Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
  • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
  • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]

Definitions

• the present invention aims to provide nanoparticulate water-dispersible formulations of therapeutic active agents of low aqueous solubility, or even hydrophobic.
• an asset is said to be poorly water-soluble when it exhibits a solubility in pure water of less than 100 ⁇ g / ml at ambient temperature, that is to say a temperature of approximately 25 ° C.
• solubilization in an aqueous medium may, for some of them, be acquired subject to formulating them as organic or inorganic salts.
• cyclosporines As an illustration of these therapeutically insoluble assets, there may be mentioned cyclosporines, taxoids, taxanes and peptide molecules such as insulin image. Cyclosporins are anti-fungal compounds, usually with immunosuppressive activity. These cyclosporines most often exhibit an aqueous solubility not exceeding 25 ⁇ g / ml, a value that is approximately one hundred times lower than that required for regular absorption by the body. To obtain an acceptable bioavailability of cyclosporin, conventional formulations generally employ dispersion systems combining a hydrophilic phase, a hydrophobic phase and a surfactant.
• taxoids or taxanes are diterpene substances.
• Paclitaxel which is a natural taxoid, and its semi-synthetic derivative, docetaxel, are widely used for the treatment of tumors.
• the taxane derivatives generally have a lower aqueous solubility than cyclosporins. Thus, this very low aqueous solubility required the development of specific formulations.
• the formulation of paclitaxel distributed under the trademark TAXOL ® by BRISTOL MEYERS SQUIBB and dedicated to a systemic administration is a sterile form, non pyrogenic disposable single dose containing 30 mg of anhydrous docetaxel.
• each dose contains polyoxyethylene glycol triricinolate, Cremophore EL® and ethanol.
• this type of formulation requires to be diluted in sterile, pyrogen-free and isotonic infusion solution (sodium chloride 0.9%, glucose 5%, etc.) before administration.
• Cremophore EL ® and ethanol which are necessary to overcome the lack of solubility of paclitaxel, are unfortunately likely to cause adverse effects.
• Cremophore EL ® and ethanol which are necessary to overcome the lack of solubility of paclitaxel, are unfortunately likely to cause adverse effects.
• it is generally a premedication of the subject to be treated with dexamethasone orally, three days before the initiation of chemotherapy.
• paclitaxel implements paclitaxel bound to albumin.
• This specific formulation is marketed under the name Abraxane ® .
• Abraxane ® a specific formulation of paclitaxel bound to albumin.
• this alternative is also not satisfactory insofar as it requires the formation of this derivative, by covalent bonding of an albumin molecule to a paclitaxel molecule.
• this coupling affects the therapeutic activity of paclitaxel.
• this lack of bioavailability of poorly water-soluble active becomes an exacerbated handicap when the asset is intended to be administered orally, another particularly popular route for the administration of therapeutic assets.
• the present invention aims precisely at preparing a novel mode of formulation of therapeutically weak water-soluble active agents making it possible to compensate for the drawbacks of conventional formulations of such active agents.
• the present invention results more particularly from the observation by the inventors that it is possible to formulate these therapeutically active agents, which are poorly water-soluble or even hydrophobic, in the form of nanoparticles in suspension in an aqueous medium and of reduced size, in particular compatible. for administration by injection, provided that these assets are associated with a squalene derivative or the like.
• the present invention relates to a hydrodispersible derivative of a therapeutic active agent of low aqueous solubility formed of at least one molecule of said active agent covalently coupled to at least one molecule of a hydrocarbon compound with a squalene structure or similar.
• the present invention relates to a aforementioned hydrodispersible derivative in which the therapeutic active agent has a solubility of less than 100 ⁇ g / ml in pure water measured at ambient temperature, in particular less than 25 ⁇ g / ml, in particular less than 20 ⁇ g / ml, or even less than 10 ⁇ g / ml and more particularly less than 5 ⁇ g / ml.
• the therapeutic active agent has a solubility of less than 100 ⁇ g / ml in pure water measured at ambient temperature, in particular less than 25 ⁇ g / ml, in particular less than 20 ⁇ g / ml, or even less than 10 ⁇ g / ml and more particularly less than 5 ⁇ g / ml.
• such a derivative may also be named
• the present invention relates, in another aspect, to water-dispersible nanoparticles of at least one therapeutic active agent of low aqueous solubility in which said active agent is present in a form associated with at least one hydrocarbon compound with a squalene structure or similar.
• a “squalene structure” is intended to designate a linear hydrocarbon structure, consisting of isoprene units, and more particularly 6, in the image of squalene, the formula of which is the following:
• hydrocarbon compounds As an illustration of these hydrocarbon compounds, mention may more particularly be made of squalenic acid and its derivatives.
• this squalene structure is particularly important in the context of the present invention because it manifests spontaneously, when placed in the presence of a polar medium and more particularly water, a compacted conformation.
• analogous means a hydrocarbon compound on the one hand, capable of reproducing the behavior of a squalene derivative when it is placed in the presence of a polar medium and, on the other hand, capable of reproducing this ability when bound to a molecule of a therapeutic active of low aqueous solubility.
• This definition covers in particular the substituted forms of squalene derivatives and in particular squalene acid and its derivatives, in particular substitution derivatives.
• Such a derivative can be, for example, in the image of 1, 1 ', 2-troinsorsenic acid, squalenoylacetic acid, 1, 1', 2-tris-norsqualenamine, 1, 1 ', 2-tris-norsqualenol, 1,1 ', 2-tris-norsqualenethiol, squalenacetic acid, squalenylethanol, squalenylethanethiol or squalenylethylamine
• At least one hydrocarbon molecule with a squalene structure is covalently bound to a molecule of a therapeutic active agent of low aqueous solubility.
• the number of hydrocarbon derivative molecules capable of interacting with a therapeutic active molecule may be greater than 1.
• a derivative according to the invention may comprise at least two radicals with identical or different squalene structure.
• This hydrocarbon compound is generally carrying a function capable of reacting with a function present on the molecule of the active agent in order to establish a covalent bond between the two entities, for example of the ester, ether, thioether, disulfide, phosphate or amide.
• a function present on the molecule of the active agent for example of the ester, ether, thioether, disulfide, phosphate or amide.
• it is a carboxylic function.
• the squalene-structured hydrocarbon derivative is squalene acid or one of its derivatives such as, for example, squalenoyl N-hydroxy succinimidyl ester.
• the covalent link existing between the two types of molecules may be represented by a "linker" or linker.
• Such an arm may especially be useful when the functions respectively present on the squalene structure compound and the low solubility therapeutic active have no reaction affinity with each other and therefore are not likely to form the expected covalent bond.
• Such an arm makes it possible precisely to introduce via each of the two ends of its skeleton the appropriate functions, ie possessing respectively the expected reaction affinity, one for the function present on the derivative with a squalene structure and the other for the present function. on the asset in question.
• this linker also has at its skeleton a labile function, which is suitable for subsequent separation of the squalene-structured compound from the therapeutic active agent. It may for example be a peptide motif recognizable by an enzyme.
• linkage type patterns are well known to those skilled in the art and their implementation clearly falls within its competence.
• a "covalent bond” preferably represents a covalent bond especially as specified above, but also covers a covalent bond represented by a linker as defined above.
• a therapeutic active agent of low aqueous solubility is a compound having a solubility of less than 100 ⁇ g / ml in pure water, measured at ambient temperature, that is to say approximately 25 ° C., in particular less than 25 ⁇ g / ml, in particular less than 20 ⁇ g / ml, in particular less than 15 ⁇ g / ml or even less than 10 ⁇ g / ml and more particularly less than 5 ⁇ g / ml.
• a pure water is a water of pH close to neutrality (between pH5 and pH8) and devoid of any other compound such as organic or inorganic salts for example.
• the therapeutic active agents more particularly considered may be chosen from the substances of groups 2 and 4 of the biopharmaceutical classification.
• immunosuppressants especially antitumour agents, such as taxoids, doxorubicin, also known as adriamycin and its isomer, epirubicin, antiangiogenic agents, antivirals, antibacterials, may be mentioned.
• antibiotics and antiparasitic agents substances affecting the metabolism of sugars, peptides, lipids, agents acting on calcium channels, non-steroidal antifiogistics and peptide compounds such as insulin.
• the form of the active agent coupled to at least one molecule with squalene structure is generally a neutral form, that is to say nonionic or non-salified unless it is itself has low water solubility.
• Immunosuppressants are hydrophobic compounds and include N-methylated cyclic undecapeptides. Among this family of active ingredients, cyclosporins are more particularly considered. These include cyclosporins A and G. However, other macroides can also be considered according to the invention. It may also be an immunosuppressant such as deoxyspergualine, rapamycin or ascomycin.
• the therapeutic assets of low solubility are more particularly taxanes and taxoids.
• taxanes and taxoids are described in particular in application WO 2005/013968.
• docetaxel paclitaxel or a derivative thereof.
• these substances are functionalized with at least one molecule of a hydrocarbon compound with a squalene structure or the like.
• the therapeutic active agents considered according to the present invention can comprise two derivatizations, three derivatizations, or even more, these being able to be identical or different.
• reaction necessary for the establishment of at least one covalent bond between a molecule of a therapeutic active agent of low aqueous solubility and at least one molecule of a hydrocarbon derivative of squalene structure or the like can be carried out according to standard conditions and its realization therefore clearly falls within the knowledge of those skilled in the art.
• This reaction is generally carried out in solution in the presence and in excess of at least one compound having a squalene structure with respect to the weakly water-soluble active ingredient considered, for example at the rate of two equivalents, according to the standard conditions required for the two functions to interact specific ones carried by each of the molecules.
• hydrodispersible derivatives are: - squalenoyl-insulin of formula as follows:
• the present invention relates to an aqueous dispersion of at least one derivative as defined above.
• the covalent coupling of a therapeutic active agent with at least one molecule of a hydrocarbon compound with a squalene structure is such as to confer on the active agent thus functionalized with at least one squalenoyl radical an ability to organize under a compacted form in a polar solvent medium, thus leading to the formation of nanoparticles.
• the nanoparticles thus obtained have an average size ranging from 30 to 650 nm, in particular from 30 to 500 nm, and in particular from 50 to 250 nm, or even from 100 to 200 nm, measured by light scattering. using the Coulter ® N4MD nanosizer, Coulter Electronics, Hialeah, USA.
• a weakly water-soluble therapeutic active agent as considered according to the invention with a hydrocarbon derivative according to the invention, and more particularly with squalene acid or one of its derivatives, for example, the ester squalenoyl N-hydroxysuccinimidyl, gives said therapeutic substance physicochemical characteristics sufficient to impart an ability to form particles whose size is compatible for parenteral administration and especially intravenous.
• the present invention relates, according to another of its aspects, to a process for the preparation of these nanoparticles, characterized in that it comprises: the solubilization of a derivative according to the invention, that is to say formed preliminary by coupling of at least one molecule of a hydrocarbon compound with a squalene structure or a molecule analog of a therapeutic active agent of low aqueous solubility, in at least one organic solvent, for example an alcohol such as ethanol, at a concentration sufficient to obtain, during the addition of the resulting mixture, with stirring, and generally at dropwise, to an aqueous phase, the instantaneous formation of nanoparticles of said derivative in suspension in said aqueous phase, and, where appropriate, the isolation of said nanoparticles.
• a derivative according to the invention that is to say formed preliminary by coupling of at least one molecule of a hydrocarbon compound with a squalene structure or a molecule analog of a therapeutic active agent of low aqueous solubility, in at least one organic
• the reaction can generally be carried out at room temperature. Whatever it is, the reaction temperature must not affect the activity of the asset under consideration.
• the process for preparing the nanoparticles according to the invention is particularly advantageous insofar as it does not require the presence of surfactants.
• the coupling between the squalene-structured hydrocarbon derivative and the active molecule can be direct or via a linker.
• the nanoparticles according to the invention are formulated in the form of aqueous dispersion with a view to their administration generally through the systemic route.
• this aqueous dispersion contains less than 5% by weight, or even less than 2% by weight of C 2 to C 4 alcohol such as, for example, ethanol.
• this aqueous dispersion contains less than 5% by weight, or even less than 2% by weight and more particularly is devoid of surfactant or the like such as for example polyethylene glycols, polyglycerol and their derivatives, such as esters for example.
• surfactant or the like such as for example polyethylene glycols, polyglycerol and their derivatives, such as esters for example.
• this aqueous dispersion contains less than 5% by weight, or even less than 2% by weight and more particularly is devoid of polyoxyethylated castor oil such as for example that marketed under the name Cremophore EL ® .
• this aqueous dispersion intrinsically has a viscosity compatible with intravenous administration.
• the formulation in aqueous medium of the taxoid such as Paclitaxel ® using squalenic acid in the form of water-dispersible nanoparticles advantageously makes it possible to obtain a suspension of nanoparticles with no other additive than the 5% dextrose required. to obtain the isotonicity of the injectable suspension. It is thus possible to: (i) dispense with the use of the toxic Cremophore, (ii) have a directly injectable aqueous formulation and (iii) administer higher concentrations of the product (up to 4 mg / ml).
• the present invention also relates, according to another of its aspects, the use of these derivatives and nanoparticles in pharmaceutical compositions.
• composition comprising, as active ingredient, at least one derivative according to the present invention, especially in the form of nanoparticles.
• the derivatives according to the present invention may also be administered by any conventional route. However, as specified previously, given the small size of their particles, they can be administered in the form of an aqueous suspension intravenously and therefore compatible with vascular microcirculation.
• Another aspect of the invention therefore relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least, as an active ingredient, a compound according to the present invention, in particular in the form of nanoparticles.
• the derivatives in accordance with the present invention may be associated therewith with at least one pharmaceutically acceptable vehicle.
• the compounds When the compounds are used in dispersion in an aqueous solution, they can be combined with sequestering or chelating agent excipients, antioxidant, pH modifying agents and / or buffering agents.
• the nanoparticles according to the invention are of course capable of carrying on the surface a multitude of reactive functions, such as hydroxyl or amine functions, for example. It is therefore conceivable to fix these functions all kinds of molecules, including covalent bonds.
• this type of molecule capable of being associated with the nanoparticles mention may be made especially of marker-type molecules, compounds capable of providing a targeting function, as well as any compound capable of conferring on them characteristics. specific pharmacokinetics.
• a surface coating based on such a compound is in advantageous effect for conferring increased vascular remanence due to a significant reduction in the uptake of nanoparticles by hepatic macrophages.
• compositions according to the invention may contain preserving agents, wetting agents, solubilizing agents and coloring agents.
• a derivative according to the present invention for systemic taxoid treatment intended for an adult patient, it may be envisaged to administer a derivative according to the present invention at a dose of about 0.1 to 150 mg / kg of body weight. and per day and more particularly from 1 to 40 mg / kg per day.
• anticancer or cytostatic molecules or macromolecules for example platinum salts, antracyclines, mitotic spindle poisons, topoisomerase inhibitors.
• kinases or metalloproteases for example anti-inflammatory agents of the corticoid (for example dexamethasone) or non-corticoid type or molecules with immunoadjuvant activity (for example antibodies with anticancer activity).
• the association with hyperthermia used in certain chemotherapies may be considered.
• the derivatives according to the present invention may also be combined with surgical and / or radiation therapies for the treatment of cancer.
• the present invention also relates, in another of its aspects, to a method of therapeutic treatment comprising the administration to a patient of an effective amount of at least one derivative and / or nanoparticles according to the invention, optionally in combination with another active ingredient and / or therapeutic means (hyperthermia, radiation) and / or surgical therapies, as defined above
• Figure 1 Accumulation of tubulin after incubation of KB cells with nanoparticles of squalenoylpaclitaxel (IB) against untreated KB cells (IA).
• FIG. 2 Evaluation of the anti-cancer activity in vivo of nanoparticles of a squalenoyldiglycolyl-paclitaxel derivative obtained according to Example 11, by characterization of the volume of a tumor implanted on a mouse (in mm 3 ) as a function of time (number of days after tumor implantation).
• Figure 3 Evaluation of the weight variations of mice bearing L1210 leukemia, treated or not treated with the metastasis-induced squalenoyl-doxorubicin nanoparticles, as a function of time (number of days after intravenous injection of L1210 leukemic cells).
• Figure 4 Evaluation of the survival rate of mice with L1210 leukemia, treated or not treated with squalenoyl-doxorubicin nanoparticles, as a function of time (number of days after intravenous injection of L1210 leukemic cells).
• 450 mg of Paclitaxel (0.526 mmol) in dichloromethane (DCM) are reacted with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCA, 2.5 molar equivalents relative to Paclitaxel), dimethylamino pyridine (DMAP, 0.5 molar equivalent relative to Paclitaxel) and squalene acid (2 molar equivalents relative to Paclitaxel) previously dissolved in DCM at room temperature.
• EDCA 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
• DMAP dimethylamino pyridine
• squalene acid 2 molar equivalents relative to Paclitaxel
• squalenoylpaclitaxel 4 mg are dissolved in 1.5 ml of ethanol (2.5 mg / ml) and added dropwise with continuous stirring (500 rpm speed) to 1 ml of an aqueous solution containing 5% of dextrose.
• Squalenoylpaclitaxel self-assembles in the form of nanoparticles with a size between 100 and 200 ⁇ m.
• the ethanol is then completely evaporated under reduced pressure using a rotary evaporator to obtain a nanoparticulate ® suspension squalénoylpaclitaxel at a final concentration of 4 mg / ml.
• Variable quantities (specified in Table 1 below) of squalenoylpaclitaxel are dissolved in 0.25 ml of tetrahydrofuran (THF). The solutions obtained are added dropwise and with continuous stirring (500 rpm speed) to 0.25 ml of water. Squalenoylpaclitaxel precipitates in the form of nanoparticles with a size of between 100 and 200 ⁇ m. The THF is then completely evaporated under reduced pressure using a rotavapor ® to obtain nanoparticulate suspensions of squalenoylpaclitaxel whose size varies according to the concentration (see table below)
• the nanoparticulate suspension of squalenoylpaclitaxel as prepared in Example 3 (4 mg / ml) is directly injected intravenously into mice (C57BL6) at a dose of 4 ⁇ 50 mg / kg (Paclitaxel equivalent); the injections take place on days 0, 5, 9 and 14. The injection is easy and the animals show no sign of toxicity. No mortality is recorded (within a period of at least one month).
• Direct intravenous injection of the TAXOL solution (without dilution in 0.9% NaCl) resulted in the immediate death of 100% of the treated mice. When the TAXOL solution is previously diluted in 0.9% NaCl, the maximum dose that can be injected is 20 mg / kg.
• KB 3.1 cells from human cervical carcinoma, and HT-29 cells corresponding to a human colon cancer cell line are plated on 6-well plates at a density of 20000 cells / well (final volume 2 ml RPMI plus FCS). The cells are incubated at a temperature of 37 ° C. and at 5 ° C.
• tubulin clusters As illustrated by FIG. 1B, the formation of tubulin clusters is clearly visible by fluorescence microscopy, which shows the spindle poison activity of the squalenoylpaclitaxel nanoparticles. Untreated KB cells are presented as a control ( Figure IA).
• the squalenoyl N-hydroxysuccinimidyl esters are characterized by NMR and by mass spectrometry.
• b) Obtaining the squalenoyl-insulin conjugate 50 mg of insulin (bovine pancreas origin and meeting USP test criteria) are dissolved in 5 ml of dry dimethylsulfoxide (> 99% purity) and added to 30 ⁇ l of N, N diisopropylethylamine in a vial containing 13.2 mg of Sq-CO-NHS (in a 3: 1 molar excess). The reaction is carried out at room temperature for 2 hours and the solution is intensively dialyzed to remove organic solvents and small molecular impurities and then lyophilized. The solid is then washed with DCM and filtered. 46 mg of white powder are obtained.
• the conjugation reaction is monitored by analytical HPLC using a Waters RP C18 column (150.9 mm, 5 ⁇ m, 300 ⁇ ) and methanol / water (gradient plus 0.1% TFA) as the eluting solvent.
• Squalene conjugate of insulin was performed by MALDI-TOF and Orbi-Trap MS analyzes.
• the compound has the following structural formula:
• Deoxyspergualine is first prepared by purification of the commercial product called Gusperimus (Spanidine). After two chromatographic processes (ion exchange and gel filtration), the basic form of deoxyspergualine (poorly soluble in water) is reacted with the squalenoyl N-hydroxysuccinimidyl ester. For this purpose, 24 mg deoxyspergualine is rapidly dissolved in 1 ml of dry DMF which is added to 29 mg of squalenoyl N-hydroxysuccinimide ester (molar excess: 1.3) previously dissolved in the same solvent. 10 ⁇ l of N, N-diisopropylethylamine are then added.
• the squalenoyl-deoxyspergualine conjugate has the following structural formula:
• Step 1 Paclitaxel is dissolved in 4-dimethylaminopyridine (0.1 eq) and succinic anhydride (2 eq) and dried under vacuum for 2 hours. After addition of dry pyridine, the reaction mixture is stirred for 3 hours at room temperature. After removal of the solvent, the crude mixture is dissolved in dichloromethane (DCM) and rinsed with brine. No additional purification step of paclitaxel-2'-succinate is necessary. NMR analysis to detect the absence of 2 'OH, the conversion of 2' CH (at 5.51) and the quantitative presence of 7 CH in the normal position (4.48) shows the total transformation of the starting material into succinate derivative.
• DCM dichloromethane
• N-Hydroxysuccinimido-diphenyl phosphate (SDPP) is prepared from diphenylphosphoryl chloride, N-hydroxysuccinimide, and triethylamine (TEA) in DCM.
• the crude SDPP is triturated in ether, dissolved in ethyl acetate, rinsed with water, dried and concentrated in vacuo to obtain SDPP. Characterization by mass spectrometry (MS) confirms a molecular peak at 348.
• paclitaxel-2 '-succinate is added SDPP (1.5 eq.) In acetonitrile with TEA (4 eq.). The reaction mixture is stirred for 6 hours at room temperature and then concentrated in vacuo. The crude reaction is dissolved in ethyl acetate and extracted with brine. The formation of paclitaxel-2'-succinyl-NHS is monitored by TLC and HPLC assays, as described above, which does not require an additional purification step.
• Paclitaxel-2'-succinyl-NHS dissolved in dry dimethylformamide (DMF) is reacted with the amine squalene (1 eq.) In the presence of triethylamine (1 eq.). After 8 hours at room temperature and overnight at 5 0 C, the mixture is extracted with brine and purified on an SiO 2 gel, eluted with DCM-ethyl acetate. The main product (squalenoylsuccinyl-paclitaxel) is eluted with 30-50% ethyl acetate and its purity is monitored by HPLC analysis on an RP-18 column.
• DMF dry dimethylformamide
• Step 1 The squalene, in alcoholic form, is mixed with diglycolic anhydride (2.5 eq) in dry pyridine at room temperature overnight, with stirring. The solvent is removed and the residue is extracted from dilute hydrochloric acid and brine with DCM. The conversion of the expected product is controlled by TLC. The product thus obtained is dried under vacuum and the acid is used without an additional purification step.
• step 1 The squalenediglycolic acid (2 eq.) Described in step 1 is dissolved in DCM, then Paclitaxel (1 eq.) And 4-N, N-dimethylaminopyridine (3 eq.), Previously dissolved in DCM, are added. . After 10 minutes, the EDCI (1.3 eq) is added and the solution is stirred at room temperature for 2 hours. After removing the solvent, the crude product is passed through a silica gel column using a DCM / ethanol gradient to obtain the purified product, the purity of which is monitored by HPLC analysis on an RP-18 column.
• Paclitaxelsuccinyl-NHS (see Example 10, step 3) is dissolved in DCM, and the tBoc-NH-PEG3-NH 2 solution (1 eq.) In DCM is added to the previous solution with stirring. Triethylamine (0.5 eq) is added at a temperature of 40 ° C., and the reaction is maintained at a temperature of 20 ° C. for 5 hours. The crude reaction mixture is extracted with 0.1N HCl and then with brine. The product thus obtained is used in the next step without further purification.
• Paclitaxel-succinyl-Peg3-NH-tBoc is dissolved in dry DCM, and trifluoroacetic acid (TFA) is added with stirring at 20 ° C. After 2 hours, the reaction is complete and the mixture is extracted with sodium acetate solution, then with brine until neutral.
• Squalene N-hydroxysuccinimide ester (1.2 eq.), Previously prepared by reaction of squalene acid and NHS, in the presence of EDCI, is added to the Paclitaxel-succinyl-Peg3-amine (1 eq.).
• Triethylamine 0.3 eq. Is added and the reaction mixture is stirred at room temperature for 6 hours. After removing the solvent, the crude product is purified through a silica gel column using a DCM / ethanol gradient to obtain the purified product, the purity of which is monitored by HPLC analysis on an RP-18 column.
• Nanoparticles of different actives are prepared by nanoprecipitation.
• the ethanolic solution containing the squanenoyl / paclitaxel conjugate base solution 5-10 mg / ml
• base solution 5-10 mg / ml
• stirring 500 rpm
• Precipitation of nanoparticles occurs spontaneously.
• Organic solvents are totally evaporated using a Rotavapor ® to obtain an aqueous suspension of pure nanoparticles.
• the average size of the nanoparticles and the polydispersity index of the nanoparticles are determined at a temperature of 20 ° C. by light scattering with a zetasizer (Malvern Instruments, UK). The measurements are carried out after dilution of the suspension of the nanoparticles in MilliQ ® water.
• the size of the nanoparticles of the squalenoyl derivatives of paclitaxel and doxorubicin and their polydispersity index are listed in the table below:
• M109 Madison's 109 murine lung tumor cell line.
• M109 cells are cultured in RPMI 1640, supplemented with 10% fetal calf serum, 50 U.ml- 1 penicillin and 50 ⁇ g.ml- 1 streptomycin and 2 mM L-glutamine.
• This assay is performed using the 3- [4,5-dimethylthiazol-2-yl] -3,5-diphenyl tetrazolium bromide (MTT) assay, measuring the mitochondrial dehydrogenase activity.
• MTT 3- [4,5-dimethylthiazol-2-yl] -3,5-diphenyl tetrazolium bromide
• the cells in the exponential growth phase are seeded on a 96-well plate and preincubated for 24 hours at 37 ° C. in a humidified atmosphere with 5% CO 2 in the air.
• Different dilutions of paclitaxel nanoparticles are added to the cells in the culture medium. Each dilution is tested three times.
• 200 ⁇ l of MTT solution in the cell culture medium 0.5 mg / ml
• the culture medium is removed and the obtained formazan crystals are dissolved in 200 ⁇ l of an extraction solution (dimethyl sulfoxide).
• the absorbency of the transformed dyestuff which is proportional to the number of viable cells, is measured at 570 nm using a microplate reader (Metertech® 960, Fisher Bioblock, Illkirch, France). The percentage of surviving cells is calculated as the ratio of absorbency between treated and untreated cells.
• the in vitro anti-cancer activity of squalenoyl-doxorubicin nanoparticles is evaluated on a L1210 WT murine leukemia cell line.
• Cells are cultured in RPMI 1640 supplemented with 10% fetal calf serum, 50 U.ml- 1 penicillin and 50 ⁇ g.ml- 1 streptomycin and 2 mM L-glutamine. The evaluation is carried out according to the protocol described above.
• IC 50 50% inhibition concentrations
• CD2F1 mice (4-5 weeks old) weighing about 15-18 g were used for this study.
• the mice are fed standard mouse feed and ad libitum water.
• the M 109 subcutaneous tumor model (murine lung tumor) is developed by injecting the exponentially growing M 109 cells (1x106 cells) in suspension, under the skin, into the lower portion of the abdomen of the mice.
• a palpable tumor (approximately 50-100 mm3) is allowed to grow at the injection site.
• Tumor-bearing mice are divided into 2 groups of 5-6, ie untreated and treated with squalenoyldiglycolyl-paclitaxel nanoparticles 160 mg.kg- 1 (injected intravenously for 5 consecutive days). controlled to check the difference in tumor volume, and thus evaluate the anti-cancer efficacy.
• the squalenoyldiglycolyl-paclitaxel nanoparticles demonstrate anti-cancer activity by controlling the progression of M 109 tumors implanted hypodermically in mice.
• mice (4-5 weeks old) weighing about 15-18 g were used for this study.
• the mice are fed standard mouse food and ad libitum water.
• the L1210 aggressive metastatic leukemia model (murine leukemia) is developed by injecting L1210 exponential growth cells (0, IxIO 6 cells) intravenously. suspension, in mice.
• mice are divided into 2 groups of 5-6, namely untreated and treated with squalenoyl-doxorubicin nanoparticles (13 mg.kg- 1 , injected intravenously on days 1, 7 and 14, After injection of the tumor cells.) After treatment, the mice are checked regularly to verify the differences in weight and the survival rate, parameters that make it possible to evaluate the effectiveness of the anti-cancer activity.

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