Formulas for diagnosing and treating hormone-dependent cancers and cancers of the organs responsible for steroid hormone synthesis

Abstract

The present invention relates to a formulation of a therapeutic or diagnostic agent in the form of a nano-emulsion, to its preparation method and to the use of this formulation for treating or diagnosing hormone-dependent cancers or cancers of organs synthesizing steroidal hormones.

Claims

1. A formulation of a therapeutic or diagnostic agent in the form of a nano-emulsion, comprising a continuous aqueous phase and at least one dispersed oily phase, wherein: the oily phase comprises: at least one amphiphilic lipid, at least one solubilizing lipid, at least one compound of the following formula (I):
(R-L).sub.n-A(I), wherein : n represents an integer from 1 to 5 L represents a simple bond or a divalent group selected from O, COO, OOC, CONR, NRCO, S, NRCONR, OCOO, wherein R and R represent independently H or a linear or branched alkyl with 1 to 20 carbon atoms and R represents a linear or branched alkyl comprising at least 11 carbon atoms or a linear or branched alkenyl comprising at least 11 carbon atoms, and A represents a steriodic or sterolic group, and the aqueous phase includes at least one co-surfactant comprising at least one chain consisting of alkylene oxide units; the proportion of the compound of formula (I) being from 0.05% to 3% by weight based on the weight of the dispersed oily phase, the weight of the dispersed oily phase including the weight of the co-surfactant.

2. The formulation of a therapeutic or diagnostic agent according to claim 1, wherein the oily phase comprises at least one therapeutic agent for treating hormone-dependent cancers or cancers of organs synthesizing steroidal hormones, or one diagnostic agent.

3. The formulation of a therapeutic or diagnostic agent according to claim 1, wherein the group A is a group having one of the following formulae: ##STR00005## wherein one or several of the carbon atoms are substituted with an -L-R group as defined in claim 1.

4. The formulation of a therapeutic or diagnostic agent according to claim 1, wherein L represents O or COO.

5. The formulation of a therapeutic or diagnostic agent according to claim 1, wherein the amphiphilic lipid is a phospholipid.

6. The formulation of a therapeutic or diagnostic agent according to claim 1, wherein the solubilizing lipid comprises at least one glyceride of fatty acids.

7. The formulation of a therapeutic or diagnostic agent according to claim 6, wherein the solubilizing lipid comprises a mixture of saturated fatty acid glycerides including: at least 10% by weight of C12 fatty acids, at least 5% by weight of C14 fatty acids, at least 5% by weight of C16 fatty acids, and at least 5% by weight of C18 fatty acids.

8. The formulation of a therapeutic or diagnostic agent according to claim 1, wherein the oily phase further includes at least one oil.

9. The formulation of a therapeutic or diagnostic agent according to claim 1, wherein the co-surfactant includes at least one chain consisting of ethylene oxide or ethylene oxide and propylene oxide units.

10. The formulation of a therapeutic agent according to claim 1, comprising a therapeutic agent for treating hormone-dependent cancers or cancers of organs synthesizing steroidal hormones selected from the group consisting of: agonists of gonadotropin releasing hormone, antagonists of aromatase, anti-estrogens, and anti-androgens.

11. The formulation of a diagnostic agent according to claim 1, wherein the diagnostic agent is selected from the group consisting of a liphophilic fluorophore, fatty acid analogs, phospholipids functionalized with a fluorescent group, amphiphilic derivatives of dialkylcarbocyanines, fluorescent probes derived from sphingolipids, steroids or lipopolysaccharides, amphiphilic derivatives of cyanines, rhodamines, fluoresceins or cumarins, and diphenylhexatriene and its derivatives.

12. A method for preparing a formulation of a therapeutic or diagnostic agent in the form of a nano-emulsion according to claim 1, comprising at least one continuous aqueous phase and at least one dispersed oily phase, comprising: (i) preparing the oily phase comprising at least one solubilizing lipid, at least one amphiphilic lipid, optionally at least one therapeutic or diagnostic agent and at least one compound of formula (I); (ii) preparing an aqueous phase comprising a polyalkoxylated co-surfactant; (iii) dispersing the oily phase in the aqueous phase under the action of sufficient shearing for forming a nano-emulsion; and (iv) recovering the thereby formed nano-emulsion.

13. A therapeutic treatment method for hormone-dependent cancers, or cancers of organs synthesizing steroidal hormones comprising administering to a mammal in need thereof a therapeutically effective amount of the formulation of a therapeutic agent according to claim 1.

14. A method for diagnosing hormone-dependent cancers or cancers of organs synthesizing steroidal hormones comprising administering to a subject a formulation of a diagnostic agent according to claim 1.

15. The formulation of a therapeutic agent according to claim 10, comprising a therapeutic agent for treating hormone-dependent cancers or cancers of organs synthesizing steroidal hormones selected from the group consisting of estrogen, progestins, Formestane, Exemestane, Aminoglutethimide, Anastrozole, Fadrozole, Letrozole, Megestrol acetate, Medroxyprogesterone acetate, Mifepristone, Tamoxifene, Fulvestrant, abiraterone acetate and cyproterone acetate.

16. The formulation of a diagnostic agent according to claim 11, wherein the diagnostic agent is indocyanine green or 1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine perchlorate.

17. The therapeutic treatment method according to claim 13, wherein the method is used to treat breast cancer, prostate cancer, cancer of the ovaries, or cancer of the adrenal glands.

18. The method for diagnosing according to claim 14, wherein the method is used to diagnose breast cancer, prostate cancer, cancer of the ovaries, or cancer of the adrenal glands.

Description

EXAMPLES

Example 1

Preparation of Formulations According to the Invention

(1) The following preparation method was followed: (i) Preparation of the Oily Phase:
Soy oil, Suppocire NC and lecithin were weighed and then mixed with dichloromethane before being heated to 60 C. in order to obtain a homogeneous viscous solution. Dichloromethane gives the possibility of promoting solubilization. The dichloromethane is then evaporated in vacuo. The compound(s) of formula (I) was (were) then added into the obtained oily phase. (ii) Doping the Oily Phase:
A solution of the diagnostic agent (fluorophore) in ethanol was incorporated to the oily phase and the ethanol was then evaporated under a nitrogen flow for 5 mins. The mixture was maintained viscous via a water bath at 40 C./50 C. (iii) Preparation of the Aqueous Phase:
During the phase for evaporating the ethanol, the aqueous phase was prepared. In a 5 ml Eppendorf, the co-surfactant, the glycerol and the aqueous PBS solution were mixed and then dissolved in a bath at 75 C. (iv) Mixing Both Phases:
The oily phase was at about 40 C. (in viscous form) and the aqueous phase at about 70 C. (at the outlet of the bath). The aqueous phase was poured into the oily phase. (v) Emulsification:
The flask containing both phases was attached in the sonication chamber. The base of the flask (about 1 cm) was positioned in a water bath at 15 C. The conical sonotrode is immersed in the flask, the latter being placed at half-height (over about 1 cm in the solution) for better homogeneity for making the emulsion. Sonication was then carried out with an AV505 Sonicator (Sonics, Newtown USA). The effective sonication duration was 10 mins. (vi) Purification:
Purification was carried out by dialysis in a large volume of PBS 1 overnight. The glycerol is removed during this step.
The purified nano-emulsion sample was taken and the volume was adjusted by adding sterile PBS 1 to the desired volume (13 ml). The whole of the sample was sterilized by filtration on a 0.22 m filter.

Example 1.1.

A trebly Labelled Formulation Comprising Two Compounds of Formula (I) (Labelled Cholesteryl Oleate and Labelled Cholesteryl Hexadecyl Ether) and a Fluorophore as a Diagnostic Agent (DiD)

(2) A formulation of the following composition was prepared:

(3) TABLE-US-00003 TABLE 1 Composition of the formulation of Example 1.1. % by mass of the dispersed phase (the co-surfactant Amount belonging to Raw (2 ml % by mass of the total the dispersed material batch) formulation phase) oily amphiphilic Lipoid 17 mg 0.85 i.e. i.e. oily 8.42% phase lipid 5.4% pre solubilizing Suppocire 68 mg 3.4 mix = 33.66% lipid 5.50% oil Soy oil 23 mg 1.15 11.39% compound [3H]CHE 348 Ci 0.045 i.e. 0.45% of formula 0.10% (I) compound [14C]CO 88 Ci 0.015 0.15% of formula (I) diagnostic DiD 400 M 0.04 0.40% agent aqueous co- PEG 92 mg 4.6 45.53% phase surfactant aqueous PBS 1X 1800 L 90 solution Dispersed 202.22 mg 10.1 100% phase i.e. (rounded 100.11 mg/ml to 10) (rounded to 100 mg/ml)

(4) DiD is a fluorophore which may be detected by fluorescence.

(5) The compounds [3H]CHE and [14C]CO are labelled cholesteryl hexadecylether and labelled cholesteryl oleate respectively and respectively have the following formulae:

(6) ##STR00004##

(7) As these compounds of formula (I) are labelled, they may be localized by measuring radioactivity.

(8) The localization of the nano-emulsion of Example 1.1. may therefore be monitored by fluorescence or radioactivity.

Example 1.2.

A Formulation Comprising Cholesteryl Stearate as a Compound of Formula (I) and a Fluorophore as a Diagnostic Agent (DiD)

(9) Formulations were prepared with the same compositions as those of Table 1, except that [3H]CHE and [14C]CO are replaced with:

(10) 0.6 wt % based on the total of the dispersed phase (considering that the co-surfactant belongs to the dispersed phase) of cholesteryl stearate (Example 1.2.1), or 2 wt % based on the total of the dispersed phase (considering that the co-surfactant belongs to the dispersed phase) of cholesteryl stearate (Example 1.2.2).
Cholesteryl stearate is designated by CHST hereafter.

(11) In the following examples, the radioactivity was counted with the TRiCarb Counter (Ref: Packard, Liquid Scintillation Analyzers; Models 2200CA).

(12) Fluobeam 700 (Fluoptics) is the apparatus with which the whole of the organs from the biodistribution were analyzed in fluorescence as well as the one with which the biodistribution kinetics of the nano-emulsions were established in vivo. This imaging system allows viewing of the fluorescence in spectral bands from 700 to 850 nm for a 650 nm excitation. The apparatus consists of an electric case containing the laser of class 3B, its power supply and an optical head containing the CCD camera.

(13) In all the tables below, MEAN and STD respectively mean the average and the standard deviation of the measurements.

Example 2

In Vivo Result of the Biodistribution of the Formulations According to the Invention in FVB Female and Male Mice

(14) The nano-emulsions of Example 1 were injected into FVB female and male mice. As a comparison, a mixture of three tracers ([3H]CHE, [14C]CO and DiD) (not formulated as a nano-emulsion) was injected in order to observe the distribution of the tracers in the absence of droplets being used as a distribution carrier.

Example 2.1.

Biodistribution Result of the Formulation of Example 1.1.

Comparative Example: Non-Encapsulated Free Tracers

(15) Tables 2 to 5 provide the results of the biodistribution of each tracer ([3H]CHE, [14C]CO and DiD) expressed as a percentage of the injected dose (for ([3H]CHE and [14C]CO) per gram of tissue or as fluorescence intensity (expressed as a number of photons per pixel and reduced to 100 ms of integration of the signal, and this in all the tables of the present application comprising fluorescence intensity values) (for DiD) depending on the organ of the mouse, 2 hours (Table 2), 6 hours (Table 3), 16 hours (Table 4) and 24 hours (Table 5) after injecting into an FVB female mouse a mixture of the three tracers ([3H]CHE, [14C]CO and DiD) non-formulated as a nano-emulsion (comparative data).

(16) TABLE-US-00004 TABLE 2 2 h [3H]CHE [14C]CO DiD MEAN STD MEAN STD MEAN STD Liver 1.76 0.80 4.79 1.51 2477.20 949.88 Intestine 0.03 0.04 0.76 0.28 451.58 98.27 Lung 29.10 14.16 72.86 30.69 848.08 299.20 Spleen 1.55 0.40 3.65 0.61 1162.07 528.53 Kidney 0.09 0.05 0.89 0.41 862.35 55.97 Fat 0.02 0.02 0.69 0.49 280.30 82.36 Ovary 0.04 0.03 1.57 0.78 954.70 149.20 Adrenal 0.17 0.16 3.25 1.07 730.45 392.63 glands Uterus 0.02 0.01 0.49 0.05 694.28 549.88 Brain 0.00 0.00 0.23 0.08 439.56 61.39 Heart 0.08 0.08 0.59 0.09 487.37 101.42 Muscle 0.04 0.00 0.38 0.09 211.84 73.35 Pancreas 0.05 0.04 0.47 0.10 466.58 82.71 Salivary 0.01 0.02 0.74 0.38 399.58 178.76 gland

(17) TABLE-US-00005 TABLE 3 6 h [3H]CHE [14C]CO DiD MEAN STD MEAN STD MEAN STD Liver 3.23 1.88 5.53 3.71 2293.21 901.09 Intestine 0.15 0.11 1.21 0.37 363.09 127.53 Lung 83.99 53.84 142.66 81.34 433.52 111.50 Spleen 3.35 1.64 3.53 2.11 3224.12 645.77 Kidney 0.41 0.17 1.36 0.49 573.78 97.58 Fat 0.05 0.10 1.05 0.37 234.61 88.10 Ovary 0.07 0.08 2.12 0.20 250.27 338.83 Adrenal 0.36 0.23 3.56 0.36 208.14 196.51 glands Uterus 0.04 0.01 0.46 0.09 506.03 78.54 Brain 0.02 0.02 0.26 0.04 116.17 78.03 Heart 0.30 0.18 1.21 0.59 346.63 60.44 Muscle 0.03 0.03 0.53 0.16 108.34 29.11 Pancreas 0.09 0.04 0.76 0.13 191.73 50.40 Salivary 0.09 0.01 1.15 0.14 232.98 151.37 gland

(18) TABLE-US-00006 TABLE 4 16 h [3H]CHE [14C]CO DiD MEAN STD MEAN STD MEAN STD Liver 6.95 0.79 11.86 2.81 2954.59 420.30 Intestine 0.13 0.05 1.09 0.33 505.69 190.99 Lung 124.35 61.28 261.87 130.59 370.94 27.96 Spleen 4.17 0.17 5.32 1.21 3403.29 953.61 Kidney 0.53 0.06 1.82 0.26 382.29 194.01 Fat 0.02 0.02 1.39 0.38 295.69 118.05 Ovary 0.19 0.16 1.64 0.39 729.53 604.29 Adrenal 0.47 0.22 4.24 1.01 1683.36 399.48 gland Uterus 0.03 0.02 1.00 0.27 665.40 284.46 Brain 0.02 0.02 0.30 0.07 24.23 53.01 Heart 0.35 0.17 1.87 0.13 186.72 102.04 Muscle 0.05 0.01 0.43 0.15 93.88 56.25 Pancreas 0.04 0.04 1.00 0.07 101.87 104.38 Salivary 0.05 0.05 1.29 0.22 271.01 172.82 gland

(19) TABLE-US-00007 TABLE 5 24 h [3H]CHE [14C]CO DiD MEAN STD MEAN STD MEAN STD Liver 1.12 0.93 3.29 3.51 2663.45 439.54 Intestine 0.07 0.04 0.81 0.28 354.94 252.57 Lung 6.37 6.75 29.91 34.49 300.36 72.13 Spleen 10.63 15.66 2.27 1.90 3880.70 147.54 Kidney 1.12 1.69 1.02 0.43 397.90 234.64 Fat 1.42 2.36 1.64 0.39 344.99 37.37 Ovary 0.08 0.07 1.40 0.51 989.32 137.18 Adrenal 0.31 0.25 2.60 0.77 653.68 140.78 gland Uterus 0.34 0.53 0.62 0.12 636.69 101.11 Brain 0.03 0.05 0.22 0.03 57.68 51.05 Heart 0.12 0.05 0.82 0.37 285.67 92.94 Muscle 0.11 0.01 0.79 0.27 289.03 383.41 Pancreas 0.03 0.02 0.95 0.63 158.45 65.37 Salivary 0.14 0.13 0.86 0.50 187.28 176.21 gland

(20) Tables 2 to 5 show a strong accumulation of the free radioactive tracers ([3H]CHE and [14C]CO) in the lungs. Strong accumulation in the liver and the spleen is observed for the tracer DiD alone.

Nano-emulsion According to Example 1.1. Injected into an FVB Female Mouse

(21) Tables 6 to 14 provide the biodistribution results of each tracer ([3H]CHE, [14C]CO and DiD) expressed as a percentage of the dose injected per gram of tissue (or [3H]CHE and [14C]CO) or as fluorescence intensity (for DiD) depending on the organ of the mouse, 15 minutes (Table 6), 2 hours (Table 7), 4 hours (Table 8), 6 hours (Table 9), 16 hours (Table 10), 24 hours (Table 11), 72 hours (Table 12), 120 hours (Table 13) and 168 hours (Table 14), after injection into an FVB female mouse of the nano-emulsion of Example 1.1. (nano-emulsion according to the invention comprising the three tracers ([3H]CHE, [14C]CO and DiD).

(22) TABLE-US-00008 TABLE 6 15 min [3H]CHE [14C]CO DiD Organs MEAN STD MEAN STD MEAN STD Liver 8.26 1.27 8.86 1.85 979 174 Intestine 1.71 0.87 1.75 0.81 300 262 Lung 3.91 1.65 3.92 1.81 618 391 Spleen 3.63 0.88 3.58 0.82 727 182 Kidney 4.09 2.70 4.20 2.76 228 125 Fat 1.10 0.54 1.15 0.51 147 130 Ovary 5.38 3.33 4.59 2.62 484 616 Adrenal 10.60 3.57 10.18 2.89 457 366 glands Uterus 0.90 0.29 0.93 0.29 178 142 Brain 0.476 0.047 0.499 0.052 145 55 Heart 8.303 6.540 8.676 6.814 451 136 Muscle 0.675 0.677 0.744 0.824 1 11 Pancreas 1.233 0.298 1.262 0.244 216 145 Salivary 1.157 0.562 1.057 0.312 59 190 glands

(23) TABLE-US-00009 TABLE 7 2 h [3H]CHE [14C]CO DiD Organs MEAN STD MEAN STD MEAN STD Liver 31.09 4.34 19.41 3.24 1421 142 Intestine 2.44 0.13 2.33 0.19 637 377 Lung 4.44 1.49 4.42 1.25 727 406 Spleen 5.10 1.10 2.87 0.42 805 304 Kidneys 3.83 0.29 3.91 0.23 473 70 Fat 0.76 0.17 3.85 1.04 247 102 Ovary 29.28 14.72 22.72 12.69 1807 661 Adrenal 21.12 4.59 18.50 3.42 1050 340 glands Uterus 2.23 0.11 2.21 0.20 814 50 Brain 0.26 0.09 0.29 0.08 250 120 Heart 4.64 1.18 4.41 1.17 389 157 Muscle 0.45 0.12 0.48 0.09 213 28 Pancreas 1.43 0.10 1.68 0.15 399 59 Salivary 1.42 0.70 1.74 0.56 288 221 glands

(24) TABLE-US-00010 TABLE 8 4 h [3H]CHE [14C]CO DiD Organs MEAN STD MEAN STD MEAN STD Liver 37.43 5.51 13.69 1.62 1845 245 Intestine 2.46 0.56 1.80 0.39 974 152 Lungs 4.02 0.34 3.09 0.19 925 81 Spleen 5.00 0.93 1.97 0.22 955 19 Kidney 2.51 0.32 2.27 0.34 614 108 Fat 1.40 1.17 1.46 0.44 522 311 Ovary 39.41 11.65 25.14 6.94 2781 181 Adrenal 28.13 3.23 21.37 3.98 1754 385 glands Uterus 1.69 1.42 1.39 1.11 712 538 Brain 0.19 0.02 0.23 0.04 279 74 Heart 4.94 1.05 3.75 0.49 550 87 Muscle 0.41 0.11 0.38 0.09 236 64 Pancreas 1.01 0.26 1.17 0.18 527 138 Salivary 1.09 0.19 1.47 0.13 516 114 glands

(25) TABLE-US-00011 TABLE 9 16 h [3H]CHE [14C]CO DiD Organs MEAN STD MEAN STD MEAN STD Liver 47.67 7.58 8.68 3.35 1661 342 Intestine 4.87 0.93 2.53 0.37 1139 290 Lungs 4.63 1.97 2.80 0.94 592 169 Spleen 7.48 0.51 1.89 0.19 978 434 Kidney 2.06 0.15 1.75 0.08 627 79 Fat 1.36 0.86 8.06 1.53 532 248 Ovary 52.26 2.35 21.45 2.85 2915 184 Adrenal 43.16 14.73 30.64 9.04 2355 328 glands Uterus 3.91 1.01 2.76 0.98 1759 1216 Brain 0.13 0.01 0.13 0.01 259 42 Heart 3.49 0.66 2.00 0.41 444 123 Muscle 0.76 0.26 1.59 0.48 289 43 Pancreas 1.17 0.44 1.19 0.35 564 233 Salivary 1.27 0.79 2.02 1.05 641 238 glands

(26) TABLE-US-00012 TABLE 10 24 h [3H]CHE [14C]CO DiD Organs MEAN STD MEAN STD MEAN STD Liver 48.76 7.87 5.39 1.95 1675 299 Intestine 2.78 1.00 1.22 0.20 777 215 Lung 3.21 0.55 1.30 0.29 667 124 Spleen 7.50 2.34 1.10 0.11 774 85 Kidney 1.50 0.40 1.03 0.22 599 47 Fat 0.88 0.43 2.28 1.08 553 472 Ovary 38.06 14.16 9.58 2.88 3173 370 Adrenal 39.68 14.88 13.05 4.09 2624 460 glands Uterus 1.82 0.87 0.96 0.48 971 474 Brain 0.09 0.01 0.11 0.01 239 40 Heart 3.53 0.62 1.31 0.25 288 54 Muscle 0.46 0.17 1.01 0.53 161 52 Pancreas 0.64 0.13 0.67 0.04 446 113 Salivary 1.40 0.61 1.76 0.07 411 186 glands

(27) TABLE-US-00013 TABLE 11 48 h [3H]CHE [14C]CO DiD Organs MEAN STD MEAN STD MEAN STD Liver 58.43 2.25 1.53 0.37 1868 258 Intestine 4.43 0.38 1.01 0.04 481 77 Lungs 3.91 0.21 1.28 0.10 212 39 Spleen 12.32 2.48 1.03 0.05 342 71 Kidney 1.63 0.19 0.77 0.08 555 305 Fat 1.05 0.26 3.46 2.81 208 31 Ovary 36.42 8.45 6.46 1.12 4018 668 Adrenal 42.26 1.49 10.83 0.49 1484 607 glands Uterus 3.75 2.06 1.24 0.51 951 697 Brain 0.08 0.01 0.11 0.00 51 47 Heart 3.52 0.23 0.87 0.04 122 72 Muscle 0.36 0.05 0.34 0.18 32 39 Pancreas 1.00 0.29 0.70 0.21 202 102 Salivary 1.09 0.16 1.20 0.61 268 103 glands

(28) TABLE-US-00014 TABLE 12 72 h [3H]CHE [14C]CO DiD Organs MEAN STD MEAN STD MEAN STD Liver 39.73 8.94 0.75 0.11 1136 392 Intestine 3.58 1.96 0.62 0.23 297 115 Lungs 2.95 0.54 0.88 0.18 120 169 Spleen 7.65 3.13 0.75 0.21 330 172 Kidney 1.27 0.30 0.66 0.09 365 129 Fat 0.85 0.14 5.01 3.51 159 51 Ovary 25.11 6.58 3.04 0.81 2397 325 Adrenal 36.92 14.04 9.36 5.06 1135 586 glands Uterus 5.63 5.79 1.00 0.66 514 552 Brain 0.08 0.02 0.10 0.02 43 35 Heart 2.98 0.88 0.64 0.19 113 26 Muscle 0.22 0.04 0.28 0.05 3 47 Pancreas 0.74 0.20 0.50 0.12 102 38 Salivary 1.10 0.27 1.16 0.43 237 114 glands

(29) TABLE-US-00015 TABLE 13 120 h [3H]CHE [14C]CO DiD Organs MEAN STD MEAN STD MEAN STD Liver 39.13 3.21 0.42 0.03 996 280 Intestine 3.60 0.24 0.28 0.03 279 24 Lungs 3.30 0.75 0.57 0.05 53 55 Spleen 9.69 1.34 0.45 0.04 229 98 Kidney 1.29 0.12 0.40 0.05 383 59 Fat 0.68 0.10 2.42 1.32 176 65 Ovary 16.92 11.11 1.24 0.50 2319 815 Adrenal 41.92 8.51 6.43 0.63 1688 136 gland Uterus 4.99 3.67 0.62 0.41 563 507 Brain 0.08 0.00 0.08 0.00 90 36 Heart 2.59 0.54 0.37 0.03 80 32 Muscle 0.27 0.02 0.30 0.05 254 348 Pancreas 1.00 0.34 0.40 0.16 297 49 Salivary 2.14 0.84 1.78 1.01 120 339 glands

(30) TABLE-US-00016 TABLE 14 168 h [3H]CHE [14C]CO DiD Organs MEAN STD MEAN STD MEAN STD Liver 32.70 6.86 0.28 0.01 676.60 14.849 Intestine 2.80 0.80 0.18 0.02 214.02 23.794 Lungs 1.70 0.14 0.25 0.06 1.35 0.120 Spleen 10.71 0.49 0.28 0.00 171.93 88.862 Kidneys 1.44 0.19 0.28 0.03 391.45 128.679 Fat 0.99 0.14 5.02 2.61 164.55 115.690 Ovary 20.61 4.67 0.74 0.08 1614.45 387.463 Adrenal 29.30 3.35 2.73 0.04 660.62 704.034 glands Uterus 4.86 0.63 0.37 0.03 697.92 378.217 Brain 0.07 0.02 0.07 0.00 40.28 1.563 Heart 2.19 0.44 0.22 0.03 117.29 17.600 Muscle 0.37 0.19 0.32 0.08 16.21 54.384 Pancreas 0.79 0.12 0.23 0.00 125.11 68.066 Salivary 2.01 0.93 1.14 0.19 216.84 196.130 glands

(31) A comparison of the values of Tables 2 to 5 and of Tables 6 to 14 shows that the biodistribution is different when free tracers are injected or when the nano-emulsion according to Example 1.1 is injected. In the case of the use of a nano-emulsion according to the invention, the three tracers go to the same location at the same moment after an elapsed period of about 12h post-injection, which demonstrates the carrier effect (targeting) of the droplets of the nano-emulsion according to the invention.

(32) An accumulation of droplets of the nano-emulsion according to the invention is observed in the liver (metabolization area of lipid nano-droplets) as well as in steroidal organs (ovaries and adrenal glands), which are organs synthesizing steroidal hormones. These same areas synthesizing steroidal hormones are again found in cancer tissues of hormone-dependent cancers, such as for example certain breast or prostate cancers.

(33) A slight fluorescence signal is observed in the uterus. It was observed, after dissection and histology, that following the period of the cycle of the mouse, the morphology of the uterus changes as well as its self-fluorescence which becomes higher. Thus, these results would stem from self-fluorescence of the uterus.

(34) As a preferential accumulation in the ovaries and adrenal glands was observed, fluorescence images were produced in cryohistology. The fluorescence images at 680 nm confirmed the accumulation of the droplets of the nano-emulsion in these organs. The histological images of the ovaries show accumulation in the corpus luteum, which has the function of secreting progesterone. The histological images of the adrenal glands show accumulation in the cortical area which ensures secretion of the steroids.

Nano-emulsion According to Example 1.1. Injected into an FVB Male Mouse

(35) Tables 15 to 21 provide the results of the biodistribution of each tracer ([3H]CHE, [14C]CO and DiD) expressed as a percentage of the dose injected per gram of tissue (for [3H]CHE and [14C]CO) or as fluorescence intensity (for DiD) depending on the organ of the mouse, 15 minutes (Table 6), 2 hours (Table 7), 4 hours (Table 8), 6 hours (Table 9), 16 hours (Table 10), 24 hours (Table 11), 72 hours (Table 12), 120 hours (Table 13) and 168 hours (Table 14), after injection into an FVB male mouse of the nano-emulsion of Example 1.1. comprising the three tracers ([3H]CHE, [14C]CO and DiD).

(36) TABLE-US-00017 TABLE 15 2 h [3H]CHE [14C]CO DiD Mean STD Mean STD Mean STD Liver 22.67 1.50 11.13 1.40 831.9 234.8 Intestine 2.44 0.17 2.05 0.25 392.1 78.3 Lung 5.08 0.80 4.45 0.90 559.9 124.5 Spleen 7.15 0.49 2.97 0.36 455.7 216.8 Kidney 5.29 0.90 6.10 1.35 601.1 79.7 Fat 1.14 0.19 1.62 0.44 184.0 31.0 Testicle 1.36 0.12 1.34 0.12 639.9 141.0 Adrenal 10.16 2.47 8.63 3.25 779.1 21.6 gland Seminal 0.79 0.28 0.80 0.28 175.3 64.6 glands Brain 0.35 0.03 0.40 0.05 186.5 47.7 Heart 9.84 1.09 9.28 1.36 427.6 55.3 Muscle 0.99 0.24 0.94 0.27 176.7 54.4 Pancreas 1.64 0.15 1.72 0.09 255.1 99.4 Salivary 1.53 0.25 1.99 0.37 310.0 97.2 gland

(37) TABLE-US-00018 TABLE 16 4 h [3H]CHE [14C]CO DiD Mean STD Mean STD Mean STD Liver 17.25 5.85 5.76 1.18 813.0 71.6 Intestine 1.98 0.68 1.49 0.50 336.2 108.5 Lung 2.98 1.44 2.30 1.04 446.4 105.1 Spleen 5.31 1.82 2.08 0.41 407.4 151.7 Kidney 3.14 0.96 3.36 1.13 505.0 127.1 Fat 0.74 0.20 0.71 0.15 128.3 55.5 Testicle 1.35 0.33 1.26 0.29 470.9 283.7 Adrenal 16.96 7.65 15.01 5.05 536.1 177.9 gland Seminal 0.50 0.17 0.52 0.16 179.5 39.8 gland Brain 0.23 0.07 0.24 0.06 150.6 28.3 Heart 7.57 2.16 6.07 1.93 306.9 52.4 Muscle 0.56 0.06 0.51 0.02 152.3 56.0 Pancreas 1.50 0.40 1.54 0.43 295.0 58.7 Salivary 1.26 0.20 1.70 0.18 297.8 130.0 gland

(38) TABLE-US-00019 TABLE 17 6 h [3H]CHE [14C]CO DiD Mean STD Mean STD Mean STD Liver 22.609 3.289 5.926 0.706 847.4 301.5 Intestine 2.549 0.564 1.778 0.434 378.0 54.4 Lung 3.771 0.541 2.790 0.325 426.0 181.2 Spleen 6.453 1.102 2.025 0.135 488.6 129.1 Kidney 3.336 0.460 3.841 0.775 427.7 68.0 Fat 1.234 0.574 1.217 0.633 221.1 69.1 Testicle 2.000 0.632 1.839 0.642 723.1 112.0 Adrenal 13.399 2.214 10.940 1.302 721.9 38.7 gland Seminal 0.54 0.16 0.59 0.19 261.0 32.1 gland Brain 0.24 0.04 0.28 0.04 235.9 42.1 Heart 8.52 0.69 6.10 0.23 253.8 75.3 Muscle 0.77 0.10 0.58 0.04 169.3 40.2 Pancreas 1.67 0.32 1.55 0.38 347.0 90.3 Salivary 2.10 1.24 1.95 0.52 488.5 50.8 gland

(39) TABLE-US-00020 TABLE 18 16 h [3H]CHE [14C]CO DiD Mean STD Mean STD MEAN STD Liver 40.91 1.28 3.55 0.34 1157.2 433.7 Intestine 5.19 0.59 2.13 0.21 640.4 298.4 Lung 5.31 0.91 2.14 0.42 423.5 94.4 Spleen 9.19 1.06 1.25 0.14 373.3 96.6 Kidney 2.40 0.31 2.01 0.26 343.0 61.3 Fat 1.70 1.13 2.24 0.88 117.0 6.5 Testicle 2.95 0.50 2.14 0.41 525.4 119.1 Adrenal 32.89 1.11 17.48 5.45 1086.2 242.6 gland Seminal 0.64 0.21 0.77 0.33 163.0 128.4 gland Brain 0.18 0.01 0.17 0.01 134.7 73.7 Heart 8.50 0.94 3.22 0.21 281.5 85.1 Muscle 0.97 0.25 0.72 0.20 281.9 83.9 Pancreas 1.92 0.60 1.35 0.37 332.6 131.2 Salivary 2.68 0.17 2.11 0.35 838.8 227.5 gland

(40) TABLE-US-00021 TABLE 19 24 h [3H]CHE [14C]CO DiD Mean STD Mean STD MEAN STD Liver 30.93 7.18 2.05 0.42 1152.7 120.1 Intestine 3.41 0.80 1.18 0.22 621.0 64.0 Lung 2.88 0.80 1.08 0.03 406.0 28.6 Spleen 6.65 1.60 0.80 0.12 638.0 197.6 Kidney 1.80 0.26 1.11 0.11 323.1 10.6 Fat 0.83 0.50 0.60 0.33 142.1 54.5 Ovary 1.71 1.29 1.08 0.66 575.9 104.8 Adrenal 17.41 5.59 7.78 3.66 1105.9 126.9 gland Seminal 0.35 0.28 0.30 0.24 170.6 43.2 gland Brain 0.11 0.03 0.12 0.03 109.3 16.6 Heart 5.76 2.10 1.72 0.71 239.7 17.6 Muscle 0.38 0.31 0.38 0.08 144.9 28.5 Pancreas 1.31 0.12 1.14 0.57 268.9 38.7 Salivary 1.57 0.78 1.69 1.21 501.1 155.2 gland

(41) TABLE-US-00022 TABLE 20 48 h [3H]CHE [14C]CO DiD Mean STD Mean STD MEAN STD Liver 24.53 0.76 0.70 0.13 988.2 149.5 Intestine 3.50 0.61 0.55 0.03 381.6 116.6 Lung 3.02 0.02 0.67 0.10 346.5 115.9 Spleen 11.18 3.50 0.76 0.01 656.7 132.2 Kidney 1.77 0.44 0.82 0.34 168.3 34.4 Fat 0.94 0.18 0.85 0.34 185.1 62.2 Testicle 2.97 0.40 0.97 0.14 648.6 246.3 Adrenal 25.12 2.72 8.12 2.67 783.7 240.6 gland Seminal 0.35 0.28 0.30 0.24 166.7 47.5 gland Brain 0.10 0.00 0.10 0.01 160.4 25.8 Heart 6.05 0.99 0.97 0.07 178.1 102.8 Muscle 0.63 0.08 0.27 0.04 131.7 28.7 Pancreas 0.99 0.18 0.49 0.11 266.2 82.1 Salivary 2.07 0.21 1.54 0.20 489.7 53.1 gland

(42) TABLE-US-00023 TABLE 21 72 h [3H]CHE [14C]CO DiD Mean STD Mean STD MEAN STD Liver 24.97 2.51 0.61 0.08 648.0 103.1 Intestine 3.47 0.62 0.49 0.12 267.8 33.8 Lung 3.04 0.41 0.67 0.06 286.6 104.7 Spleen 6.40 2.62 0.48 0.15 278.0 68.4 Kidney 1.82 0.39 0.78 0.07 160.3 23.1 Fat 0.70 0.12 1.87 0.55 78.0 11.3 Testicle 4.34 1.62 1.43 0.47 481.2 105.0 Adrenal 46.41 8.98 14.65 0.95 717.3 268.8 gland Seminal 0.58 0.03 0.47 0.03 74.4 1.3 gland Brain 0.11 0.02 0.10 0.01 14.8 8.6 Heart 7.83 3.47 0.88 0.35 142.3 43.0 Muscle 0.49 0.20 0.35 0.21 109.2 23.0 Pancreas 1.12 0.36 0.63 0.19 160.6 22.1 Salivary 2.36 0.42 1.38 0.66 430.2 8.2 gland

(43) A comparison of the values of Tables 6 to 14 and of Tables 15 to 21 shows that the biodistribution in vivo between females and males is identical as regards the accumulation in the liver and in the adrenal glands.

Example 2.2.

Biodistribution Result of the Formulation of Example 1.2.

(44) Table 22 provides the biodistribution results of DiD expressed as fluorescence intensity depending on the organ of the mouse 4 hours after injection into a FVB female mouse of the nano-emulsion of Example 1.2.1. or 1.2.2. comprising the tracer DiD and cholesteryl stearate (CHST) as a compound of formula (I).

(45) TABLE-US-00024 TABLE 22 0% (compound % of without the cholesteryl compound of stearate in formula (I)) 0.6% CHST 2% CHST the (comparative (Example (Example dispersed example) 1.2.1.) 1.2.2.) phase MEAN STD MEAN STD MEAN STD Brain 249.8 62.0 303 86 280 79 Heart 619.6 69.1 549 134 624 38 Liver 1585.5 270.9 1598 294 1992 305 Fat 299.4 52.9 426 166 342 70 Intestine 532.4 68.1 627 249 548 92 Muscle 198.9 22.7 235 87 136 43 Ovary 1286.6 47.2 2440 491 2671 659 Pancreas 353.8 89.9 472 120 284 7 Lung 602.8 115.5 960 213 878 123 Spleen 730.2 142.5 737 99 669 48 Kidney 796.8 106.8 886 185 747 8 Salivary 707.7 258.7 404 181 320 63 gland Adrenal 1780.2 560.2 1498 527 1476 899 gland Uterus 1379.6 680.5 1177 45 1165 534

(46) Like in Example 2.2., accumulation is observed in the liver (metabolization area of lipid nano-droplets) as well as in the steroidal organs (ovaries and adrenal glands), organs synthesizing steroidal hormones. These same areas synthesizing steroidal hormones are again found in cancer tissues of hormone-dependent cancers, such as for example certain breast or prostate cancers.

Example 3

Comparison of the Formulations According to the Invention and of Formulations Comprising Cholesterol Instead of the Compound of Formula (I)

(47) Stability Tests

(48) Stability tests at 4 C. were conducted for formulations either comprising or not a diagnostic agent (DiD) and either comprising cholesteryl stearate (compound of formula (I)) (0.6%, 2% and 3% by weight based on the weight of the dispersed phase), or cholesterol (0.6% 2%, 5% or 10% by weight based on the weight of the dispersed phase) instead of the compound of formula (I).

(49) In the absence of a diagnostic agent, beyond 90 days, increases in the size of the dispersed phase droplets and in the polydispersity index (DPI) were observed for formulations comprising 2%, 5% and 10% of cholesterol based on the weight of the dispersed phase, which indicates a lack of stability of these formulations. On the other hand, the size of the dispersed phase droplets and the polydispersity index of the formulations according to the invention remain constant beyond 150 days, which indicates that these formulations are stable.

(50) In the presence of the diagnostic agent, for the formulation comprising cholesterol, it was observed that the more the cholesterol level increases, the more the encapsulation level of the diagnostic agent decreases. There again, destabilization of these formulations beyond 90 days at 4 C. was observed. On the other hand, no encapsulation difference of DiD and no stability problem were observed for formulations comprising cholesteryl stearate (formulations according to the invention).

(51) Thus, a formulation comprising more than 0.6% by weight of cholesterol based on the weight of the dispersed phase is not stable beyond 90 days at 4 C., while formulations according to the invention comprising a compound of formula (I) are stable beyond 150 days for all the tested concentrations and allow better encapsulation of the diagnostic agent and formulations comprising cholesterol.

(52) Without intending to be bound to a particular theory, it seems that the cholesterol is located at the interface of the lipid phase droplets and destabilizes them, while the compounds of formula (I), which are more liphophilic than the cholesterol by the group R, are located in the core of the droplets and do not affect the stability of the nano-emulsion.

(53) In Vivo Biodistribution Test, 4 Hours after Injection into an FVB Female Mouse.

(54) Tables 23 to 28 provide the biodistribution results (fluorescence intensity depending on the organ of the mouse) 4 hours after injection into an FVB female mouse of nano-emulsions comprising DiD (fluorescent tracer) and: without any cholesterol or one of its derivatives (control nano-emulsion), comprising cholesterol (comparative nano-emulsions), or comprising a compound of formula (I) (nano-emulsions according to the invention, according to Examples 1.1., 1.2.1. or 1.2.2.).

(55) TABLE-US-00025 TABLE 23 fluorescence intensity according to the organ of the mouse 4 hours after injection into an FVB female mouse of a nano-emulsion comprising DiD (tracer) and 0 (control), 0.6%, 2%, 5% or 10% of cholesterol based on the weight of the dispersed phase and (comparative nano-emulsions). % of Cholesterol-fluorescence intensity-4 h post injection into an FVB female mouse cholesterol 0 0.6 2 5 10 in the dispersed (control) (comparative) (comparative) (comparative) (comparative) phase MEAN STD MEAN STD MEAN STD MEAN STD MEAN STD Brain 249.8 62.0 116.6 44.7 288.9 12.5 308.0 132.5 149.3 79.4 Heart 619.6 69.1 419.3 73.7 493.2 63.4 273.9 31.9 291.2 137.2 Liver 1585.5 270.9 1146.5 129.9 1392.6 262.6 1487.4 17.0 1459.8 670.9 Fat 299.4 52.9 355.5 98.5 257.6 31.7 237.1 60.1 261.3 79.3 Intestine 532.4 68.1 651.3 98.8 612.7 72.1 559.3 114.2 467.6 219.1 Muscle 198.9 22.7 140.3 43.0 207.9 45.0 301.0 263.2 120.7 71.2 Ovary 1286.6 47.2 718.4 230.7 1427.5 472.0 1540.8 247.9 1659.4 702.6 Pancreas 353.8 89.9 260.0 35.2 332.7 12.1 270.5 88.9 202.3 147.8 Lung 602.8 115.5 561.9 56.5 483.9 76.7 385.9 134.5 660.1 236.5 Spleen 730.2 142.5 632.0 57.6 700.6 117.2 747.4 185.9 467.6 185.0 Kidney 796.8 106.8 647.7 129.9 657.9 77.8 546.1 48.9 402.9 257.9 Salivary gland 707.7 258.7 209.6 170.6 368.9 54.6 240.9 144.0 111.6 204.8 Adrenal gland 1780.2 560.2 1316.0 213.9 941.0 126.1 1039.5 558.4 1445.5 627.3 Uterus 1379.6 680.5 842.4 173.6 1016.1 303.0 1122.6 603.6 497.2 463.1

(56) TABLE-US-00026 TABLE 24 the ratio of fluorescence intensity in an organ of the mouse over the fluorescence intensity in the muscles 4 hours after injection into an FVB female mouse of a nano-emulsion comprising DiD (tracer) and 0 (control), 0.6%, 2%, 5% or 10% of cholesterol based on the weight of the dispersed phase and (comparative nano-emulsions). % of Cholesterol-organ/muscle ratio-4 h post-injection into an FVB female cholesterol mouse in the 0 0.6 2 5 10 dispersed (control) (comparative) (comparative) (comparative) (comparative) phase MEAN STD MEAN STD MEAN STD MEAN STD MEAN STD Brain 1.2 0.3 0.8 0.1 1.4 0.3 2.0 0.8 1.3 0.1 Heart 3.1 0.3 3.3 1.7 2.5 0.8 1.8 0.1 2.6 0.6 Liver 8.3 1.2 8.9 3.8 7.0 2.4 10.1 1.5 12.8 1.8 Fat 1.5 0.2 2.8 1.3 1.3 0.2 1.6 0.2 2.8 2.0 Intestine 2.8 0.3 4.9 1.4 3.1 0.8 3.9 1.3 4.1 0.6 Muscle 1.0 0.0 1.0 0.0 1.0 0.0 1.0 0.0 1.0 0.0 Ovary 6.7 0.7 4.4 1.8 6.8 1.4 10.6 3.0 14.8 2.7 Pancreas 1.8 0.4 2.0 0.9 1.7 0.5 1.8 0.6 1.7 0.5 Lung 3.2 0.6 4.2 1.0 2.5 1.0 2.5 0.5 6.1 1.6 Spleen 3.8 0.6 4.8 1.3 3.5 0.7 5.0 1.0 4.2 0.9 Kidney 4.1 0.4 5.2 3.0 3.2 0.6 3.7 0.3 3.3 0.2 Salivary gland 3.8 1.4 1.3 1.0 1.8 0.3 1.6 1.0 0.5 1.1 Adrenal gland 10.1 4.4 12.4 6.8 4.6 0.4 6.7 2.6 12.8 2.2 Uterus 7.7 3.7 6.3 1.5 4.8 0.7 7.2 2.8 3.3 2.3

(57) TABLE-US-00027 TABLE 25 fluorescence intensity depending on the organ of the mouse 4 hours after injection into an FVB female mouse of the nano-emulsion comprising DiD (tracer) and 0 (control), 0.6%, 2%, 5% or 10% of cholesterol based on the weight of the dispersed phase and (comparative nano-emulsions). Cholesterol (CH) or cholesteryl stearate (CHST)-fluorescence intensity- 4 h post-injection into an FVB female mouse % of CH or 0.6% CHST 2% CHST of CHST In 0% CH 0.6% 2% CH (Example (Example dispersed (control) (comparative) (comparative) 1.2.1.) 1.2.2.) phase MEAN STD MEAN STD MEAN STD MEAN STD MEAN STD Brain 249.8 62.0 116.6 44.7 288.9 12.5 303 86 280 79 Heart 619.6 69.1 419.3 73.7 493.2 63.4 549 134 624 38 Liver 1585.5 270.9 1146.5 129.9 1392.6 262.6 1598 294 1992 305 Fat 299.4 52.9 355.5 98.5 257.6 31.7 426 166 342 70 Intestine 532.4 68.1 651.3 98.8 612.7 72.1 627 249 548 92 Muscle 198.9 22.7 140.3 43.0 207.9 45.0 235 87 136 43 Ovary 1286.6 47.2 718.4 230.7 1427.5 472.0 2440 491 2671 659 Pancreas 353.8 89.9 260.0 35.2 332.7 12.1 472 120 284 7 Lung 602.8 115.5 561.9 56.5 483.9 76.7 960 213 878 123 Spleen 730.2 142.5 632.0 57.6 700.6 117.2 737 99 669 48 Kidney 796.8 106.8 647.7 129.9 657.9 77.8 886 185 747 8 Salivary 707.7 258.7 209.6 170.6 368.9 54.6 404 181 320 63 gland Adrenal 1780.2 560.2 1316.0 213.9 941.0 126.1 1498 527 1476 899 gland Uterus 1379.6 680.5 842.4 173.6 1016.1 303.0 1177 45 1165 534

(58) TABLE-US-00028 TABLE 26 ratio of the fluorescence intensity in an organ of the mouse over the fluorescence intensity in the muscles 4 hours after injection into an FVB female mouse of a nano-emulsion comprising DiD (tracer) and 0 (control), 0.6% or 2% of cholesterol based on the weight of the dispersed phase (comparative nano-emulsions) or 0.6% or 2% of cholesteryl stearate based on the weight of the dispersed phase (nano-emulsions according to the invention). Cholesterol (CH) or cholesteryl stearate (CHST)-organ/muscle ratio- 4 h post -injection into FVB female mouse % of CH or 0.6% CH 2% CH 0.6% CHST 2% CHST of CHST In 0 % (control) (comparative) (comparative) (Example 1.2.1.) (Example 1.2.2.) dispersed phase MEAN STD MEAN STD MEAN STD MEAN STD MEAN STD Brain 1.2 0.3 0.8 0.1 1.4 0.3 1.3 0.2 2.1 0.1 Heart 3.1 0.3 3.3 1.7 2.5 0.8 2.4 0.4 4.8 1.2 Liver 8.3 1.2 8.9 3.8 7.0 2.4 7.3 2.6 15.1 2.6 Fat 1.5 0.2 2.8 1.3 1.3 0.2 1.8 0.3 2.7 1.4 Intestine 2.8 0.3 4.9 1.4 3.1 0.8 2.7 0.1 4.4 2.1 Muscle 1.0 0.0 1.0 0.0 1.0 0.0 1.0 0.0 1.0 0.0 Ovary 6.7 0.7 4.4 1.8 6.8 1.4 10.9 3.0 19.9 1.5 Pancreas 1.8 0.4 2.0 0.9 1.7 0.5 2.3 1.2 2.2 0.6 Lung 3.2 0.6 4.2 1.0 2.5 1.0 4.4 1.9 7.0 3.1 Spleen 3.8 0.6 4.8 1.3 3.5 0.7 3.4 1.2 5.1 1.3 Kidney 4.1 0.4 5.2 3.0 3.2 0.6 4.1 1.4 5.8 1.9 Salivary gland 3.8 1.4 1.3 1.0 1.8 0.3 1.7 0.1 2.6 1.3 Adrenal gland 10.1 4.4 12.4 6.8 4.6 0.4 6.5 1.6 10.3 3.3 Uterus 7.7 3.7 6.3 1.5 4.8 0.7 7.6 2.2 7.6 2.2

(59) TABLE-US-00029 TABLE 27 fluorescence intensity according to the organ of the mouse 4 hours after injection into an FVB female mouse of the nano-emulsion comprising DiD (tracer) and 0 (control), 0.6% or 2% of cholesteryl stearate based on the weight of the dispersed phase (nano- emulsion according to the invention) or 0.6% of the set ([3H]CHE/[14C]CO) based on the weight of the dispersed phase (nano-emulsions according to the invention). [3H]CHE and [14C]CO or cholesteryl stearate-fluorescence intensity- 4 h post-injection into an FVB female mouse 0.45% of % of [3H]CHE cholesteryl and 0.15% of 0.6% CHST 2% CHST derivative in [14C]CO 0 (Example (Example the dispersed (Example 1.1.) (control) 1.2.1.) 1.2.2.) phase MEAN STD MEAN STD MEAN STD MEAN STD Brain 279 74 249.8 62.0 303 86 280 79 Heart 550 87 619.6 69.1 549 134 624 38 Liver 1845 245 1585.5 270.9 1598 294 1992 305 Fat 522 311 299.4 52.9 426 166 342 70 Intestine 974 152 532.4 68.1 627 249 548 92 Muscle 236 64 198.9 22.7 235 87 136 43 Ovary 2781 181 1286.6 47.2 2440 491 2671 659 Pancreas 527 138 353.8 89.9 472 120 284 7 Lung 955 19 602.8 115.5 960 213 878 123 Spleen 614 108 730.2 142.5 737 99 669 48 Kidney 925 81 796.8 106.8 886 185 747 8 Salivary gland 516 114 707.7 258.7 404 181 320 63 Adrenal gland 1754 385 1780.2 560.2 1498 527 1476 899 Uterus 712 538 1379.6 680.5 1177 45 1165 534

(60) TABLE-US-00030 TABLE 28 ratio of the fluorescence intensity in an organ of the mouse over the fluorescence intensity in the muscles 4 hours after injection into an FVB female mouse of a nano-emulsion comprising DiD (tracer) and 0 (control), 0.6% or 2% of cholesteryl stearate based on the weight of the dispersed phase (nano-emulsions according to the invention) or 0.6% of the set ([3H]CHE/[14C]CO) based on the weight of the dispersed phase (nano-emulsion according to the invention). [3H]CHE and [14C]CO or cholesteryl stearate-organ/muscle ratio-4 h post-injection into FVB female mouse 0,45% of % of [3H]CHE cholesterol And 0.15% of 0,6% CHST 2% CHST In the [14C]CO 0 (Example (Example Dispersed (Example 1.1.) (control) 1.2.1.) 1.2.2.) phase MEAN STD MEAN STD MEAN STD MEAN STD Brain 1.2 0.1 1.2 0.3 1.3 0.2 2.1 0.1 Heart 2.4 0.6 3.1 0.3 2.4 0.4 4.8 1.2 Liver 8.1 1.4 8.3 1.2 7.3 2.6 15.1 2.6 Fat 2.1 0.8 1.5 0.2 1.8 0.3 2.7 1.4 Intestine 4.5 1.8 2.8 0.3 2.7 0.1 4.4 2.1 Muscle 1.0 0.0 1.0 0.0 1.0 0.0 1.0 0.0 Ovary 12.3 2.8 6.7 0.7 10.9 3.0 19.9 1.5 Pancreas 2.3 0.4 1.8 0.4 2.3 1.2 2.2 0.6 Lung 4.3 1.3 3.2 0.6 4.4 1.9 7.0 3.1 Spleen 2.7 0.3 3.8 0.6 3.4 1.2 5.1 1.3 Kidney 4.1 0.9 4.1 0.4 4.1 1.4 5.8 1.9 Salivary gland 2.2 0.4 3.8 1.4 1.7 0.1 2.6 1.3 Adrenal gland 8.0 3.8 10.1 4.4 6.5 1.6 10.3 3.3 Uterus 3.6 3.7 7.7 3.7 7.6 2.2 7.6 2.2 * = trebly labelled

(61) An increase in the intensity of the signal in the ovaries is observed at cholesterol encapsulation levels of more than 5% based on the weight of the dispersed phase. It is therefore necessary to use at least 5% by weight of cholesterol based on the weight of the dispersed phase in order to observe a more specific targeting towards the steroidal organs than towards the other organs. It should be noted that at low cholesterol levels, the signal is even less than that of the formulation without any cholesterol (0%).

(62) Now, as shown above in stability tests, a formulation of at least 5% of cholesterol based on the weight of the dispersed phase (required proportion for beginning to observe specific targeting of hormone-dependent cancers) is not stable. Now, stability is an essential condition for industrial application.

(63) By comparison, the in vivo biodistribution tests at 4 h post-injection of formulations comprising 0.6% and 2% by weight of cholesteryl stearate based on the weight of the dispersed phase and of DiD in healthy FVB mice (formulation according to the invention) shown in Example 2 above show that these formulations specifically target ovaries, even with a low cholesteryl stearate level (0.6% by weight based on the weight of the dispersed phase).

(64) The comparison of the biodistribution results show that the same targeting intensity is obtained in the ovaries for concentrations of 5% by weight of cholesterol based on the weight of the dispersed phase and 0.6% by weight of cholesteryl stearate based on the weight of the dispersed phase, and that the signal with 2% by weight of cholesteryl stearate based on the weight of the dispersed phase is greater than that of the formulation comprising 10% by weight of cholesterol based on the weight of the dispersed phase. For a same concentration of diagnostic agents, at least five times more, generally eight times more cholesterol than of a compound of formula (I) are required for obtaining the same intensity for targeting steroidal organs.

(65) The formulation according to the invention comprising a compound of formula (I) therefore allows much more specific targeting of steroidal organs than a formulation comprising cholesterol.

Example 4

Comparison of the Specificity of the Targeting between a Hormone-Dependent Cancer (Breast Cancer) and a Non-Hormone-Dependent Cancer (Brain CancerGlioma Model)

(66) Two types of cancer models were studied: the glioma model (brain cancer) and the PyMT model (breast cancer). The kinetics of the accumulation of droplets of the nano-emulsion according to Example 1.1. in the tumor of an FVB female mouse were evaluated.

(67) In the FVB-implanted PyMT (breast cancer) model, fast accumulation of the signal was observed in the tumoral area. As soon as 5h post-injection, a tumor/muscle ratio of 3.5 is observed. The fluorescence intensity maximum is observed at 21 h post-injection with a tumor/muscle signal ratio of 9. After 21h, the signal begins to decrease.

(68) On the other hand, the accumulation in the tumoral areas is much slower in the model of the glioma. The maximum fluorescence intensity is observed 48h post-injection versus 21h post-injection for the PyMT FVB model.

(69) These results show that the nano-emulsion preferentially targets hormone-dependent cancers relatively to non-hormone-dependent cancers.

Example 5

Influence of the Weight Proportion of the Compound of Formula (I) Based on the Weight of Dispersed Phase on the Possibility of Formulating the Nano-Emulsion

(70) An attempt was made for preparing nano-emulsions with mass proportions of cholesteryl stearate based on the total weight of the dispersed phase, greater than those of the nano-emulsions according to Examples 1.2.1.and 1.2.2. described above (comprising 0.6% and 2% of cholesteryl stearate respectively based on the total weight of the dispersed phase), i.e. with 3%, 6%, 16% and 33% of cholesteryl stearate based on the total weight of the dispersed phase.

(71) TABLE-US-00031 TABLE 29 composition of formulations and possibility of formulating the nano-emulsion according to the mass proportion of cholesteryl stearate based on the total weight of the dispersed phase. Raw Ex Ex Ex Ex Ex Ex material 1.2.1. 1.2.2. 5.1. 5.2. 5.3. 5.4. Oily Amphiphilic Lipoid 17 mg phase lipid Solubilizing Suppocire 68 mg lipid oil Soy oil 23 mg Compound CHST 1.2 mg 4 mg 6 mg 12 mg 40 mg 100 mg of formula (I) Mass % age of the 0.60% 1.96% 2.91% 5.66% 16.67% 33.33% compound of formula (I) in the dispersed phase (considering that the co-surfactant belongs to the dispersed phase) Aqueous co- PEG 92 mg phase surfactant aqueous PBS 1X 1800 L solution Possibility of formulating the yes yes yes no no no nano-emulsion

(72) As illustrated in Table 29, it was not possible to formulate nano-emulsions for cholesteryl stearate proportions greater than 3% based on the total weight of the dispersed phase.