cancer, an infectious disease and a metabolic disorder.

In the context of the present invention, at least one nanoparticle and at least one carrier containing the compound(s) of interest are suitably administered to a subject in need of said compound(s) of interest at an interval of from more than 5 minutes to about 72 hours from each other, typically from more than 5 minutes to about 24 hours, preferably from more than 5 or 30 minutes to about 12 hours, to optimize the pharmaceutical efficacy of the compound(s).

In the present invention, when at least one nanoparticle and at least one carrier containing the compound(s) of interest are suitably administered to a subject in need of said compound at an interval of more than 5 minutes to about 72 hours from each other, the absolute value of the surface charge of the at least one biocompatible nanoparticle is at least 10 mV (|10 mV|).

In a particular embodiment of the present invention, when at least one nanoparticle and at least one carrier containing the compound(s) of interest are suitably administered to a subject in need of said compound at an interval of from more than 5 minutes to about 24 hours from each other, the absolute value of the surface charge of the at least one biocompatible nanoparticle is advantageously at least 15 mV (|15 mV|).

In another specific embodiment of the present invention, when at least one nanoparticle and at least one carrier containing a compound of interest are suitably administered to a subject in need of said compound at an interval of from more than 5 minutes to about 12 hours from each other, the absolute value of the surface charge of the at least one biocompatible nanoparticle is advantageously at least 20 mV (|20 mV|).

Also described herein is a method of preventing or treating a subject suspected of being predisposed to, or suffering from, a disease such as those described herein, said method comprising administering to said subject a pharmaceutical composition of the invention, typically at least one biocompatible nanoparticle and at least one carrier comprising at least one compound of interest as described herein. Any at least one nanoparticle or at least one carrier comprising the compound(s) of interest may be administered to the subject first, as long as the at least one biocompatible nanoparticle and the at least one carrier comprising the compound(s) are administered separately, at an interval of from more than 5 minutes to about 72 hours. The administration of said at least one nanoparticle or at least one carrier comprising the compound(s) of interest may be a single administration of each, multiple administrations of each, such as several sequential administrations of each. The biocompatible nanoparticle may be administered once, and at least one carrier containing the compound(s) of interest may be administered more than once, and vice versa.

In a particular embodiment, at least one biocompatible nanoparticle is at least administered at the beginning of a protocol comprising multiple administrations of at least one carrier containing the compound(s) of interest, i.e. at least at the first administration of said at least one carrier, and before or after its administration.

In another specific embodiment, the biocompatible nanoparticle is not administered at the beginning

- 8 047692 protocol comprising multiple administrations of at least one carrier containing the compound(s) of interest, and not administered until the second or third administration of said at least one carrier, and before or after its administration.

In the context of these last two embodiments, the at least one biocompatible nanoparticle may also be administered together (before or after, as described previously) with at least one carrier containing the compound(s) of interest, during part or all of the subsequent administrations of said at least one carrier.

The biocompatible nanoparticle(s) of the pharmaceutical composition according to the invention can be administered by any route, such as intravenous (IV), intraarterial, intraperitoneal, intradermal, upper respiratory (inhalation), intramuscular and/or oral (per os). The preferred route of administration is intravenous.

The carrier(s) containing the compound(s) of interest of the pharmaceutical composition of the invention can be administered by any route selected from the subcutaneous route, the intravenous (IV) route, the intradermal route, the intraarterial route, via the upper respiratory tract (inhalation), the intraperitoneal route, the intramuscular route, the oral route (per os) and several different routes among those previously mentioned.

The appropriate route(s) will be chosen by the practitioner depending on the disease or disorder to be detected, prevented or treated.

The following examples illustrate the invention without limiting its scope.

Brief description of the drawings

Fig. 1. Schematic representation of carriers, free of any sterically stabilizing agent, containing at least one compound of interest. The carrier may be a solid carrier (a, b) or a hollow carrier (c, d). The compound of interest is typically captured or impregnated (a, c) or grafted (linked) to the carrier with or without a linker (b, d).

Fig. 2. a) Schematic representation of a carrier containing at least one compound of interest. The surface of the carrier is modified with a sterically stabilizing agent.

b) a schematic representation of a pharmaceutical composition according to the invention comprising a combination of (i) at least one biocompatible nanoparticle and (ii) at least one carrier comprising at least one compound of interest, wherein the carrier does not comprise any sterically stabilizing agent.

Fig. 3 Chemical formula of 1,5-dihexadecyl ester of N-(3-carboxy-1-oxopropyl)L-glutamic acid (SA-lipid)

Examples

Example 1. Synthesis No. 1 of liposomes as biocompatible nanoparticles.

Liposomes are obtained using the lipid film rehydration method.

a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a stream of nitrogen. Rehydration of the lipid film using HEPES 20 mM and NaCl 140 mM at pH 7.4 is performed at 50°C so that the lipid concentration is 5 mM.

The following lipid composition was used to obtain charged liposomes: DPPC (dipalmitoylphosphatidylcholine): 86 mol.%; MPPC (monopalmitoylphosphatidylcholine): 10 mol.%; DSPE-PEG (distearylphosphatidylethanolamine[methoxy(polyethyleneglycol)-2000]): 4 mol.%.

b) Freeze-thaw cycles are then performed 6 times by successively immersing the sample in liquid nitrogen and in a water bath controlled at 50°C.

c) A thermal cylinder extruder (LIPEX™ extruder, Northern Lipids) was used to calibrate the liposome size under controlled temperature and pressure. In all cases, extrusion was performed at 50°C under a pressure of 10 bar.

The size distribution of freshly prepared liposomes was determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern instrument) with a 633 nm HeNe laser at 90°. The liposome suspension was diluted 100-fold in HEPES 20 mM and NaCl 140 mM at pH 7.4. The liposome size (i.e., hydrodynamic diameter) was approximately 170 nm (intensity distribution) with a polydispersity index (PDI) of approximately 0.1.

As is understood by one skilled in the art, the desired surface charge was obtained by the selected lipid composition, and its value was confirmed by measuring the zeta potential using a Zetasizer Nano ZS instrument (Malvern instrument).

The liposomes were diluted 100-fold in water and the pH of the resulting suspension was adjusted to pH 7.4. The surface charge of the liposomes was approximately -14 mV at pH 7.4.

Example 2. Synthesis No. 2 of liposomes as biocompatible nanoparticles.

Liposomes are obtained using the lipid film rehydration method.

a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a stream of nitrogen.

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Rehydration of the lipid film using HEPES 20 mM and NaCl 140 mM at pH 7.4 was performed at 65°C so that the lipid concentration was 25 mM.

The following lipid composition was used to obtain liposomes: DSPC (distearoylphosphatidylcholine):DSPG (distearoylphosphatidylglycerol):Chol (cholesterol) in a molar ratio of 7:2:1.

b) Freeze-thaw cycles are then performed 6 times by successively immersing the sample in liquid nitrogen and in a water bath controlled at 65°C.

c) A thermal cylinder extruder (LIPEX™ extruder, Northern Lipids) was used to calibrate the liposome size under controlled temperature and pressure. First, 5 passes were performed through a 0.45 μm polyethersulfone (PES) membrane at 5 bar, then 10 passes through a 0.22 μm PES membrane at 10 bar, and finally 10 passes through a 0.1 μm polyvinylidene fluoride (PVDF) membrane at 15 bar.

The size distribution of freshly prepared liposomes was determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern instrument) with a 633 nm HeNe laser at 90°. The liposome suspension was diluted 100-fold in HEPES 20 mM and NaCl 140 mM at pH 7.4. The liposome size (i.e., hydrodynamic diameter) was approximately 145 nm (intensity distribution) with a polydispersity index (PDI) of approximately 0.1.

The desired surface charge, which is typically below 10 mV, was obtained by the chosen lipid composition and its value was confirmed by measuring the zeta potential using a Zetasizer Nano ZS instrument (Malvern instrument).

Example 3. A method for increasing the efficacy and/or decreasing the toxicity after administration to a subject of a compound of interest included in a pharmaceutical composition according to the invention, compared to the same dose of the compound of interest in monotherapy.

The pharmaceutical composition according to claim 1, containing a combination of (i) at least one biocompatible nanoparticle and (ii) at least one carrier containing doxorubicin, is administered to nude mice bearing a xenografted MDA-MB-231-lucD3H2LN tumor, as follows:

a) administration of Dox-NP® (pegylated liposomal formulation of doxorubicin) to the first group of nude mice (by intravenous injection);

administration of doxorubicin to the second group of nude mice (by intravenous injection);

administration of biocompatible nanoparticles to a third group of nude mice (by intravenous injection);

administering to a fourth group of nude mice (by intravenous injection) biocompatible nanoparticles and, at an interval of from more than 5 minutes to about 72 hours after the administration of the biocompatible nanoparticles to the fourth group of nude mice, administering (by intravenous injection) to said fourth group of nude mice a vehicle containing doxorubicin, wherein the vehicle does not contain any sterically stabilizing agent;

b) evaluation of any clinical sign of toxicity in nude mice following administration of Dox-NP® (first group), doxorubicin (second group), biocompatible nanoparticles (third group) and pharmaceutical composition (fourth group);

c) measurement of tumor regrowth delay after administration of Dox-NP® (first group), doxorubicin (second group), biocompatible nanoparticles (third group) and pharmaceutical composition (fourth group).

Example 4. Synthesis No. 3 of liposomes as biocompatible nanoparticles.

Liposomes are obtained using the lipid film rehydration method.

a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a stream of nitrogen to form a lipid film on the walls of a Pyrex tube. Rehydration of the lipid film using HEPES 25 mM and NaCl 150 mM at pH 7.4 is performed at 60°C so that the lipid concentration is 50 mM.

The following lipid composition was used to obtain charged liposomes: DPPC (dipalmitoylphosphatidylcholine): 58 mol.%; HSPC (hydrogenated soy phosphatidylcholine): 21 mol.%; Chol (cholesterol): 16 mol.%; POPS (1-palmitoyl-2-oleoylphosphatidylserine): 5 mol.%.

b) Freeze-thaw cycles are then performed 6 times by successively immersing the sample in liquid nitrogen and in a water bath controlled at 60°C. Ultrasonication of the liposome solution is performed for 30 s every 3 freeze-thaw cycles and immediately prior to extrusion.

c) A thermal cylinder extruder (LIPEX™ extruder, Northern Lipids) is used to calibrate the liposome size under controlled temperature and pressure. Extrusion is carried out at 60°C. Ten passes through a 0.1 µm polyvinylidene fluoride (PVDF) membrane at a pressure of 10 bar are used.

The size distribution of freshly prepared liposomes was determined using dynamic

- 10 047692 direct light scattering (DLS) using a Zetasizer Nano ZS (Malvern instrument) with a 633 nm HeNe laser at 173°. The liposome solution was diluted 200-fold in HEPES 25 mM and NaCl 150 mM at pH 7.4. The liposome size (i.e., hydrodynamic diameter) was approximately 170 nm (intensity distribution) with a polydispersity index (PdI) of approximately 0.2.

As will be appreciated by those skilled in the art, the desired surface charge is obtained by the selected lipid composition and its value is confirmed by measuring the zeta potential using a Zetasizer Nano ZS (Malvern instrument). The liposome solution is diluted 200-fold in a 1 mM sodium chloride solution and the pH of the solution is adjusted to pH 7. The surface charge of the liposomes is approximately -40 mV at pH 7, NaCl 1 mM.

The final lipid concentration in the liposome solution is measured by colorimetric analysis (Bartlett's method). The method is based on the determination of the total amount of phosphorus by acid digestion of the phospholipid. The released inorganic phosphate reacts with ammonium molybdate, the complex producing a dark blue color. The lipid concentration is approximately 50 mM.

Example 5. Synthesis No. 4 of liposomes as biocompatible nanoparticles.

Liposomes are obtained using the lipid film rehydration method.

a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a stream of nitrogen to form a lipid film on the walls of a Pyrex tube. Rehydration of the lipid film using HEPES 25 mM and NaCl 150 mM at pH 7.4 is performed at 60°C so that the lipid concentration is 50 mM.

The following lipid composition was used to obtain charged liposomes: DPPC (dipalmitoylphosphatidylcholine): 45.15 mol%; Chol (cholesterol): 45.15 mol%; DSPE-PEG (distearylphosphatidylethanolamine[methoxy(polyethyleneglycol)-2000]): 0.60 mol%; 1,5-dihexadecyl ester of N-(3-carboxy-1-oxopropyl)-L-glutamic acid (SA-lipid): 9.10 mol%. SA-lipid brings COOH groups to the surface of liposomes.

b) Freeze-thaw cycles are then performed 6 times by successively immersing the sample in liquid nitrogen and in a water bath controlled at 60°C.

c) A thermal cylinder extruder (LIPEX™ extruder, Northern Lipids) is used to calibrate the liposome size under controlled temperature and pressure. Extrusion is carried out at 60°C. Seven passes through a 0.45 μm PVDF membrane at 3 bar and ten passes through a 0.22 μm PVDF membrane at 10 bar are used. The size distribution of freshly prepared liposomes is determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern instrument) with a 633 nm HeNe laser at 173°. The liposome solution is diluted 200-fold in HEPES 25 mM and NaCl 150 mM at pH 7.4. The liposome size (i.e. hydrodynamic diameter) is approximately 230 nm (intensity distribution) with a polydispersity index (PdI) of approximately 0.2.

As will be appreciated by those skilled in the art, the desired surface charge is obtained by the selected lipid composition and its value is confirmed by measuring the zeta potential using a Zetasizer Nano ZS (Malvern instrument). The liposome solution is diluted 200-fold in a 1 mM sodium chloride solution and the pH of the solution is adjusted to pH 7. The surface charge of the liposomes is approximately -60 mV at pH 7, NaCl 1 mM.

The final lipid concentration in the liposome solution is measured by colorimetric analysis (Bartlett's method). The method is based on the determination of the total amount of phosphorus by acid digestion of the phospholipid. The released inorganic phosphate reacts with ammonium molybdate, the complex producing a dark blue color. The lipid concentration is approximately 50 mM.

Example 6. Synthesis No. 5 of liposomes as biocompatible nanoparticles

Liposomes are obtained using the lipid film rehydration method.

a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under nitrogen flow to form a lipid film on the walls of a Pyrex tube. Rehydration of the lipid film using HEPES 25 mM and NaCl 150 mM at pH 7.4 is performed at 60°C, and the lipid concentration is 50 mM. The following lipid composition was used to obtain charged liposomes: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine): 60 mol%, Chol (cholesterol): 35 mol% and succinyl PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-succinyl): 5 mol%.

b) Freeze-thaw cycles are then performed 6 times by successively immersing the sample in liquid nitrogen and in a water bath controlled at 60°C. Ultrasonication of the liposome solution is performed for 30 s every 3 freeze-thaw cycles and before extrusion.

c) A thermal cylinder extruder (LIPEX™ extruder, Northern Lipids) is used to calibrate the liposome size under controlled temperature and pressure. Extrusion is carried out at 60°C. Twelve passes through a 0.22 µm PVDF membrane are used under

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Claims (6)

pressure of 12 bar. d) Conjugation of p-aminophenyl-a-O-mannopyranoside (MAN) to succinyl PE liposome: The surface of succinyl PE liposome is modified with a mannose derivative, a ligand of p-aminophenyl-a-O-mannopyranoside (MAN), using a carbodiimide coupling to form a mannose-conjugated liposome. MAN is covalently linked by its amino group to the carboxyl group of succinyl PE present on the surface of the preformed succinyl PE liposome. Briefly, EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (succinyl PE/EDC molar ratio of 1:10) and N-hydroxysuccinimide (NHS) (NHS/EDC molar ratio of 1:2.5) were added to the pre-formed succinyl PE liposome solution. Then, the pH of the suspension was adjusted to 6 with 1 M NaOH, and the resulting suspension was stirred for 15 min at room temperature. Then, the pH of the solution was adjusted to 7 with 1 M NaOH, and an aqueous solution of MAN (succinyl PE/MAN molar ratio of 1:2) was added to the solution. The pH was adjusted to 7 with 1 M NaOH, and the suspension was stirred for additional 2 hours at room temperature. Excess unbound MAN, EDC and NHS molecules are removed by three dialysis steps with a dilution factor (x500, x500, x500) using a 50 kDa cellulose membrane. It should be noted that due to possible dilution during dialysis, the liposome solution can be concentrated by centrifugation (typically a Sigma 3-15K centrifuge at 5°C, 1200 rpm) using membrane ultrafiltration on Vivaspin concentrators with a polyethylenesulfone (PES) membrane and a cut-off of 300 kDa. The size distribution of freshly prepared liposomes is determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern instrument) with a HeNe laser of 633 nm at an angle of 173°. The liposome solution is diluted 200-fold in HEPES 25 mM and NaCl 150 mM at pH 7.4. The size of the liposomes (i.e., the hydrodynamic diameter) is approximately 230 nm (intensity distribution) with a polydispersity index (PDI) of approximately 0.2. As will be appreciated by one skilled in the art, the desired surface charge is obtained due to the selected lipid composition, and its value is confirmed by measuring the zeta potential using the Zetasizer Nano ZS instrument (Malvern instrument). The liposome solution is diluted 200-fold in a 1 mM sodium chloride solution at pH 7. The surface charge of the liposomes is approximately -70 mV at 1 mM NaCl, pH 7. The final lipid concentration in the liposome solution is measured by colorimetric analysis (Bartlett's method). The method is based on the determination of the total amount of phosphorus by acid digestion of the phospholipid. The released inorganic phosphate reacts with ammonium molybdate, the complex yielding a dark blue color. The lipid concentration is approximately 50 mM. CLAUSE OF INVENTION 1. A method for treating cancer, comprising the step of administering at least one carrier containing at least one pharmaceutical compound to a subject in need of said at least one pharmaceutical compound, and a separate step of administering at least one biocompatible nanoparticle to said subject, wherein the pharmaceutical compound is a chemotherapeutic agent, wherein the at least one carrier is a lipid-based nanoparticle and the surface of the carrier does not comprise a polymer selected from dextran, polysialic acid (PSA), hyaluronic acid, chitosan, heparin, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyacrylamide, poly(ethylene glycol) (PEG) and a PEG-based copolymer; wherein the largest dimension of the at least one biocompatible nanoparticle is from about 4 to about 500 nm, as measured by dynamic light scattering (DLS), the surface charge value of the at least one biocompatible nanoparticle is a negative surface charge value below -10 mV, and the at least one biocompatible nanoparticle is not used as such as a pharmaceutical compound, and said at least one biocompatible nanoparticle, which is a lipid-based nanoparticle, is administered to a subject at an interval of more than 5 minutes to about 24 hours to at least one carrier containing at least one pharmaceutical compound. 2. The method according to claim 1, wherein the nanoparticle is additionally coated with a biocompatible coating. 3. The method according to claim 1, wherein the carrier is a continuous carrier. 4. The method according to claim 1, wherein the carrier is a hollow carrier. 5. The method according to claim 1, wherein the carrier is a liposome and the surface of the carrier does not contain any polyethylene glycol (PEG) polymer. 6. The method according to claim 1 or 5, wherein the carrier is a liposome and the liposome comprises 62 mol.% dipalmitoylphosphatidylcholine (DPPC), 22 mol.% hydrogenated soy phosphatidylcholine (HSPC) and 16 mol.% cholesterol (Chol); 90 mol.% dipalmitoylphosphatidylcholine (DPPC) and 10 mol.% monopalmitoylphosphatidylcholine (MPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a molar ratio of 3:1 and equal to -