Pharmaceutical composition, preparation and uses thereof

Abstract

The present invention relates to a pharmaceutical composition comprising the combination of (i) a biocompatible nanoparticle and of (ii) a pharmaceutical compound of interest, to be administered to a subject in need of such a compound of interest, wherein the nanoparticle potentiates the compound 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 invention also relates to such a composition for use for administering the compound of interest to a subject in need thereof, wherein the nanoparticle and the compound of interest are to be administered to said subject between more than 5 minutes and about 72 hours from each other.

Claims

1. A method comprising a step of intravenously administering a pharmaceutical compound of interest to a subject in need thereof and a distinct step of intravenously administering a biocompatible nanoparticle to said subject, wherein the longest dimension of the biocompatible nanoparticle is between about 4 nm and about 500 nm, and the surface charge value of the biocompatible nanoparticle is a negative charge below −10 mV, said nanoparticle being intravenously administered to the subject between more than 5 minutes and about 72 hours before the pharmaceutical compound of interest, and the biocompatible nanoparticle is an organic nanoparticle free of an additional therapeutic, prophylactic or diagnostic agent, and wherein the combined intravenous administration of the biocompatible nanoparticle and the compound of interest maintains the therapeutic benefit of the compound of interest with reduced toxicity, or increases the therapeutic benefit of the compound of interest with equivalent or reduced toxicity for the subject when compared to therapeutic benefit and toxicity induced by the standard therapeutic dose of said compound of interest.

2. The method according to claim 1, wherein the nanoparticle is selected from a lipid-based nanoparticle, a protein-based nanoparticle, a polymer-based nanoparticle, a copolymer-based nanoparticle, a carbon-based nanoparticle, and a virus-like nanoparticle.

3. The method according to claim 1, wherein the nanoparticle is further covered with a biocompatible coating.

4. The method according to claim 1, wherein the combined intravenous administration of the biocompatible nanoparticle and the compound of interest allows for a reduction of at least 10% of the administered compound therapeutic dose when compared to the standard therapeutic dose of said compound of interest while maintaining the same therapeutic benefit with equivalent or reduced toxicity for the subject, or while increasing the therapeutic benefit with equivalent or reduced toxicity for the subject.

5. The method according to claim 1, wherein the nanoparticle is cleared from the subject to whom it has been administered within one hour and six weeks after its administration.

6. The method according to claim 1, wherein the compound of interest is an organic compound.

7. The method according to claim 6, wherein said organic compound is a biological compound, a small-molecule targeted therapeutic, or a cytotoxic compound.

8. The method according to claim 6, wherein the compound of interest is selected from an antibody, an oligonucleotide, and a synthesized peptide.

9. The method according to claim 1, wherein the compound of interest is an inorganic compound selected from a metallic nanoparticle, a metal oxide nanoparticle, a metal sulfide nanoparticle or any mixture thereof.

10. The method according to claim 1, wherein the compound of interest is encapsulated in a carrier.

11. The method according to claim 1, wherein the compound of interest is bound to a carrier.

12. The method according to claim 1, wherein the nanoparticle is administered to the subject between more than 5 minutes and about 24 hours before the pharmaceutical compound of interest.

13. The method according to claim 1, wherein the nanoparticle has a surface charge value below −12 mV.

14. The method according to claim 1, wherein the nanoparticle has a surface charge value below −15 mV.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Schematic view of possible routes for therapeutic compounds removal from blood circulation depending on the compound's size (longest dimension).

(2) FIG. 2: Schematic representation of the treatment schedule for the pharmaceutical composition comprising (i) the biocompatible nanoparticles of Example 3 and (ii) the Dox-NP® in MDA-MB-231-lucD3H2LN xenografts.

(3) FIG. 3: Tumor re-growth delay of the pharmaceutical composition comprising the biocompatible nanoparticles of Example 3 and the Dox-NP® in MDA-MB-231-lucD3H2LN xenografts (mean RTV±SD).

EXAMPLES

Example 1: Synthesis No. 1 of Liposomes as Biocompatible Nanoparticles

(4) Liposomes are prepared using the lipidic film re-hydration method:

(5) 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.

(6) The following lipidic composition was used to prepare charged liposomes: DPPC

(7) TABLE-US-00001 (DiPalmitoylPhosphatidylCholine): 86% mol; MPPC (MonoPalmitoylPhosphatidylcholine): 10% mol; DSPE-PEG (DiStearylPhosphatidylEthanolamine-[methoxy(PolyElthyleneGlycol)-2000]): 4% mol.
b) Freeze-thaw 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.

(8) Size distribution of the as-prepared liposomes was determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern Instruments) with a 633 nm HeNe laser at an angle of 90° C. The liposome 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 with a polydispersity index (PDI) equal to about 0.1.

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

(10) 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: Method Allowing a Reduction of at Least 10% of the Dose of Therapeutic Compound to be Administered to a Subject for an Equivalent Therapeutic Efficacy Thereof in the Subject

(11) A pharmaceutical composition according to claim 1 comprising a biocompatible nanoparticle and an activable oxide nanoparticle for anti-cancer therapy (used as “the compound” or “pharmaceutical compound”) which can generate electron and/or high energy photon when exposed to ionizing radiations such as X-rays, is administered to nude mice bearing a xenografted tumor in the following manner: a) administering to each nude mouse (by intravenous injection) the biocompatible nanoparticles; b) between more than 5 minutes and 72 hours following step a), administering (by intra venous injection) the therapeutic compound to each mouse of step a) at a lower dose (10%) when compared to the dose currently used; c) measuring the therapeutic compound concentration in blood or plasma samples of each mouse to obtain the pharmacokinetic parameters of the therapeutic compound, said concentration being measured once or preferably several times between 1 minute and 24 hours following the therapeutic compound administration; d) assessing any clinical sign of toxicity after the administration of the pharmaceutical composition; and e) measuring the tumor accumulation of the therapeutic compound 24 hours after its intravenous (IV) administration.

Example 3: Synthesis No. 2 of Liposomes as Biocompatible Nanoparticles

(12) Liposomes are prepared using the lipid film re-hydration method:

(13) 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 60° C., so that the lipid concentration is 25 mM.

(14) The following lipid composition was used to prepare charged liposomes: DPPC (DiPalmitoylPhosphatidylCholine) 62% mol; HSPC (Hydrogenated Soybean PhosphatidylCholine) 20% mol; CHOL (Cholesterol) 16% mol; POPS (1-Palmitoyl-2-Oleoyl Phosphatidyl Serine) 1% mol; DSPE-PEG (Di StearylPhosphatidylEthanolamine-[methoxy(PolyElthyleneGlycol)-2000]) 1% mol.

(15) 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.

(16) 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 60° C., under a pressure of 5 bars, with a 0.1 μm pore size polyvinylidene fluoride (PVDF) membrane.

(17) Size distribution of the as-prepared liposomes was determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern Instruments) with a 633 nm HeNe laser at an angle of 90° C. The liposome 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 with a polydispersity index (PDI) equal to about 0.1.

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

(19) The liposomes were diluted 100 times in a sodium chloride solution at 1 mM and the pH of the resulting suspension was adjusted to pH 7.4. The liposomes' surface charge was equal to about −25 mV at pH 7.4, NaCl 1 mM.

Example 4: Tumor Re-Growth Delay of the Pharmaceutical Composition Comprising the Biocompatible Nanoparticle Suspension of Example 3 and the Dox-NP® in MDA-MB-231-lucD3H2LN Xenografts (FIGS. 2 and 3)

(20) This study was performed to investigate the efficacy of the pharmaceutical composition comprising (i) the biocompatible nanoparticle from Example 3 and (ii) Dox-NP® (Liposomal Encapsulated Doxorubicin) as the therapeutic compound of interest, in MDA-MB-231-luc-D3H2LN tumor model xenografted on NMRI nude mice.

(21) The human breast adenocarcinoma MDA-MB-231-luc-D3H2LN cell line was purchased from Caliper Life Science (Villepinte, France). The cells were cultured in Minimum Essential Medium with Earle's Balanced Salt Solution (MEM/EBSS) medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% L-glutamine, and 1% sodium pyruvate (Gibco).

(22) NMRI nude mice, 6-7 weeks (20-25 g) were ordered from Janvier Labs (France). Mice were subjected to a total body irradiation of 3Gy with the Cesium-137 irradiation device one day before the inoculation of the cancer cells for xenograft.

(23) MDA-MB-231-luc-D3H2LN tumors were obtained by subcutaneous injection of 4×10.sup.6 cells in 50 μL in the lower right flank of the mouse. The tumors were grown until reaching a volume around about 100 mm.sup.3. Tumor diameter was measured using a digital caliper and the tumor volume in mm.sup.3 was calculated using the formula:

(24) $\mathrm{Tumor}\mathrm{volume}\left({\mathrm{mm}}^3\right)=\frac{\mathrm{length}\left(\mathrm{mm}\right)\times {\left(\mathrm{width}\right)}^2\left({\mathrm{mm}}^2\right)}{2}$

(25) Mice were randomized into separate cages and identified by a number (paw tattoo). Four groups were treated as illustrated in FIG. 2.

(26) Group 1: Sterile Glucose 5% (Control (Vehicle) Group)

(27) Four (4) mice were intravenously (IV) injected with a sterile glucose 5% solution on day 1, day 7 and day 14. Each time (day), two injections of glucose 5% were performed. The first injection of glucose 5% solution was performed 4 hours before the second injection.

(28) Group 2: Biocompatible Nanoparticles from Example 3 (Control Group)

(29) Four (4) mice were intravenously (IV) injected with a sterile glucose 5% solution and the biocompatible nanoparticles from Example 3 (10 ml/kg) on day 1, day 7 and day 14. Each time (day), the injection of biocompatible nanoparticles from Example 3 was performed 4 hours before injection of the glucose 5% solution.

(30) Group 3: Dox-NP® (3 mg/kg Doxorubicin) (Treatment Group)

(31) Five (5) mice were intravenously (IV) injected with a sterile glucose 5% solution and Dox-NP® (3 mg/kg doxorubicin) on day 1, day 7 and day 14. Each time (day), the injection of sterile glucose 5% solution was performed 4 hours before the injection of Dox-NP® (3 mg/kg doxorubicin).

(32) Group 4: Pharmaceutical Composition, i.e. the Combination of (i) the Biocompatible Nanoparticles from Example 3 and of (ii) Dox-NP® (3 mg/kg Doxorubicin) (Treatment Group)

(33) Five (5) mice were intravenously (IV) injected with the biocompatible nanoparticles from Example 3 (10 ml/kg) and with the Dox-NP® (3 mg/kg doxorubicin) on day 1, day 7 and day 14. Each time (day), the injection of biocompatible nanoparticles from Example 3 was performed 4 hours before the injection of Dox-NP® (3 mg/kg doxorubicin).

(34) The Dox-NP® (Avanti Polar Lipids; liposomal formulation of 2 mg/ml doxorubicin HCl at pH 6.5-6.8, in 10 mM histidine buffer, with 10% w/v sucrose) was injected without additional dilution at a volume required to obtain 3 mg/kg of injected doxorubicin.

(35) The biocompatible nanoparticle suspension from Example 3 was used without any additional dilution.

(36) The Dox-NP® and the biocompatible nanoparticles from Example 3 were administrated by intravenous injection (IV) via lateral tail vein with a 100 U (0.3 ml) insulin syringe (Terumo, France).

(37) Mice were followed up for clinical signs, body weight and tumor size.

(38) The tumor volume was estimated from two dimensional tumor volume measurements with a digital caliper using the following formula:

(39) $\mathrm{Tumor}\mathrm{volume}\left({\mathrm{mm}}^3\right)=\frac{\mathrm{length}\left(\mathrm{mm}\right)\times {\left(\mathrm{width}\right)}^2\left({\mathrm{mm}}^2\right)}{2}$

(40) In each group, the relative tumor volume (RTV) was expressed as Vt/V.sub.0 ratio (Vt being the tumor volume on a given day during the treatment and V.sub.0 being the tumor volume at the beginning of the treatment).

(41) The treatment efficacy was determined using the specific growth delay (SGD) over two doubling times (one doubling time being the amount of time it takes for the tumor to double in volume) and the optimal percent T/C value (% T/C).

(42) The SGD was calculated over two doubling times as follows:

(43) $\mathrm{SGD}=\frac{T4d\mathrm{treated}-T4d\mathrm{control}}{T4d\mathrm{control}}$$\mathrm{with}T4d\mathrm{being}\mathrm{the}\mathrm{time}\mathrm{required}\mathrm{for}\mathrm{the}\mathrm{tumor}\mathrm{to}\mathrm{double}\mathrm{twice}\mathrm{in}\mathrm{volume}\left(\mathrm{mean}\mathrm{RTV}\mathrm{from}100{\mathrm{mm}}^3\mathrm{up}\mathrm{to}400{\mathrm{mm}}^3\right).$

(44) The Percent T/C value (“% T/C”) was calculated by dividing the median of the relative tumor volume of treated groups (groups 2, 3, 4) versus control group (group 1) at days 1, 3, 7, 10, 13, 15, 18, 21 and 24, and by multiplying the result of said division by 100 (see Table 2). The lowest % T/C values obtained within 2 weeks following treatment injection (with or without biocompatible nanoparticles as used in the context of the present invention) correspond to the optimal % T/C values.

(45) FIG. 3 shows the mean relative tumor volume (mean RTV) for all groups as obtained (in the conditions previously described) after IV injections of: vehicle (sterile glucose 5%) on days 1, 7 and 14 (group 1); biocompatible nanoparticles from Example 3, 4 hours prior to each vehicle (sterile glucose 5%) injection on days 1, 7 and 14 (group 2); Dox-NP® (3 mg/kg doxorubicin) on days 1, 7 and 14 (group 3); or biocompatible nanoparticles from Example 3, 4 hours prior to the Dox-NP® (3 mg/kg doxorubicin) injection on days 1, 7 and 14 (group 4).

(46) As shown in FIG. 3, a marked tumor growth inhibition is observed after the first injection of the pharmaceutical composition comprising the combination of (i) the biocompatible nanoparticles from Example 3 and (ii) the Dox-NP® (3 mg/kg doxorubicin), when compared to the Dox-NP® (3 mg/kg doxorubicin) alone.

(47) The time required (expressed in days) for each tumor to double twice in volume (T4d) was calculated (as a measurement of the duration of the treatment effects). T4d for the pharmaceutical composition was estimated to about 31 days versus about 14 days for the Dox-NP® alone (Table 1). In addition the Specific Growth Delay (SGD) estimated from the tumors growth over two doubling time (starting from a mean RTV of 100 mm.sup.3 up to 400 mm.sup.3) was equal to about 2 for the pharmaceutical composition versus about 0 for the Dox-NP® alone (Table 1).

(48) TABLE-US-00002 TABLE 1 Table 1: Time for the tumor to double twice in volume (T4d) and Specific Growth Delay (SGD) estimated from the tumors growth over two doubling times. Td4 represents the number of days to reach two doubling times (mean RTV from 100 mm.sup.3 up to 400 mm.sup.3). The control group is the vehicle (glucose 5%) alone (-). T4d (in days) between 100 and Groups 400 mm.sup.3 (mean RTV) SGD Group 1: vehicle (control group) 11 — Group 2: Biocompatible nanoparticles 11 0 from Example 3 Group 3: Dox-NP ® alone (3 mg/Kg) 14 0 Group 4: Pharmaceutical composition 31 2 comprising (i) the biocompatible nanoparticle from Example 3 and (ii) Dox-N ® (3 mg/Kg)

(49) Furthermore, the percent T/C (% T/C) (calculated until the day of sacrifice of group 1) decreased faster for the pharmaceutical composition than for Dox-NP® alone. This demonstrates a marked impact of the pharmaceutical composition. The optimal % T/C of 25 observed at day 24 was indeed obtained for the pharmaceutical composition, i.e. the combination of (i) the biocompatible nanoparticles from Example 3 and (ii) the Dox-NP® (3 mg/kg doxorubicin), whereas the optimal % T/C of 38 observed at day 21 was obtained for the group Dox-NP® alone (Table 2).

(50) TABLE-US-00003 TABLE 2 Table 2: percent T/C (% T/C) is calculated by dividing the median of the relative tumor volume of treated groups (groups 2, 3, 4) versus control group (group 1) at days 1, 3, 7, 10, 13, 15, 18, 21 and 24, and by multiplying the result of said division by 100. Control group is group 1 (vehicle sterile glucose 5% alone). % T/C is calculated until day 24 which corresponds to the day of sacrifice of group 1 (control group). Optimal % T/C is indicated for each group as **. Group 4: Pharmaceutical composition comprising (i) the Group 2: biocompatible Group 3: Dox-NP ® biocompatible nanoparticle and (ii) Days nanoparticles alone alone (3 mg/kg) Dox-NP ® (3 mg/Kg) 1 100 100 100 3 104 126 121 7  90 106  80 10    87 **  76  60 13 103  80  55 15  98  74  45 18  98  56  43 21  87    38 **  33 24  98  40    25 **

(51) Overall, those results showed an advantageous tumor growth delay when using the pharmaceutical composition of the present invention [corresponding to the combination of (i) the biocompatible nanoparticles from Example 3 and of (ii) the Dox-NP® (3 mg/kg doxorubicin)], which is not observed when the Dox-NP® (3 mg/kg doxorubicin) is used alone (i.e. in the absence of the biocompatible nanoparticles used in the context of the present invention). This tumor growth delay was observed when the biocompatible nanoparticles from Example 3 and the compound of interest (the Dox-NP®) were administered sequentially, the biocompatible nanoparticle being administered to the subjects 4 hours before the Dox-NP®.

(52) Inventors are reproducing this experiment to confirm that the same result is observed so long as the compound of interest and the biocompatible nanoparticles are administered to the subject between more than 5 minutes and about 72 hours from each other.