PHARMACEUTICAL COMPOSITION, PREPARATION AND USES THEREOF

Abstract

The present invention relates to a pharmaceutical composition comprising the combination of (i) at least one biocompatible nanoparticle and (ii) at least one carrier comprising at least one pharmaceutical compound, to be administered to a subject in need of such a pharmaceutical compound, wherein the combination of the at least one biocompatible nanoparticle and of the at least one carrier comprising the pharmaceutical compound(s) potentiates the compound(s) of interest efficiency. The longest dimension of the biocompatible nanoparticle is typically between about 4 and about 500 nm and its absolute surface charge value is of at least 10 mV (|10 mV|). The carrier is in addition devoid of any surface sterically stabilizing agent. The invention also relates to such a composition for use for administering the pharmaceutical compound(s) in a subject in need thereof, wherein the at least one biocompatible nanoparticle and the at least one carrier comprising the at least one pharmaceutical compound are to be administered separately in a subject in need of said pharmaceutical compound, typically between more than 5 minutes and about 72 hours one from each other.

Claims

1-20. (canceled)

21. A therapeutic, prophylactic or diagnostic method comprising a step of administering at least one carrier comprising at least one pharmaceutical compound to a subject in need of said at least one pharmaceutical compound and a distinct step of administering at least one biocompatible nanoparticle to said subject, wherein the at least one carrier is devoid of any surface sterically stabilizing agent and wherein the longest dimension of the at least one biocompatible nanoparticle is between about 4 nm and about 500 nm, the absolute surface charge value of the at least one biocompatible nanoparticle is of at least |10 mV|, the at least one biocompatible nanoparticle is not used as a pharmaceutical compound, and said at least one biocompatible nanoparticle is administered to the subject between 5 minutes and about 72 hours before or after the at least one carrier comprising at least one pharmaceutical compound.

22. The method according to claim 21, wherein the nanoparticle has an absolute surface charge value of more than |10 mV|, said charge being a negative charge.

23. The method according to claim 21, wherein the nanoparticle is an organic nanoparticle.

24. The method according to claim 23, wherein the nanoparticle is selected from a lipid-based nanoparticle, a protein-based nanoparticle, a polymer-based nanoparticle, a co-polymer-based nanoparticle, a carbon-based nanoparticle, and a virus-like nanoparticle.

25. The method according to claim 21, wherein the nanoparticle is an inorganic nanoparticle and the longest dimension of said nanoparticle is less than about 7 nm.

26. The method according to claim 21, wherein the nanoparticle is an inorganic nanoparticle, the longest dimension of said nanoparticle is at least 10 nm, and the inorganic material of the nanoparticle is selected from (i) one or more divalent metallic elements, (ii) one or more trivalent metallic element, and (iii) one or more tetravalent metallic element comprising Si.

27. The method according to claim 26, wherein the inorganic material is selected from calcium carbonate (CaCO.sub.3), magnesium carbonate (MgCO.sub.3), magnesium hydroxide (Mg(OH).sub.2), iron hydroxide (Fe(OH).sub.2), iron oxyhydroxide (FeOOH), iron oxide (Fe.sub.3O.sub.4 or Fe.sub.2O.sub.3), aluminium oxide (Al.sub.3O.sub.4), aluminium hydroxide (Al(OH).sub.3), aluminium oxyhydroxide (AlOOH) and silicium oxide (SiO.sub.2).

28. The method according to claim 21, wherein the nanoparticle is further covered with a biocompatible coating.

29. The method according to claim 21, wherein the carrier is a plain carrier.

30. The method according to claim 21, wherein the carrier is a hollow carrier.

31. The method according to claim 21, wherein the carrier's surface is devoid of any hydrophilic polymer.

32. The method according to claim 21, wherein the carrier's surface is devoid of polyethylene glycol (PEG) polymer.

33. The method according to claim 21, wherein the administration of the at least one biocompatible nanoparticle and of the at least one carrier comprising the pharmaceutical compound(s) maintains the therapeutic benefit of said pharmaceutical compound(s) and reduces toxicity, or increases the therapeutic benefit of said pharmaceutical compound(s) for an equivalent or reduced toxicity, for the subject, when compared to therapeutic benefit and toxicity induced by the standard therapeutic dose(s) of said compound(s) in the absence of any biocompatible nanoparticle and/or carrier.

34. The method according to claim 21, wherein the administration of the at least one biocompatible nanoparticle and of the at least one carrier comprising the pharmaceutical compound(s) allows a reduction of at least 10% of the administered pharmaceutical compound(s) therapeutic dose(s) when compared to the standard therapeutic dose(s) of said compound(s) while maintaining the same therapeutic benefit for an equivalent toxicity or a reduced toxicity for the subject or while increasing the therapeutic benefit for an equivalent or reduced toxicity for the subject in the absence of any biocompatible nanoparticle and/or carrier.

35. The method according to claim 21, wherein the nanoparticle is cleared from the subject to whom it has been administered within one hour and six weeks after its administration to a subject in need of said at least one pharmaceutical compound.

36. The method according to claim 21, wherein the pharmaceutical compound is selected from a small molecule, a targeted small molecule, a cytotoxic compound and a transition metal coordination complex.

37. The method according to claim 21, wherein the pharmaceutical compound is encapsulated in the carrier, impregnated in the carrier or bound to the carrier.

38. The method according to claim 21, wherein the pharmaceutical compound is released from the carrier by temporal-controlled diffusion, carrier erosion and/or carrier degradation.

39. The method according to claim 21, wherein the pharmaceutical compound is released from the carrier in response to an intracellular or an extracellular stimulus.

40. The method according to claim 21, wherein the pharmaceutical compound is released from the carrier when said carrier is exposed to electromagnetic radiation, ultrasound or a magnetic field.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0075] FIG. 1: Schematic representation of carriers devoid of any sterically stabilizing agent comprising at least one compound of interest. The carrier can be a plain carrier (a, b) or a hollow carrier (c, d). The compound of interest is typically entrapped or impregnated (a, c) or grafted (bound) to the carrier with the help of a linker or in the absence of any linker (b, d).

[0076] FIG. 2: a) Schematic representation of a carrier comprising at least one compound of interest. The surface of the carrier is modified by a sterically stabilizing agent. b) schematic representation of a pharmaceutical composition according to the invention comprising the combination of (i) at least one biocompatible nanoparticle and of (ii) at least one carrier comprising at least one compound of interest, the carrier being devoid of any sterically stabilizing agent.

[0077] FIG. 3: Chemical formula of L-Glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA-lipid)

EXAMPLES

Example 1: Synthesis no 1 of Liposomes as Biocompatible Nanoparticles

[0078] Liposomes are prepared using the lipidic film re-hydration method:

a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow. Re-hydration of the lipidic film with HEPES 20 mM and NaCl 140 mM at pH 7.4 is performed at 50° C., so that the lipidic concentration is 5 mM.

[0079] The following lipidic composition was used to prepare charged liposomes: DPPC (DiPalmitoylPhosphatidylCholine): 86% mol; MPPC (MonoPalmitoylPhosphatidylcholine): 10% mol; DSPE-PEG (DiStearylPhosphatidylEthanolamine-[methoxy(PolyElthyleneGlycol)-2000]): 4% mol.

b) Freeze-thaws cycles are then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 50° C.
c) A thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) was used to calibrate the size of the liposomes under controlled temperature and pressure. In all cases, extrusion was performed at 50° C., under a pressure of 10 bars.

[0080] Size distribution of the as-prepared liposomes was determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 90° C. The liposomes suspension was diluted 100 times in HEPES 20 mM and NaCl 140 mM at pH 7.4. Liposome size (i.e. hydrodynamic diameter) was equal to about 170 nm (distribution by intensity) with a polydispersity index (PDI) equal to about 0.1.

[0081] As understandable by the skilled person, the desired surface charge was obtained thanks to the selected lipidic composition, and its value is confirmed by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument).

[0082] The liposomes were diluted 100 times in water and the pH of the resulting suspension was adjusted to pH 7.4. The liposome surface charge was equal to about −14 mV at pH 7.4.

Example 2: Synthesis n° 2 of Liposomes as Biocompatible Nanoparticles

[0083] Liposomes are prepared using the lipid film re-hydration method:

a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow. Re-hydration of the lipid film with HEPES 20 mM and NaCl 140 mM at pH 7.4 is performed at 65° C., so that the lipid concentration is 25 mM.

[0084] The following lipid composition was used to prepare liposomes: DSPC (DiStearoylPhosphatidylCholine): DSPG (DiStearoylPhosphatidylGlycerol): CHOL (Cholesterol) in a 7:2:1 molar ratio.

b) Freeze-thaw cycles are then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 65° C.
c) A thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) was used to calibrate the size of the liposomes under controlled temperature and pressure. First, 5 passages were performed through a polyethersulfone (PES) 0.45 μm-pores sized membrane at 5 bars, then 10 passages through a PES 0.22 μm-pores sized membrane at 10 bars, and finally 10 passages through a polyvinylidene fluoride (PVDF) 0.1 μm-pores sized membrane at 15 bars.

[0085] Size distribution of the as-prepared liposomes was determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 90° C. The liposomes suspension was diluted 100 times in HEPES 20 mM and NaCl 140 mM at pH 7.4. Liposome size (i.e. hydrodynamic diameter) was equal to about 145 nm (distribution by intensity) with a polydispersity index (PDI) equal to about 0.1. The desired surface charge, which is typically below −10 mV, was obtained thanks to the selected lipidic composition, and its value is confirmed by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument).

Example 3: Method Allowing an Improved Efficacy and/or a Reduced Toxicity Following the Administration to a Subject of a Compound of Interest Included in the Pharmaceutical Composition According to the Invention when Compared to the Same Dose of the Compound of Interest Alone

[0086] A pharmaceutical composition according to claim 1 comprising the combination of (i) at least one biocompatible nanoparticle and of (ii) at least one carrier comprising doxorubicin, is administered in nude mice bearing a MDA-MB-231-lucD3H2LN xenografted tumor in the following manner: [0087] a)—administering to a first group of nude mice (by intra venous injection) the Dox-NP® (a PEGylated liposomal formulation of doxorubicine); [0088] administering to a second group of nude mice (by intra venous injection) the doxorubicine; [0089] administering to a third group of nude mice (by intra venous injection) the biocompatible nanoparticles; [0090] administering to a fourth group of nude mice (by intra venous injection) the biocompatible nanoparticles and, between more than 5 minutes and 72 hours following the administration of the biocompatible nanoparticles to the fourth group of nude mice, administering (by intra venous injection) to said fourth group of nude mice a carrier comprising the doxorubicin wherein the carrier is devoid of any sterically stabilizing agent; [0091] b) assessing any clinical sign of toxicity in nude mice after the administration of the Dox-NP® (first group), the doxorubicin (second group), the biocompatible nanoparticles (third group) and the pharmaceutical composition (fourth group); and [0092] c) measuring the tumor re-growth delay after the administration of the Dox-NP® (first group), the doxorubicin (second group) the biocompatible nanoparticles (third group) and the pharmaceutical composition (fourth group).

Example 4: Synthesis n° 3 of Liposomes as Biocompatible Nanoparticles

[0093] Liposomes are prepared using the lipid film re-hydration method: [0094] a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow to form a lipid film on the Pyrex tube walls. Re-hydration of the lipid film with HEPES 25 mM and NaCl 150 mM at pH 7.4 is performed at 60° C., so that the lipid concentration is 50 mM.

[0095] The following lipid composition was used to prepare charged liposomes: DPPC (DiPalmitoylPhosphatidylCholine) 58% mol; HSPC (Hydrogenated Soybean PhosphatidylCholine) 21% mol; CHOL (Cholesterol) 16% mol; POPS (1-Palmitoyl-2-Oleoyl PhosphatidylSerine) 5% mol. [0096] b) Freeze-thaw cycles are then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C. Ultra-sonication of the liposomes solution is performed during 30 s every 3 freeze-thaw cycles and just before extrusion. [0097] c) A thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) is used to calibrate the size of the liposomes under controlled temperature and pressure. Extrusion is performed at 60° C. Ten passages are applied through a 0.11 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 10 bars.

[0098] Size distribution of the as-prepared liposomes is determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 173° C. The liposomes solution is diluted 200 times in HEPES 25 mM and NaCl 150 mM at pH 7.4. Liposomes size (i.e. hydrodynamic diameter) is equal to about 170 nm (distribution by intensity) with a polydispersity index (PdI) equal to about 0.2.

[0099] As understandable by the skilled person, the desired surface charge is obtained thanks to the selected lipid composition, and its value is confirmed by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument). The liposomes are diluted 200 times in a sodium chloride solution at 1 mM and the pH of the solution is adjusted to pH 7. The liposomes surface charge is equal to about −40 mV at pH 7, NaCl 1 mM.

[0100] The final lipid concentration of the liposomes solution is measured by a colorimetric assay (Bartlett method). The method is based on total phosphorus determination through an acidic digestion of phospholipid. The released inorganic phosphate is reacted with ammonium molybdate, the complex giving a strong blue color. Lipids concentration is equal to about 50 mM.

Example 5: Synthesis n° 4 of Liposomes as Biocompatible Nanoparticles

[0101] Liposomes are prepared using the lipid film re-hydration method: [0102] a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow to form a lipid film on the Pyrex tube walls. Re-hydration of the lipid film with HEPES 25 mM and NaCl 150 mM at pH 7.4 is performed at 60° C., so that the lipid concentration is 50 mM.

[0103] The following lipid composition was used to prepare the charged liposomes: DPPC (DiPalmitoylPhosphatidylCholine) 45.15% mol; CHOL (Cholesterol) 45.15% mol; DSPE-PEG (DiStearylPhosphatidylEthanolamine-[methoxy(PolyElthyleneGlycol)-2000]) 0.60% mol; L-Glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA-lipid) 9.10% mol. The SA-lipid brings COOH groups on the liposomes surface. [0104] b) Freeze-thaw cycles are then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C. [0105] c) A thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) is used to calibrate the size of the liposomes under controlled temperature and pressure. Extrusion is performed at 60° C. Seven passages are applied through a 0.451 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 3 bars and ten passages through a 0.221 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 10 bars. Size distribution of the as-prepared liposomes is determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 173° C. The liposomes solution is diluted 200 times in HEPES 25 mM and NaCl 150 mM at pH 7.4. Liposomes size (i.e. hydrodynamic diameter) is equal to about 230 nm (distribution by intensity) with a polydispersity index (PdI) equal to about 0.2.

[0106] As understandable by the skilled person, the desired surface charge is obtained thanks to the selected lipid composition, and its value is confirmed by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument). The liposomes solution is diluted 200 times in a sodium chloride solution at 1 mM and the pH of the solution is adjusted to pH 7.

[0107] The liposomes surface charge is equal to about −60 mV at pH 7, NaCl 1 mM. The final lipid concentration of the liposomes solution is measured by a colorimetric assay (Bartlett method). The method is based on total phosphorus determination through an acidic digestion of phospholipid. The released inorganic phosphate is reacted with ammonium molybdate and the complex giving a strong blue color. Lipids concentration is equal to about 50 mM.

Example 6: Synthesis n° 5 of Liposomes as Biocompatible Nanoparticles

[0108] Liposomes are prepared using the lipid film re-hydration method: [0109] a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow to form a lipid film on the Pyrex tube walls. Re-hydration of the lipid film with 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 prepare the charge 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. [0110] b) Freeze-thaw cycles are then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C. Ultra-sonication of the liposomes solution is performed during 30 s, every 3 freeze-thaw cycles and just before extrusion. [0111] c) A thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) is used to calibrate the size of the liposomes under controlled temperature and pressure. Extrusion is performed at 60° C. Twelve passages are applied through a 0.22 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 12 bars. [0112] d) Conjugation of p-aminophenyl-α-D-mannopyranoside (MAN) to Succinyl PE liposome:

[0113] The succinyl PE liposome surface are modified with a mannose derived ligand p-aminophenyl-α-D-mannopyranoside (MAN), using carbodiimide coupling to develop mannose conjugated liposome. MAN is covalently coupled by its amino group to the carboxylic acid group of Succinyl PE, present on the surface of preformed Succinyl PE liposome. Briefly, to the preformed Succinyl PE liposome solution are added EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride), (Succinyl PE/EDC 1:10 molar ratio) and N-hydroxysuccinimide (NHS) (NHS/EDC 1:2.5 molar ratio). The pH of the suspension is then adjusted at 6 with NaOH 1M and the resulting suspension is stirred for 15 minutes at room temperature. Subsequently, the pH of the solution is adjusted at 7 with NaOH 1M and the aqueous MAN solution is added (Succinyl PE/MAN 1:2 molar ratio) to the solution. pH is readjusted at 7 using NaOH 1M and the suspension is stirred for 2 additional hours at room temperature. Excessive unbound MAN, EDC and NHS molecules are removed by 3 steps of dialysis with dilution factor (×500; ×500; ×500) using a 50 KDa cellulose membrane.

[0114] Of note, due to possible dilution upon dialysis, the liposomes solution can be concentrated by centrifugation (typically a Sigma 3-15K centrifuge at 5° C.; 1,200 rpm) using membrane ultrafiltration on Vivaspin concentrators with a polyethylene sulfone (PES) membrane and a cut-off 300 KDa.

[0115] Size distribution of the as-prepared liposomes is determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 173° C. The liposomes solution is diluted 200 times in HEPES 25 mM and NaCl 150 mM at pH 7.4. Liposomes size (i.e. hydrodynamic diameter) is about 230 nm (distribution by intensity) with a polydispersity index (PDI) around 0.2. As understandable by the skilled person, the desired surface charge is obtained thanks to the selected lipid composition, and its value is confirmed by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument). The liposomes solution is diluted 200 times in a sodium chloride solution at 1 mM and at pH 7. The liposomes surface charge is around −70 mV at NaCl 1 mM, pH 7. The final lipid concentration of the liposomes solution is measured by a colorimetric assay (Bartlett method). The method is based on total phosphorus determination through an acidic digestion of phospholipid. The released inorganic phosphate is reacted with ammonium molybdate and the complex giving a strong blue color. Lipids concentration is equal to about 50 mM.