WO2017046037A1

WO2017046037A1 - Nanoparticles loaded with active ingredients, their process of preparation and their uses

Info

Publication number: WO2017046037A1
Application number: PCT/EP2016/071468
Authority: WO
Inventors: Christelle Zandanel
Original assignee: Onxeo
Priority date: 2015-09-14
Filing date: 2016-09-12
Publication date: 2017-03-23
Also published as: AR106004A1; TW201720447A

Abstract

The present invention concerns nanoparticles of one or more active ingredient, such as doxorubicin with controlled average diameter, their process of preparation, their formulations and their therapeutic uses.

Description

NANOPARTICLES LOADED WITH ACTIVE INGREDIENTS, THEIR PROCESS OF

PREPARATION AND THEIR USES

FIELD OF THE INVENTION

The present invention concerns the field of pharmaceuticals, in particular the field of pharmaceutical nanoparticles and more specifically nanoparticles of doxorubicin.

BACKGROUND OF THE INVENTION

W099/43359 describes nanoparticles loaded with active ingredients such as chemotherapeutic agents. The nanoparticles are prepared by mixing (i) an active ingredient with (ii) a complexing agent such as a cyclodextrin, and (iii) a monomer such as an alkyl cyanoacrylate monomer. While W099/43359 notes that a surfactant or stabilizing agent, such as dextran or a poloxamer, can be used in the preparation of the nanoparticles, W099/43359 also notes that such agents are not necessary because said cyclodextrins have a sufficient stabilizing effect on the nanoparticles for the surfactant agent to be omitted. The polymerization of said monomer is then conducted to obtain nanoparticles loaded with the active ingredient. According to W099/43359, the complexing agent complexes the active ingredient during preparation of the nanoparticle so as to protect it against chemical reactions occurring during the formation of the nanoparticle. Therefore, the active ingredient is advantageously associated in a non- covalent manner with the nanoparticle and protected from reactions or degradation. WO2012/131018 discloses the use of said nanoparticles loaded with doxorubicin for treating cancer, by intravenous or intra-arterial infusion, for at least 2 hours.

These nanoparticles of doxorubicin are currently developed under the trademark name Livatag® for the treatment of Hepatocellular Carcinoma (HCC) in particular.

HCC is known as hypervascular solid liver cancer characterized by a high degree of drug resistance. The mechanisms of this chemoresistance in HCC are multiple. The most common mechanism is related to the multidrug resistance (MDR) transporters, P-gp and MRP pumps. These transporters or pumps allow tumor cells to efflux different types of chemotherapeutic agents into the extracellular environment However, said nanoparticles display original mechanisms to bypass multi-drug resistance that can be summarized as follow: The nanoparticles loaded with doxorubicin adsorb to the surface of tumor cells and release the entrapped doxorubicin close to the cell membrane which leads to a high local gradient concentration. The nanoparticles then degrade and release soluble polycyanoacrylic acid which might interact with the plasma membrane and contribute to improve the intracellular delivery of doxorubicin. The soluble polycyanoacrylic acid could also mask the positive charge of doxorubicin thus preventing its efflux by the Pgp or MRP pumps.

The nanoparticles are thus believed to fight against chemotherapy resistance, a major mechanism responsible of the failure of some anticancer drugs. Hence, doxorubicin can more efficiently exert its cytotoxic effect when encapsulated in said nanoparticles.

Moreover, encapsulated in the form of nanoparticles, doxorubicin can specifically reach the liver, its therapeutic target.

A Phase I I clinical trial has been completed with Livatag® in patients suffering from HCC. Further this Phase I I trial, an international open randomised Phase I I I clinical trial is underway, aiming to recruit 390 patients with advanced stage HCC, and test Livatag® after failure or intolerance to sorafenib.

The tensioactive poloxamer is a synthetic block copolymer of ethylene oxide and propylene oxide of formula:

With a and b representing, respectively, the number of ethyleneoxide (EO) and propyleneoxide (PO) units, and where a is generally comprised between 2 and 130 and b is generally comprised between 15 and 70. In the State of the Art, ethyleneoxide (EO) can also be named oxyethylene and propyleneoxide (PO) can also be named oxypropylene.

Poloxamers are available in several types, depending on the content of EO and PO (i.e., and thus the a and l values), with various molecular weights and weight percentages of ethyleneoxide / propyleneoxide (EO / PO). One particular poloxamer is poloxamer 188. According to the US Pharmacopea, poloxamer 188 is solid, has an average molecular weight between 7680 and 9510 Da and an average EO weight percentage of 81 .8 ± 1 .9, i.e. an EO weight percentage comprised between 79.9 and 83.7%.

Poloxamer 188 is commercially available from BASF under the trade names Kolliphor® P188, Pluronic® F68, and, in the past, Lutrol® F68. According to the BASF manufacturer's data sheets, these products have an EO weight percentage that varies between 79.9 and 83.8% and is typically 81 .9%.

According to the BASF manufacturer's data sheet, Kolliphor^® P188 has an EO weight percentage comprised between 79.9 and 83.8%. In the course of further industrial scale up production of nanoparticle chemotherapeutic drug candidates, it is essential to achieve reproducible nanoparticles with particular specifications, for example, to comply with specific manufacturing and therapeutic requirements and especially regulatory requirements. Among the several parameters to ensure regulatory conformity of said nanoparticles, nanoparticle size is one of the most important.

Indeed, it is generally known that the size of chemotherapeutic agent-loaded nanoparticles is to be such that the nanoparticles are not too small (to avoid elimination by the kidneys, which would result in loss of therapeutic effect) and not too large (to avoid blocking or obstructing blood vessels, which would lead to vessel embolization and thus toxicity).

As a result, it has been determined that nanoparticles of doxorubicin must generally have an average diameter between 100 and 300 nm (Hillaireau et al., Cellular and Molecular Life Sciences, 2009, 66:2873-2896). It is essential that this size can be controlled and reproducibly achieved through the various industrial, large scale, batch manufactures.

However, processes for making chemotherapeutic agent-loaded nanoparticles have faced reproducibility drawbacks as the size has not been fully controlled. Indeed, some nanoparticles productions had fallen outside the desired 100-300 nm average diameter, for unknown reasons.

W099/43359 and W02012/131018 teach that the size of the nanoparticles is essentially related to the concentration of cyclodextrin or the type of cyclodextrin. However, contrary to this teachings of W099/43359 and W02012/131018, it has now been surprisingly shown that poloxamer 188 is responsible for the variation of the size of nanoparticles loaded with doxorubicin and that known sources of poloxamer 188, which have varying weight percentage of EO, frequently do not lead to nanoparticles of doxorubicin with the desired 100-300 nm average diameter.

To solve this problem, the inventors have surprisingly shown that the EO weight percentage of poloxamer 188 must be within a particular range to achieve the desired 100-300 nm average diameter of nanoparticles.

Further, and contrary to the general expectation, it has also been shown that an increase of the poloxamer 188 molecular weight (Mw) does not necessarily lead to an increase of the size of the nanoparticles. SUMMARY OF THE INVENTION

According to a first object, the present invention thus concerns nanoparticles comprising (i) at least one active ingredient or any pharmaceutically acceptable salt thereof, (ii) a polymer of poly(alkylcyanoacrylate), (iii) one or more cyclodextrin(s), and (iv) poloxamer 188, wherein said poloxamer 188 has an ethyleneoxide (EO) weight percentage comprised between 80 and 81 .5%.

The EO weight percentage as used therein refers to the content of ethyleneoxide in weight with respect to the total weight of poloxamer 188, in a given sample.

The EO weight percentage may be obtained by standard procedures such as NMR, such as those disclosed in the US pharmacopea and reported in the following examples.

According to a specific embodiment, the EO weight percentage is comprised between 80 and 81 %. According to another embodiment, the EO weight percentage is comprised between 80 and 80.8%. According to a further embodiment, the EO weight percentage is comprised between 80.5 and 80.8%. According to an embodiment, the therapeutically active ingredient is a chemotherapeutic agent, such as an anthracycline, such as doxorubicin. According to an embodiment, the nanoparticles in the dispersion of the invention have an average diameter comprised between 100 and 300 nm.

DETAILED DESCRIPTION OF THE INVENTION

The average diameter of nanoparticles as used herein refers to the average value of the diameter of nanoparticles in a given sample.

The average diameter can be obtained by application of known procedures, such as Dynamic Light Scattering and reported in the following examples.

As examples of active ingredients which may enter into the composition of the nanoparticles of the invention, the following can be cited: anticancers, antivirals, antibiotics, proteins, polypeptides, polynucleotides, antisense nucleotides, vaccinating substances, immunomodulators, steroids, analgesics, antimorphinics, or antifungals. Among the anticancer substances, the invention gives particular consideration to chemotherapeutic agents. A more particular preferred chemotherapeutic agents to carry out the present invention is doxorubicin. Chemotherapeutic agents can be defined as cytotoxic drugs to treat cancer. Broadly, most chemotherapeutic agents work by impairing mitosis (cell division) or DNA synthesis, effectively targeting fast-dividing cells. As these drugs cause damage to cells they are termed "cytotoxic". According to the present invention, chemotherapeutic agents can refer to (i) anthracyclines, (ii) topoisomerase inhibitors, (iii) spindle poison plant alkaloids, (iv), alkylating agents, (v) anti-metabolites, and (vi) other chemotherapeutic agents:

(i) Anthracyclines

Anthracyclines (or anthracycline antibiotics) are derived from Streptomyces bacteria. These compounds are used to treat a wide range of cancers, including leukemias, lymphomas, and breast, uterine, ovarian, and lung cancers. Anthracyclines have three mechanisms of action:

Inhibition of DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand, thus preventing the replication of rapidly-growing cancer cells.

Inhibition topoiosomerase II enzyme, preventing the relaxing of supercoiled DNA and thus blocking DNA transcription and replication.

Creation of iron-mediated free oxygen radicals that damage the DNA and cell membranes.

Some non limitating examples of anthracyclins are: doxorubicin daunorubicin, epirubicin, idarubicin, valrubicin or their pharmaceutically acceptable salts.

Topoisomerase inhibitors

Topoisomerases are essential enzymes that maintain the topology of DNA. Inhibition of type I or type II topoisomerases interferes with both transcription and replication of DNA by upsetting proper DNA supercoiling.

Some type I topoisomerase inhibitors include camptothecins derivatives Camptothecin derivatives refer to camptothecin analogs such as irinotecan, topotecan, hexatecan, silatecan, lutortecan, karenitecin (BNP1350), gimatecan (ST1481 ), belotecan (CKD602), ... or their pharmaceutically acceptable salts. Irinotecan, its active metabolite SN38 and topotecan are preferred. Irinotecan is more preferred.

Examples of type II topoisomerase inhibitors include amsacrine, etoposide, etoposide phosphate, teniposide, These are semisynthetic derivatives of epipodophyllotoxins, alkaloids naturally occurring in the root of American Mayapple (Podophyllum peltatum).

spindle poison plant alkaloids

These alkaloids are derived from plants and block cell division by preventing microtubule function, essential for cell division.

Examples are vinca alkaloids (like vinblastine, vincristine, vindesine vinorelbine vinpocetine...) and taxanes. Taxanes include paclitaxel and docetaxel or their pharmaceutically acceptable salts. Paclitaxel was originally derived from the Pacific yew tree. Docetaxel is a semi-synthetic analogue of paclitaxel.

In contrast to the taxanes, the vinca alkaloids destroy mitotic spindles. Both, taxanes and vinca alkaloids are therefore named spindle poisons or mitosis poisons, but they act in different ways.

alkylating agents Alkylating agents are so named because of their ability to add alkyl groups to many electronegative groups under conditions present in cells. They impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules. Noteworthy, their cytotoxicity is thought to result from inhibition of DNA synthesis.

Platinum compounds like oxaliplatin, cisplatin, carboplatin are alkylating agents. Other alkylating agents are mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide.

(v) Anti-metabolites

An anti-metabolite is a chemical that inhibits the use of a metabolite, which is part of normal metabolism. Such substances are often similar in structure to the metabolite that they interfere with. The presence of anti-metabolites alters cell growth and cell division,

Purine or pyrimidine analogues prevent the incorporation of nucleotides into DNA, stopping DNA synthesis and thus cell divisions. They also affect RNA synthesis. Examples of purine analogues include azathioprine, mercaptopurine, thioguanine, fludarabine, pentostatin and cladribine. Examples of pyrimidine analogues include 5-fluorouracil (5FU), which inhibits thymidylate synthase, floxuridine (FUDR) and cytosine arabinoside (Cytarabine) .

Antifolates are drugs which impair the function of folic acids. Many are used in cancer chemotherapy, some are used as antibiotics or antiprotozoal agents. A well-known example is Methotrexate. This is a folic acid analogue, and owing to structural similarity with it binds and inhibits the enzyme dihydrofolate reductase (DHFR), and thus prevents the formation of tetrahydrofolate. Tetrahydrofolate is essential for purine and pyrimidine synthesis, and this leads to inhibited production of DNA, RNA and proteins (as tetrahydrofolate is also involved in the synthesis of amino acids serine and methionine). Other antifolates include trimethoprim, raltitrexed, pyrimethamine and pemetrexed.

(vi) Other chemotherapeutic agents

Examples above are not limitating and other chemotherapeutic agents can be described.

Among others, ellipticine and harmine can be cited.

The poly(alkylcyanoacrylate) polymer may be linear or branched, preferably branched. The alkyl group of the poly(alkylcyanoacrylate) may be linear or branched, preferably branched. In a particular embodiment, the poly(alkylcyanoacrylate) polymer is a poly(C1 -C12) alkylcyanoacrylate, preferably a poly(C4-C10) alkylcyanoacrylate, more preferably a poly(C6-C8) alkylcyanoacrylate. In a preferred embodiment, the poly(alkylcyanoacrylate) polymer is a polyisohexylcyanoacrylate (PIHCA). In a more preferred embodiment, the poly(alkylcyanoacrylate) polymer is a polyethylbutylcyanoacrylate (PEBCA). The monomer corresponding to the latter polymer is available, for instance under the trademark Monorex® by Onxeo (France).

The cyclodextrin may be neutral or charged, native (cyclodextrins α, β, γ, δ, ε), branched or polymerized, or even chemically modified, for example, by substitution of one or more hydroxypropyls by groups such as alkyls, aryls, arylalkyls, glycosidics, or by etherification, esterication with alcohols or aliphatic acids. Among the above groups, particular preference is given to those chosen from the group consisting of hydroxypropyl, methyl and sulfobutylether groups, and mixtures thereof. In a preferred embodiment, the cyclodextrin is selected from the group consisting of hydroxypropyl-beta-cyclodextrin and/or randomly methylated-beta cyclodextrin, and mixtures thereof.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, excipients, compositions or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem complications commensurate with a reasonable benefit/risk ratio.

As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, benzenesulfonic, glucoronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic and the like. Further addition salts include ammonium salts such as tromethamine, meglumine or epolamine, metal salts such as sodium, potassium, calcium, zinc or magnesium. For instance, a suitable salt of doxorubicin is the doxorubicin hydrochloride. As used in this specification, the term "about" refers to a range of values ± 10% of the specified value. For instance, "about 1 " means from 0.9 to 1 .1 when 10% is considered and from 0.95 to 1 .05 when 5% is considered. Where "about" is used in connection with numeric ranges, for example "about 1 to about 3", or "between about one and about three", preferably the definition of "about" given above for a number is applied to each number defining the start and the end of a range separately. Preferably, where "about" is used in connection with any numerical values, the "about" can be deleted.

In a particular embodiment, the nanoparticles comprise doxorubicin, at least one poly(C1 -C12 alkylcyanoacrylate), preferably a polyethylbutylcyanoacrylate (PEBCA), at least one cyclodextrin, preferably selected from the group consisting of hydroxypropyl- beta- cyclodextrin and randomly methylated-beta cyclodextrin, and mixtures thereof and poloxamer 188, wherein said poloxamer 188 has an ethyleneoxide (EO) weight percentage comprised between 80 and 81 .5%.

In a particularly preferred embodiment, the nanoparticles comprise doxorubicin, a polyethylbutylcyanoacrylate, a hydroxypropyl-beta-cyclodextrin and poloxamer 188, wherein said poloxamer 188 has an ethyleneoxide (EO) weight percentage comprised between 80 and 81 .5%.

Doxorubicin is present at a concentration from about 0.01 to about 200 mg/g of the nanoparticles, preferably from about 1 to about 50 mg/g.

The proportion of cyclodextrin is in general from about 0.1 to about 70% by weight of the nanoparticles, preferably from about 1 to about 30 %, more preferably from about 5 to about 20 %.

The proportion of doxorubicin and the proportion of cyclodextrin are generally independent from one another.

The proportion of the poly(alkylcyanoacrylate) polymer is in general from about 1 to about 25 % by weight of the nanoparticles, preferably from about 5 to about 15 %.

The nanoparticles of the invention may comprise:

- doxorubicin at a concentration from 0.01 to 200 mg/g, and/or

from 0.1 to 70 % w/w of said at least one cyclodextrin; and from 1 to 25 % w/w of said at least one poly(alkylcyanoacrylate).

In a particular embodiment, the nanoparticles of the invention may comprise one or more further active ingredient in addition to doxorubicin, such as chemotherapeutic agents.

According to an embodiment, the active ingredient is doxorubicin and the the poly(alkylcyanoacrylate) is polyisohexylcyanoacrylate (PIHCA) or polyethylbutylcyanoacrylate (PEBCA).

According to a second object, the present invention also concerns a process of preparation of nanoparticles of doxorubicin or any pharmaceutically acceptable salt thereof, said process comprising:

a) mixing at least one active ingredient or any pharmaceutically acceptable salt thereof, with at least one cyclodextrin and poloxamer 188, wherein said poloxamer 188 has an ethyleneoxide (EO) weight percentage comprised between 80 and 81 .5% in a polymerization medium;

b) adding an alkylcyanoacrylate monomer to said mixture; and

c) conducting polymerization .

The nanoparticles of the present invention may be prepared by application or adaptation on any method known by the skilled person, within the scope of the process of the invention. Such a method is disclosed, for example, in W099/43359.

Said at least one active ingredient, poloxamer, cyclodextrin and alkylcyanoacrylate monomer are defined as above.

In particular, the nanoparticles may be prepared by a method comprising the steps of

a) preparing, in an acid aqueous, a complex of doxorubicin cyclodextrin with poloxamer 188 where said poloxamer 188 has a EO weight percentage comprised between 80 and 81 .5% ;

b) gradually adding the alkylcyanoacrylate monomer, preferably the isohexylcyanoacrylate monomer, or more preferably the 2-ethylbutylcyanoacrylate, in the solution obtained at step (a); and c) conducting polymerization of this monomer, optionally in the presence of one or more surfactant and/or stabilising agents.

Preferably, the polymerization is anionic but may also be inducible by other agents, in particular by photochemical agents.

Additional surfactant agents include dextran (such as dextran 70,000) or other non- ionic surfactive agents (like polysorbate, sorbitan esters or others). According to an embodiment of the process of the invention, EO weight percentage of poloxamer 188 is comprised between 80 and 81 %. According to another embodiment, the EO weight percentage is comprised between 80 and 80.8%. According to a further embodiment, the EO weight percentage is comprised between 80.5 and 80.8%. According to an embodiment, the process further comprises recovering the nanoparticles from the polymerization medium, for example by filtering the nanoparticles.

According to an embodiment, the process further comprises resuspending the nanoparticles into a dispersion medium.

According to an embodiment, the polymerization and/or dispersion medium is water.

According to an embodiment, the polymerization is carried out in water at a pH comprised between 3 and 4.

The present invention also concerns nanoparticles obtainable by the process of the invention. According to an embodiment, the nanoparticles have an average diameter comprised between 100 and 300 nm. The invention also concerns a dispersion comprising said nanoparticles in a dispersion medium.

A "dispersion" as used herein refers to a mixture in which the nanoparticles are dispersed throughout a continuous phase, i.e. the dispersion medium. Said dispersions include suspensions, colloids and solutions. The term "dispersion medium" as used herein refers to the continuous phase of the dispersions of the invention. It may be water or a non-aqueous solvent. It may be also the polymerization medium in which the nanoparticles are synthetized, or it may be a different medium in which the nanoparticles are resuspended following synthesis and filtration from the polymerization medium.

According to an embodiment, the dispersion is an aqueous suspension, having a pH comprised between 0.5 and 5, more particularly between 3 and 4. This pH may be achieved by adding a suitable acid, base or buffer such as citric acid to the suspension medium.

The nanoparticles as described above can be administered in the form of a pharmaceutical composition comprising said nanoparticles and at least one pharmaceutically acceptable excipient.

According to a further object, the invention also concerns a pharmaceutical composition comprising the nanoparticles of the invention and at least one pharmaceutically acceptable excipient. It also concerns a method of treating cancer, consisting of administering the nanoparticles of the invention in patients in need thereof.

The nanoparticles for use in the treatment of cancer are another object of the invention.

The use of the nanoparticles for the manufacture of a medication dedicated to the treatment of cancer is also another object of the invention.

The pharmaceutical composition comprising said nanoparticles may be formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art. In particular, possible pharmaceutical compositions include those suitable for intravenous, intra-arterial and intra-tumoral administration. For these formulations, conventional excipients can be used according to techniques well known by those skilled in the art. Such compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions which can optionally be prepared immediately before use from solid or lyophilized form. Adjuvants such as a local anaesthetic, preservatives and buffering agents can be dissolved in the vehicle and a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the nanoparticles.

In addition to nanoparticles as described above, the pharmaceutical composition may further comprise at least one additional active substance, such as another chemotherapeutic agent included or not in nanoparticles.

The nanoparticles used in the present invention may be administered to a patient in need thereof to provide a therapeutically effective amount of the chemotherapeutic agent(s). As used herein, the term "patient" refers to either an animal, such as a valuable animal for breeding, company or preservation purposes, or preferably a human or a human child, which is afflicted with, or has the potential to be afflicted cancer.

As used herein, a "therapeutically effective amount" refers to an amount of a compound which is effective in preventing, reducing, eliminating, treating or controlling the symptoms of the herein-described diseases and conditions. The term "controlling" is intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the diseases and conditions described herein, but does not necessarily indicate a total elimination of all disease and condition symptoms, and is intended to include prophylactic treatment. The amount of nanoparticles to be administered has to be determined by standard procedure well known by those of ordinary skill in the art. In particular, physiological data of the patient (e.g. age, size, and weight), the type and localisation of cancer, the nature of the chemotherapeutic agent have to be taken into account to determine the appropriate dosage. In a particular embodiment, the nanoparticles are to be administered in an amount providing a dosage of doxorubicin from about 10 to about 75 mg/m², preferably from about 10 to about 60 mg/m², preferably from about 10 to about 45 mg/m², more preferably from about 10 to about 30 mg/m², from about 20 to about 30 mg/m². More preferably, the dosage of doxorubicin may be about 20mg/m² or 30 mg/m². The body surface area of a patient can easily be calculated by the skilled person from the body weight and height of the patient (e.g. body weight of about 65kg corresponds to a body surface of about 1 .8 mg/m². The identification of those subjects who are in need of treatment of herein-described diseases and conditions is well within the ability and knowledge of one skilled in the art. A clinician skilled in the art can readily identify, by the use of clinical tests, physical examination and medical/family history, those subjects who are in need of such treatment. A therapeutically effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the therapeutically effective amount, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of subject; its size, age, and general health; the specific disease involved; the degree of involvement or the severity of the disease; the response of the individual subject; the mode of administration; the bioavailability characteristic of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. The amount of nanoparticles, which is required to achieve the desired biological effect will vary depending upon a number of factors, including the dosage of the drug to be administered, the type of disease, the diseased state of the patient, and the route of administration. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient and its route of administration.

The nanoparticles can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients. Such compositions may be prepared for use in oral administration, particularly in the form of tablets or capsules; or parenteral administration, particularly in the form of liquid solutions, suspensions or emulsions; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically or via trans-dermal patches. Parenteral administration is preferred.

It includes:

• intravenous (into a vein), • intra-arterial (into an artery),

• intraosseous infusion (into the bone marrow),

• intra-muscular,

• intracerebral (into the brain parenchyma),

· intracerebroventricular (into cerebral ventricular system),

• intrathecal (an injection into the spinal canal), and

• subcutaneous (under the skin).

Liquid preparations for administration include sterile aqueous or nonaqueous solutions, dispersions, such as suspensions, and emulsions. The liquid compositions may also include binders, buffers, preservatives, chelating agents, sweetening, flavoring and coloring agents, and the like. Nonaqueous solvents include alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohols and water, buffered media, and saline. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the active compounds.

Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

As used herein, the term "intravenous administration" ("IV") refers to the infusion of liquid substances directly into a vein. This term refers to any type of intravenous access devices. In particular, this term refers to hypodermic needle. It is the simplest form of intravenous access by passing a hollow needle through the skin directly into the vein. This needle can be connected directly to a syringe or may be connected to a length of tubing and thence whichever collection or infusion system is desired.

As used herein, the term "cancer" refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. This term refers to any type of malignancy (primary or metastases). In particular, this term refers to any malignant proliferative cell disorders such as solid tumor or hematopoietic tumor, including carcinoma, sarcoma, lymphoma, stem cell tumor, blastoma. Particularly, the cancer is selected from the group consisting of hepatic cancer, in particular hepatocellular carcinoma, acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic myelogenous leukemia, Hodgkin's disease, diffuse large B-cell lymphoma, lung cancer, in particular small cell lung cancer, colorectal cancer, pancreas cancer, breast cancer, ovary cancer, uterine cancer, cervix cancer, head and neck cancer, brain cancer, blade cancer, multiple myeloma, neuroblastoma, Edwing's sarcoma, osteosarcoma, soft tissue sarcoma, thyroid cancer, prostate cancer, stomach cancer, nephroblastoma, Kaposi's sarcoma, and non-Hodgkins lymphoma. More particularly, the cancer is selected from the group consisting of hepatocellular carcinoma, acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic myelogenous leukemia, Hodgkin's disease, diffuse large B-cell lymphoma, small cell lung cancer, small cell lung cancer, colorectal cancer, pancreas cancer, uterine cancer, cervix cancer, head and neck cancer, brain cancer, breast cancer, ovary cancer, blade cancer, multiple myeloma, neuroblastoma, Edwing's sarcoma, osteosarcoma, soft tissue sarcoma, thyroid cancer, prostate cancer, stomach cancer, nephroblastoma, Kaposi's sarcoma, and non- Hodgkins lymphoma.

The following examples are given for purposes of illustration and not by way of limitation.

Example 1 : Preparation of nanoparticles loaded with doxorubicin

Livatag^® nanoparticles are presented as a sterile lyophilisate for injectable suspension that contains doxorubicin hydrochloride as active ingredient and other excipients including the polymer, polyethylbutylcyanoacrylate (PEBCA).

The doxorubicin loaded nanoparticles are obtained by aqueous emulsion polymerisation of 2-ethylbutylcyanoacrylate (EBCA) or isohexylcyanoacrylate (IHCA) monomer dropped in the bulk solution containing the active ingredient doxorubicin and the other excipients (P. Couvreur, B. Kante, M. Roland, P. Guiot, P. Baudhuin, P. Speiser, J. Pharm. Pharmacol., 1979, 31 , 331 ). At the end of polymerisation, a stable suspension of nanoparticles entrapping doxorubicin is obtained. The nanoparticles suspension is then filtered and aseptically filled in glass vials before freeze- drying. Nanoparticles loaded with Doxorubicin freeze-dried product are preferably kept protected from light and humidity and stored in a refrigerator between 2°C - 8°C, for stability purposes. Raw Materials

For 100 ml of total volume of polymerization media:

Method for preparing 100 ml of polymerization Medium (pH comprised between 3 and 4)

- Dissolution of doxorubicin in water at 10 mg/ml

- Addition of poloxamer 188 (concentration between 0,5 and 15%), under

stirring and till complete dissolution occurs

- Addition of Cyclodextrin (concentration between 0.1 and 70%)

- Adjustment to the required pH (between 3 and 4) with citric acid 0.1 M

Method for preparing nanoparticles loaded with Doxorubicin

In a 10 ml flask with magnetic stirring:

- Add 5 ml of polymerization media

- Add 50 μΙ of pure EBCA or IHCA solution gased with S02 (density of EBCA = 0,980) - Let polymerization occurs up to 2h30 period of time or more, under magnetic stirring and at room temperature

- Filtration onto 2 μηι filters

Example 1 was repeated with various samples of poloxamer 188, having different EO weight percentage (cf. example 2).

Example 2: Assessment of the nanoparticles parameters.

?.-. .. T.h.e.. EQ .. eight . percen tage . of_m poloxamer _ .188. was . obtained, according, to _m the (Plowing, procedure, in. accordance with the, US Pharmacppea: Solvent— Use deuterated water or deuterochloroform.

NMR reference— Use sodium 2,2-dimethyl-2-silapentane-5-sulfonate (for deuterated water) or tetramethylsilane (for deuterochloroform). Test preparation— Dissolve 0.1 g to 0.2 g of Poloxamer in deuterated water containing 1 % of sodium 2,2-dimethyl-2-silapentane-5-sulfonate to obtain 1 ml_ of solution, or, if the Poloxamer does not dissolve in water, use deuterochloroform containing 1 % of tetramethylsilane as the solvent.

Procedure— Transfer 0.5 ml_ to 1 .0 ml_ of the Test preparation to a standard 5-mm NMR spinning tube, and if deuterochloroform is the solvent, add 1 drop of deuterated water, and shake the tube. Proceed as directed for Relative Method of Quantitation under NMR, using the Test preparation volumes specified here, scanning the region from 0 ppm to 5 ppm, and using the calculation formulas specified here. Record as A1 the average area of the doublet appearing at about 1 .08 ppm, representing the methyl groups of the oxypropylene units, and record as A2 the average area of the composite band from 3.2 ppm to 3.8 ppm, due to the CH20 groups of both the oxyethylene and oxypropylene units and also the CHO groups of the oxypropylene units, with reference to the sodium 2,2- dimethyl-2-silapentane-5-sulfonate or tetramethylsilane singlet at 0 ppm. Calculate the percentage of oxyethylene, by weight, in the Poloxamer, taken by the formula:

3300 «/(33« + 58), in which a j_s (A2 / A1 ) - 1 . pH : between 5.0 and 7.5, in a solution (1 in 40).

?_.-_.?_.:.. T.h.e.. a yerage . diameter.. of.. the.. nanoparticles.. obtained . after . conducting.. the Procedure. of. example 1.. was calculated. according_, to. the. following. procedure:.

- Place 2.5 μΙ_ of nanoparticles suspensions into 3 ml_ of MilliQ water

- Shake gently to avoid bubbles

- Analyse the sample by Dynamic Light Scattering (T°C 20°C, 3 measurements per preparation, equilibration 120s)

- The results are given in term of mean Z-average of the 3 measurements

?.-.3_:.2 liters formulation In this experiment, nanoparticles were obtained according to example 1 following a clarifying filtration.

5 In this experiment, another EO weight percentage of poloxamer 188 has been used:

?.-.4_:.24 liters formujatjgn

In this experiment, nanoparticles were obtained according to example 1 following by a double filtration and lyophilization:

The results summarized in 2.3 and 2.4 above show that poloxamer 188 with EO weight percentage comprised between 80 and 81 .5% consistently leads to nanoparticles of average diameter comprised between 200 and 300 nm, whereas poloxamer 188 with EO weight percentage above 81 .5% leads to nanoparticles of average diameter of more than 300 nm.

Experiments were conducted with EBCA and can be carried out in a similar fashion with IHCA, by expecting similar results.

Claims

1 . Nanoparticles comprising (i) at least one active ingredient or any pharmaceutically acceptable salt thereof, (ii) a polymer of poly(alkylcyanoacrylate), (iii) one or more cyclodextrin, and (iv) poloxamer 188,

wherein said poloxamer 188 has an ethyleneoxide (EO) weight percentage comprised between 80 and 81 .5%.

2. The Nanoparticles according to claim 1 wherein the EO (weight) percentage of poloxamer 188 is comprised between 80.5 and 81 %.

3. The Nanoparticles according to anyone of the preceding claims wherein the EO (weight) percentage of poloxamer 188 is comprised between 80.5 and 80.8%.

4. The Nanoparticles according to anyone of the preceding claims wherein the at least active ingredient is doxorubicin.

5. The Nanoparticles according to anyone of the preceding claims wherein the nanoparticles have an average diameter comprised between 100 and 300 nm.

6. The Nanoparticles according to anyone of the preceding claims wherein the poly(alkylcyanoacrylate) is polyisohexylcyanoacrylate (PIHCA) or polyethylbutylcyanoacrylate (PEBCA).

7. The Nanoparticles according to anyone of the preceding claims wherein the poly(alkylcyanoacrylate) is polyethylbutylcyanoacrylate (PEBCA).

8. Process of preparation of nanoparticles of at least one active ingredient or any pharmaceutically acceptable salt thereof, said process comprising:

a) mixing said active ingredient or any pharmaceutically acceptable salt thereof, with at least one cyclodextrin and poloxamer 188, wherein said poloxamer 188 has an ethyleneoxide (EO) weight percentage comprised between 80 and 81 .5%

b) adding an alkylcyanocyanoacrylate monomer to said mixture; and

c) conducting polymerization.

9. The process according to claim 8 wherein said at least one active ingredient is doxorubicin.

10. The process according to claim 8 or 9 further comprising recovering the nanoparticles from the polymerization medium.

1 1 . The process according to claim 10, which further comprises resuspending the nanoparticles into a dispersion medium.

12. The process according to anyone of claims 8 to 1 1 , wherein the polymerization and/or dispersion medium is water.

13. A dispersion comprising the Nanoparticles according to anyone of claims 1 to 7 in a dispersion medium.

14. A pharmaceutical composition comprising the Nanoparticles according to anyone of claims 1 to 7.

15. Nanoparticles of doxorubicin or any pharmaceutically acceptable salt according to anyone of claims 1 to 7 for use in the treatment and/or prevention of cancer.