NON-DEGRADABLE EMBOLISATION MICROSPHERE

Abstract

The invention relates to non-biodegradable embolisation microspheres comprising a cross-linked matrix, the matrix being based on at least: a) from 20% to 95% of hydrophilic monomer; b) from 1% to 15% of a non-biodegradable hydrophilic cross-linking monomer; and c) from 1.5% to less than 6% of transfer agent selected from alkyl halides and cycloaliphatic or aliphatic thiols having in particular from 2 to 24 carbon atoms, and optionally having another functional group selected from amino, hydroxy and carboxy groups. The invention further relates to a pharmaceutical composition comprising non-biodegradable embolisation microspheres according to the invention in conjunction with a pharmaceutically acceptable vehicle, advantageously for parenteral administration. The invention further relates to a kit comprising a pharmaceutical composition comprising non-biodegradable embolisation microspheres according to the invention in conjunction with a pharmaceutically acceptable vehicle for parenteral administration, and at least one injection means.

Claims

1. Nonbiodegradable embolization microspheres comprising a crosslinked matrix, said matrix being based on at least: a) 20% to 95% of hydrophilic monomer selected from N-vinylpyrrolidone, and a monomer of the following formula (I): (CH.sub.2=CR.sub.1)-CO-D (I) in which: D represents O-Z or NH-Z, Z representing (C.sub.1-C.sub.6)alkyl, -(CR.sub.2R.sub.3).sub.m-CH.sub.3, —(CH.sub.2—CH.sub.2—O).sub.m—H, —(CH.sub.2—CH.sub.2—O).sub.m—CH.sub.3, -C(R.sub.4OH).sub.m or -(CH.sub.2).sub.m-NR.sub.5R.sub.6 with m representing an integer from 1 to 30; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 represent, independently of one another, H or a (C.sub.1-C.sub.6)alkyl; b) 1% to 15% of a nonbiodegradable, linear or branched hydrophilic crosslinking monomer of the following formulas (IIa) or (IIb): ##STR00023## ##STR00024## in which R.sub.7 and Rs represent, independently of one another, H or a (C.sub.1-C.sub.6)alkyl;and A represents, alone or with at least one of the atoms to which it is bound, a (C.sub.1-C.sub.6)alkylene, a polyethylene glycol (PEG), a polysiloxane, a poly(dimethylsiloxane) (PDMS), a polyglycerol ester (PGE) or a bisphenol A; and c) 1.5% to less than 6% of transfer agent selected from alkyl halides and cycloaliphatic or aliphatic thiols, and optionally having another functional group selected from the amino, hydroxy and carboxy groups, the percentages of the monomers a) and b) being given in moles relative to the total number of moles of monomers and the percentages of compound c) being given in moles relative to the number of moles of the hydrophilic monomer a).

2. The nonbiodegradable embolization microspheres of claim 1, wherein the crosslinked matrix is based on 1.5% to 4.5% in moles relative to the number of moles of the hydrophilic monomer a).

3. The nonbiodegradable embolization microspheres of claim 1, wherein the hydrophilic monomer a) is selected from the group consisting of N-vinylpyrrolidone, vinyl alcohol, 2-hydroxyethylmethacrylate, sec-butyl acrylate, n-butyl acrylate, t-butyl acrylate, t-butyl methacrylate, methylmethacrylate, N-dimethylaminoethyl(methyl)acrylate, N,N-dimethylaminopropyl (meth)acrylate, t-butylaminoethyl(methyl)acrylate, N,N-diethylaminoacrylate, poly(ethylene oxide) (meth)acrylate, methoxy poly(ethylene oxide) (meth)acrylate, butoxy poly(ethylene oxide) (meth)acrylate, poly(ethylene glycol) (meth)acrylate, methoxy poly(ethylene glycol) (meth)acrylate, butoxy poly(ethylene glycol) (meth)acrylate and mixtures thereof .

4. The nonbiodegradable embolization microspheres of claim 1, wherein the transfer agent is selected from thioglycolic acid, 2-mercaptoethanol, dodecanethiol, hexanethiol and mixtures thereof.

5. The nonbiodegradable embolization microspheres of claim 1, wherein the matrix is further based on at least one ionized or ionizable monomer of the following formula (III): ##STR00025## in which: R.sub.9 represents H or a (C.sub.1-C.sub.6) alkyl; M represents a single bond or a divalent radical having from 1 to 20 carbon atoms; E represents an ionized or ionizable group, E advantageously being selected from the group consisting of COOH, COO—, SO.sub.3H, SO.sub.3.sup.-, PO.sub.4H.sub.2, PO.sub.4H.sup.-, PO.sub.4.sup.2-, NR.sub.10R.sub.11, NR.sub.12R.sub.13R.sub.14.sup.+, R.sub.10, R.sub.11, R.sub.12, R.sub.13 and R.sub.14 represent, independently of one another, H or a (C.sub.1-C.sub.6)alkyl.

6. The nonbiodegradable embolization microspheres of claim 5, loaded with a drug, an active substance, a diagnostic agent or macromolecules, the drug, the active substance and the diagnostic agent.

7. The nonbiodegradable embolization microspheres comprising a crosslinked matrix of claim 6, wherein the drug or the active substance is selected from the group consisting of anti-inflammatory agents, local anesthetics, analgesics, antibiotics, anticancer agents, steroids, antiseptics and a mixture thereof.

8. The nonbiodegradable embolization microspheres of claim 6, wherein the macromolecules are selected from the group consisting of enzymes, antibodies, cytokines, growth factors, clotting factors, hormones, plasmids, antisense oligonucleotides, siRNA, ribozymes, DNA enzyme, aptamers, anti-inflammatory proteins, bone morphogenetic proteins (BMP), pro-angiogenic factors, vascular endothelial growth factors (VEGF), TGF-beta, angiogenesis inhibitors and mixtures thereof.

9. The nonbiodegradable embolization microspheres of claim 1, wherein the matrix is further based on at least one colored monomer of the following general formula (V): ##STR00026## in which, Z.sub.1 and Z.sub.2 represent, independently of one another, H or OR.sub.26, R.sub.26 representing H or a (C.sub.1-C.sub.6)alkyl; X represents H or a halogen; R.sub.24 represents a group selected from linear or branched (C.sub.1-C.sub.6)alkylene, (Cs-C.sub.36)arylene, (C.sub.5-C.sub.18)arylene-O-R.sub.27, (C.sub.5-C.sub.18)heteroarylene and (C.sub.5-C.sub.18)heteroarylene-O-R.sub.28, R.sub.27 and R.sub.28 representing a (C.sub.1-C.sub.6)alkyl or a (C.sub.1-C.sub.6)alkylene, R.sub.25 represents H or a (C.sub.1-C.sub.6)alkyl.

10. The nonbiodegradable embolization microspheres of claim 1, wherein the matrix is further based on 5% to 15%, of a halogenated monomer the percentage of said halogenated monomer being given in moles relative to the total number of moles of monomers.

11. The nonbiodegradable embolization microspheres comprising a crosslinked matrix comprising at least one halogenated monomer of claim 10, wherein the halogenated monomer corresponds to (triiodobenzoyl)oxo ethyl methacrylate (MAOETIB) of the following formula: ##STR00027## .

12. The nonbiodegradable embolization microspheres comprising a crosslinked matrix of claim 1, wherein the matrix is moreover based on particles visible in magnetic resonance imaging (MRI).

13. A pharmaceutical composition comprising the nonbiodegradable embolization microspheres of claim 1, in association with a pharmaceutically acceptable vehicle.

14. A kit comprising the pharmaceutical composition of claim 13, in association with a pharmaceutically acceptable vehicle for parenteral administration, and at least one means of injection.

15. A kit comprising the pharmaceutical composition of claim 13 and at least one contrast agent for imaging by X-ray, by magnetic resonance or by ultrasonography, and optionally at least one means of injection for parenteral administration, the pharmaceutical composition and the at least one contrast agent being packaged separately.

16. The nonbiodegradable embolization microspheres comprising a crosslinked matrix of claim 1, wherein the cycloaliphatic or aliphatic thiols have from 2 to 24 carbon atoms.

17. The nonbiodegradable embolization microspheres comprising a crosslinked matrix of claim 5, wherein M is a single bond.

18. The nonbiodegradable embolization microspheres comprising a crosslinked matrix of claim 9, wherein the colored monomer corresponds to formula (Vb): ##STR00028## .

19. The nonbiodegradable embolization microspheres comprising a crosslinked matrix of claim 10, wherein the halogenated monomer is of formula (IV): ##STR00029## in which Y represents O-W, (O-R.sub.16).sub.p-W, (NH-R.sub.16).sub.p-W or NH-W, W representing Ar, L-Ar, and p being an integer between 1 and 10, in which: Ar represents a (C.sub.5-C.sub.36)aryl or (C.sub.5-C.sub.36)heteroaryl group, said group being substituted with one, two or three atoms of iodine and/or of bromine, and optionally substituted with one to four groups selected from (C.sub.1-C.sub.10)alkyl, -NR.sub.aR.sub.b, -NR.sub.cCOR.sub.d, -COOR.sub.e, -ORf, -OCOR.sub.g, -CONR.sub.hR.sub.i, -OCONR.sub.jR.sub.k, -NR.sub.1COOR.sub.o, -N.sub.rCONR.sub.sR.sub.t, -OCOORu, and -COR.sub.v; L represents —(CH.sub.2).sub.n—, —(HCCH).sub.n—, —O—, —S—, —SO—, —SO.sub.2—, —OSO.sub.2.sup.-; -NR.sub.17-, —CO—, —COO—, —OCO—, —OCOO—, —CONR.sub.18—, —NR.sub.19CO—, —OCONR.sub.20—, —NR.sub.21COO— or —NR.sub.22CONR.sub.23—, n being an integer from 1 to 10; R.sub.17 to R.sub.23 and R.sub.a to R.sub.v represent, independently of one another, a hydrogen atom, a (C.sub.1-C.sub.10)alkyl, said (C.sub.1-C.sub.10)alkyl optionally being substituted with 1 to 10 OR’ groups, or a group -(CH.sub.2-CH.sub.2-O).sub.q-R′, R′ being a hydrogen atom or a -(C.sub.1-C.sub.6)alkyl, q being an integer between 1 and 10, preferably between 1 and 5; R.sub.15 represents H or a (C.sub.1-C.sub.6)alkyl; R.sub.16 represents a group selected from (C.sub.1-C.sub.36)alkylene, (C.sub.3-C.sub.36)cycloalkylene, (C.sub.2-C.sub.36)alkenylene, (C.sub.3-C.sub.36)cycloalkenylene, (C.sub.2-C.sub.36)alkynylene, (C.sub.3-C.sub.36)cycloalkynylene, (C.sub.5-C.sub.36)arylene and (C.sub.5-C.sub.36)heteroarylene, the percentage of said halogenated monomer being given in moles relative to the total number of moles of monomers.

20. The pharmaceutical composition of claim 13, wherein the pharmaceutically acceptable vehicle is for parenteral administration.

Description

DESCRIPTION OF THE FIGURES

[0254] FIG. 1: Average diameter of the microspheres (MS) after sterilization as a function of the concentration of transfer agent.

[0255] FIG. 2: Percentage of defect-free microspheres as a function of the concentration of transfer agent.

[0256] FIG. 3: Examples of microspheres. A: defect-free with 0% of hexanethiol; B: fractured with 0% of hexanethiol; C: deformed with 0% of hexanethiol; D: defect-free with 6% of hexanethiol; E: fractured with 6% of hexanethiol.

[0257] FIG. 4: Dry extract as a function of the concentration of transfer agent.

[0258] FIG. 5: Degree of swelling as a function of the concentration of transfer agent.

[0259] FIG. 6: Young’s modulus as a function of the concentration of transfer agent.

[0260] The examples presented hereunder are intended to illustrate the present invention. Hereinafter, the word “microsphere”, whether in the singular or in the plural, will generally be abbreviated to MS.

EXAMPLES

Materials and Methods

Materials

[0261] 2,2′-Azobis(2-methylpropionitrile) (AIBN), poly(ethylene glycol) methacrylate (M.sub.n=300 g.mol.sup.-1) (PEG), 1-hexanethiol (95%), methacrylic acid (99%), polyvinyl alcohol (M.sub.n=30 000-70 000 g.mol.sup.-1) (PVA), thioglycolic acid (99%), 1-dodecanethiol (98%) were purchased from Sigma-Aldrich. Toluene and acetone were purchased from VWR. NaCl was purchased from Merck. Polyethylene glycol dimethacrylate (M.sub.n=1000 g.mol.sup.-1) (PEGDMA-1000) was purchased from Polysciences Inc. The violet dyes and the iodinated monomers were synthesized in Guerbet’s R&D center. All the materials were used as received, without any additional purification.

Methods

Morphology

[0262] The morphological properties of the MS obtained were characterized with the Morphologi 4 instrument (Malvern Instruments, United Kingdom). The Morphologi 4 instrument can be used for producing databases of MS images. It is able to show the size distribution of the MS that are measured. 26 different morphological parameters could be determined by the Morphologi 4 instrument. The diameter (.Math.m) was the main parameter to be investigated.

[0263] The MS were deposited on a sample holder of the Morphologi 4 instrument. 500 MS were imaged and stored in the database of the software for more-thorough analysis. A standard operating procedure (SOP) was used in the imaging process to ensure uniformity of the measurements. After each measurement, the defective MS were excluded from the total of the MS (500 MS). The most reliable method consisted of visually examining each MS and removing them or storing them in the image databases as a function of their defects and their integrity. The histogram, the mean value of the diameter and the standard deviation were also obtained for the intact MS.

[0264] Analysis of the defects: There are various classes of defects of the microspheres (MS), listed here: [0265] Class 1: Defect-free MS [0266] Class 2: Siamese-twin MS [0267] Class 3: Double-core MS [0268] Class 4: Isolated fractured MS [0269] Class 5: Residues or residues of fractured MS [0270] Class 6: MS pile after sterilization [0271] Class 7: MS core without USPIO [0272] Class 8: Very transparent MS (phantom MS) [0273] Class 9: Stack of MS in a polymer fragment [0274] Class 10: Cracked MS [0275] Class 11: Object of oval shape containing several small MS [0276] Class 12: Deformed MS

[0277] In the investigation of defects based on the 500 MS studied, the defective MS were eliminated. The percentage of defect-free MS was therefore calculated:

[00002]$\begin{array}{l}Defect\text{-}freeMS\left(\%\right)=\\ \frac{totalquantityofMS-quantityofdefectiveMS}{totalquantityofMS}\times 100\end{array}$

Dry Extract, Degree of Swelling by Weight

[0278] The dry extract is determined as follows: 1 ml of sedimented MS is placed in a 5-ml Eppendorf vial, frozen at -80° C. and lyophilized in a lyophilizer (Heto PowerDry LL 1500, Thermo Scientific) overnight. The weight of the MS after lyophilization is then measured.

[0279] Measurement was carried out for three samples and the mean value was taken as the final value of the dry weight of the MS.

[0280] Degree of swelling: The same sample preparation as that described above was used for calculating the degree of swelling by weight of the MS:

[00003]$degreeofSwellingbyweight\left(Q\right)=\frac{m_w\left(g\right)-m_d\left(g\right)}{m_d\left(g\right)}$

where (m.sub.w) is the weight in grams of 1 mL of sedimented MS and (m.sub.d) is the weight in grams of 1 ml of sedimented microspheres that have been lyophilized. Measurement was carried out for three samples and the mean value was taken as the final value of the degree of swelling by weight.

Rheology and Compressibility

[0281] The rheological properties of the MS were measured on an HR2 Discovery rheometer (TA Instruments, USA). Young’s modulus is measured using a uniaxial compression mode.

[0282] A plane-plane type of geometry with plates with a diameter of 50 mm and an initial spacing of 1600 .Math.m was used. The temperature of the samples is maintained at 25° C. by the Peltier effect. Prior to measurement, the normal force is set at zero by the software. A uniform bed with a single layer of microspheres is then deposited on the plate.

[0283] A first measurement is performed in order to determine the point of contact with the MS as well as the linear deformation conditions.

[0284] For this, the gap between the plates is reduced from 1600 .Math.m to 700 .Math.m at a speed of 16.7 .Math.m/s (1 mm/min) and the normal force is measured. The point of contact corresponds to the gap between the plates for which a normal force begins to be exerted. On continuing to reduce this the gap between the plates, the normal force follows a linear regime as a function of the applied deformation, up to a certain point. The gap between the plates during this divergence corresponds to exit from the linear regime.

[0285] A second measurement is then carried out 3 times in succession in order to measure the mean value as well as the error of measurement of Young’s modulus. This second measurement consists of placing the upper plate directly at the point of contact and applying an axial strain up to the maximum value of exit from the linear regime. The normal force measured then varies linearly as a function of the applied strain. The slope of this curve corresponds to the Young’s modulus.

Other Method Used for Measuring Young’s Modulus (Method No. 2)

[0286] Compression tests are carried out on single microspheres with a compression machine (Synergy 800, MTS, France), using a 3D-printed piston with a diameter of 15.3 mm. The force exerted is measured with a 2 N force sensor, which provides accurate, repeatable measurements starting from 1 mN. The software TestWorks4 is an interface for controlling the piston and recording the data measured by the sensor. It is necessary to use a lamp (100 W bulb) to illuminate the cell containing the microsphere being analyzed, and to allow the camera to see the microsphere and the piston clearly. The image processing software ImageJ is used for measuring the exact size of the microspheres by measuring the number of pixels in the image. The speed of the piston is fixed at 1 mm/min and the test begins with the piston positioned about 100 .Math.m above the microsphere.

[0287] With the data collected (measurements of force, time and displacement), Young’s modulus is calculated using the Hertz model, applicable to compression of a sphere between two flat plates.

Injection of the MS

[0288] The MS were injected via a microcatheter in order to test their mechanical properties during injection. A solution made up of 30 vol% of contrast product Xenetix® 350 mg iodine/ml and 70 vol% of normal saline solution was prepared. 20 mL of this solution was taken using a 20-mL syringe, and in parallel 2 mL of sedimented MS was taken from the sterilized bottle as described above using a 3-mL syringe.

[0289] The two syringes (3 mL and 20 mL) are connected to a three-way tap. The MS are suspended in the aforementioned mixture by performing about 15 to and fro movements between the two syringes.

[0290] The 3-ml syringe is used for injecting the solution of MS in suspension in a microcatheter of type Progreat® 2.8F (Terumo) or GlideCath® 4F or 5F (Terumo).

Example 1: Synthesis by Direct Suspension Polymerization of Microspheres (MS) According to the Invention (900-1200 μm)

[0291] An aqueous solution of hydrolyzed polyvinyl alcohol and sodium chloride is poured into a reactor and heated to 50° C. The organic phase containing poly(ethylene glycol) methyl ether methacrylate (m-PEGMA) (hydrophilic monomer), poly(ethylene glycol) dimethacrylate (PEGDMA) (crosslinking agent), coloring monomer, methacrylic acid (ionized or ionizable monomer), transfer agent and AIBN (initiator) dissolved in toluene is then fed into the reactor. Stirring is applied with a stirrer of the propeller type at a suitable speed for obtaining droplets of the desired diameter. The temperature is then increased to 80° C. and stirring is continued for 12 hours. The mixture is then filtered on a 40 .Math.m sieve in order to collect the MS. The MS retained by said sieve are then washed 3 times with acetone and then 3 times with water. These washed MS are then sieved between a 900 .Math.m sieve and a 710 .Math.m sieve. The MS collected between these two sieves are then sterilized in an autoclave at 121° C. for 20 minutes, which will have the effect of swelling the MS and obtaining MS of the desired size, i.e. in this case 900 to 1200 .Math.m.

[0292] The microspheres synthesized by the method described above then have the following composition (Tables 1 and 1bis):

TABLE-US-00001 MS of size 900-1200 .Math.m Process parameters O/W oil/water volume ratio ⅙ Total volume 980 mL Stirring speed 110 rpm Aqueous phase Volume of aqueous phase 840 mL PVA 30-70 kDa 0.25 wt% relative to the aqueous phase NaCl 7 wt% relative to the aqueous phase Organic phase Ratio weight of monomers/weight of organic phase 35% m-PEGMA 84.96 mol%/total moles of monomers PEGdMA 5 mol%/total moles of monomers MA 10 mol%/total moles of monomers Transfer agent X mol%/moles of m-PEGMA Dye 0.04 mol%/total moles of monomers AIBN 1 mol%/moles of methacrylate functions

TABLE-US-00002 Batch 1 2 3 4 5 6 7 8 9 L9 Transfer agent HT HT HT HT HT HT TGA DODEC BTCM X = Ratio (in moles/moles of m-PEGMA) 0% 0.1% 0.5% 1.5% 3% 4.5% 6% 3% 3% 3% HT: 1-hexanethiol, TGA: thioglycolic acid, DODEC: 1-dodecanethiol, BTCM: bromotrichloromethane

Example 2: Size of the Microspheres From Example 1

[0293] The average diameter of the microspheres is measured after sterilization, for each of batches 1 to 9 and L9 to evaluate the effect of the concentration of transfer agent on the size of the MS.

[0294] The MS were sterilized in accordance with the procedure described above.

[0295] The average diameter after sterilization of the calibrated MS (900-1200) for each of the batches is shown in FIG. 1.

[0296] The diameter of the MS after sterilization increases from 923 .Math.m to 1259 .Math.m as the concentration of HT increases (batches 1, 2, 3, 4, 5, 6 and 7). The more transfer agent the microspheres contain, the larger their size.

Example 3: Percentage of Nondefective Microspheres from Example 1

[0297] The percentage of defective MS was calculated by the method described above on a sample of MS with a size of 900-1200 .Math.m comprising different concentrations of HT as transfer agent or transfer agents of a different kind (batches 1 to 9 and L9). The results are shown in FIG. 2.

[0298] From 0% to 1.5% of HT (batches 1 to 4), the percentage of defect-free MS varies from 89% to 91%. Starting from 1.5% of transfer agent, this percentage increases and reaches a maximum (more than 95%) at 3% of transfer agent (batches 5, 8, 9 and L9).

[0299] The type of defect differs depending on the concentration of transfer agent in the microsphere. FIG. 3 shows the two examples of morphological defects observed most often. At a concentration of HT between 0% and 1.5% of HT, the MS are most often either cracked (B), or deformed (C).

[0300] At concentrations of 6% of HT or more in the MS, there are numerous MS that have burst (see FIG. 3).

Example 4: Dry Extract of the Microspheres From Example 1

[0301] FIG. 4 shows the dry extract of the MS (mg/ml), calculated by the method described above, as a function of the concentration or nature of the transfer agent.

[0302] The dry extract (mg) for a given volume of MS decreases linearly when the concentration of HT increases (batches 1, 2, 3, 4, 5, 6 and 7). The dry extract of the MS of batch L9 is about 101 mg/mL. Without transfer agent, the dry mass of 1 mL of sedimented MS is about 159 mg. At 6% of HT, this dry extract is only about 52 mg.

Example 5: Degree of Swelling of the Microspheres From Example 1

[0303] The degree of swelling of the microspheres was determined by the method described above on batches 1 to 9 and L9 in order to evaluate the effects of the concentration and nature of the transfer agent. The results are shown in FIG. 5. It can be seen that as the concentration of HT increases, the degree of swelling obtained increases.

[0304] For a concentration of HT between 0% and 6%, the degree of swelling by weight increases from 7 to 26 g/g. It is about 8.88% for the MS of batch L9.

[0305] The MS without transfer agent have therefore swollen less and have a very high dry mass. These data show that the various transfer agents tested give a suitable degree of swelling.

Example 6: Rheology and Compressibility of the Microspheres From Example 1

[0306] The compressibility of the microspheres can be characterized by measuring Young’s modulus by the method described above. The Young’s moduli of batches 1 to 9 are shown in FIG. 6. The results obtained with method No. 2 for measurement of Young’s modulus gave quite similar results.

[0307] It can be seen that Young’s modulus decreases from about 13 kPa to about 6 kPa as the concentration of HT increases from 0% to 3% and then reaches a plateau at about 6 kPa for the higher concentrations (4.5% and 6%).

[0308] The MS containing between 0% and 0.5% of HT (batches 1 to 4) are more solid and rigid and are unsuitable for injection. Starting from 1.5% of HT, the values of Young’s modulus of the MS go below 10 kPa, which is the target limit of the present invention, and are therefore softer and more flexible. Starting from a concentration of 3%, the MS become softer and more flexible.

[0309] At equal concentration of transfer agent, here 3%, all the MS have the same plateau value of about 6 kPa.

[0310] When the concentration of transfer agent is between 1.5% and less than 6%, the compressibility of the MS makes them suitable for injection by microcatheter.

Example 7: Injection of the Microspheres From Example 1 in a Microcatheter

[0311] Each of the batches 1 to 9 and L9 of MS was injected in 4Fr and 5Fr microcatheters. No blockage was observed. For batch 7 (6% of HT), the MS did not tolerate preparation for injection, and they all broke. Therefore the mechanical properties of the MS obtained with 6% of transfer agent are not compatible for injection by microcatheter.

Example 8: Synthesis by Direct Suspension Polymerization of Microspheres Intended to Be Loaded According to the Invention (100-300 μm)

[0312] An aqueous solution of hydrolyzed polyvinyl alcohol and sodium chloride is poured into a reactor and heated to 50° C. The organic phase containing poly(ethylene glycol) methyl ether methacrylate (m-PEGMA) (hydrophilic monomer), poly(ethylene glycol) dimethacrylate (PEGDMA) (crosslinking agent), methacrylic acid (MA) (ionizable monomer), hexanethiol (HT) (transfer agent), violet dye (1-(4-((2-methacryloxyethyl)oxy)phenylamino)anthraquinone), the suspension of iron nanoparticles and AIBN (initiator) dissolved in toluene is then fed into the reactor. Stirring is applied with a stirrer of the propeller type at a suitable speed for obtaining droplets of the desired diameter. The temperature is then increased to 80° C. and stirring is continued for 8 hours. The mixture is then filtered and the microspheres are washed with acetone and then with water before being sieved and then autoclaved.

[0313] Table 2 below presents the main parameters and the composition of the organic phase and of the aqueous phase:

TABLE-US-00003 100-300 .Math.m Process parameters O/W (oil/water volume ratio) 1/11 Total volume 1060 mL Stirring speed 140 rpm Aqueous phase Volume of aqueous phase 972 mL PVA 13-23 kDa 1 wt% relative to the aqueous phase NaCl 3 wt% relative to the aqueous phase Organic phase Ratio: weight of monomers/weight of organic phase 56% m-PEGMA 64.96 mol%/total moles of monomers PEGdMA 5 mol%/total moles of monomers MA 30 mol%/total moles of monomers HT 3 mol%/moles of m-PEGMA Dye 0.04 mol%/total moles of monomers USPIO 1 vol%/volume of organic phase AIBN 1 mol%/moles of methacrylate functions

Example 8bis: Synthesis by Direct Suspension Polymerization of Microspheres (MS) Without Ionizable Monomer According to the Invention (900-1200 μm) and Evaluation of the Loading Capacity

Synthesis of the 900-1200 Μm Microspheres

[0314] An aqueous solution of hydrolyzed polyvinyl alcohol and sodium chloride is poured into a reactor and heated to 50° C. The organic phase containing poly(ethylene glycol) methyl ether methacrylate (m-PEGMA) (hydrophilic monomer), poly(ethylene glycol) dimethacrylate (PEGDMA) (crosslinking agent), coloring monomer, hexanethiol (transfer agent) and AIBN (initiator) dissolved in toluene is then fed into the reactor. Stirring is applied with a stirrer of the propeller type at a suitable speed for obtaining droplets of the desired diameter. The temperature is then increased to 80° C. and stirring is continued for 12 hours. The mixture is then filtered on a 40 .Math.m sieve in order to collect the microspheres. The microspheres retained by said sieve are then washed 3 times with acetone and then 3 times with water. These washed microspheres are then sieved between a 900 .Math.m sieve and a 710 .Math.m sieve. The microspheres collected between these two sieves are then sterilized in an autoclave at 121° C. for 20 minutes, which will have the effect of swelling the microspheres and obtaining microspheres of the desired size, i.e. in this case 900 to 1200 .Math.m. The microspheres synthesized by the method described above then have the following composition:

[0315] Table 3 below presents the main parameters and the composition of the organic phase and of the aqueous phase:

TABLE-US-00004 Microspheres 900-1200 .Math.m batch L8 Process parameters O/W (oil/water) volume ratio ⅙ Total volume 490 mL Volume of organic phase 70 mL Stirring speed 100 rev/min PVA (30-70 kDa) 0.25% (by weight relative to the aqueous phase) NaCl 7% (by weight relative to the aqueous phase) Organic phase Weight of monomer/weight of the organic phase (%) 35% Hexanethiol 3 mol%/moles of m-PEGMA AIBN 1 mol%/moles of methacrylate function m-PEGMA 94.96 mol%/total moles of monomers Monomer PEGDMA 5 mol%/total moles of monomers phase MA 0 mol%/total moles of monomers Dye 0.04 mol%/total moles of monomers

Characterizations

[0316] The dry extract (dry weight) is determined as follows: 1 ml of sedimented MS is placed in a 5-ml Eppendorf vial, frozen at -80° C. and lyophilized in a lyophilizer (Heto PowerDry® LL 1500, Thermo Scientific) overnight. The weight of the microspheres after lyophilization is then measured. The measurement was carried out for three samples and the mean value was taken as the final value of the dry mass of the MS.

[0317] The average diameter is measured by analysis of microscopy images on 2000 microspheres (Morphologi 4, Malvern).

[0318] The test of injectability in microcatheters is performed with 1 mL of sediment of microspheres suspended beforehand in 10 mL of iodinated contrast medium (70% of Optiray® 300, Guerbet, 30% of normal saline solution). A homogeneous suspension of microspheres in a 3-mL syringe is then injected in the microcatheter. The microcatheters, supplied by the Terumo company, were selected such that their inside diameter is just slightly greater than the average diameter of the microspheres. The resistance felt during injection of the microspheres in the microcatheter is recorded (Table 3Bis). Blockage during injection would indicate injection failure. After injection, the microspheres are observed in the microscope in order to verify whether the microspheres regain their spherical shape.

Results of the Characterizations

[0319] TABLE-US-00005 Batches MA (%) Dry weight per ml of wet sediment (mg/mL) Average diameter (.Math.m) Young’s modulus (method 2) (kPa) Injectability in a 4Fr microcatheter (GlideCath®) Batch L8 0 72 983 6.98 ± 0.95 No blockage, very low resistance

[0320] 100-300 .Math.m microspheres are also synthesized without methacrylic acid (same composition as the microspheres in example 8 with 0% of methacrylic acid (MA) and 94.96% of m-PEGMA) and then sterilized by autoclaving. Their capacity for loading with doxorubicin is evaluated and compared to that of microspheres as synthesized according to example 8.

[0321] Loading with doxorubicin: the target of loading is 37.5 mg of doxorubicin per ml of microspheres. For this, 3.8 mL of doxorubicin-HCl (Adriblastine®, Pfizer) in solution in water at 2.5 mg/mL is added to 250 .Math.L of wet sediment of microspheres. After mixing by inversion, the suspension is made up to 6 mM with sodium bicarbonate (Lavoisier). Loading is carried out at room temperature, with stirring for one hour. Measurement of the residual amount of doxorubicin (absorbance at 490 nm) present in the supernatants serves for determining the amount of drug loaded on the microspheres.

TABLE-US-00006 Results Doxorubicin loading (mg of doxo/mL of wet sediment) Efficiency of loading % Without methacrylic acid 29.3 82.6 With methacrylic acid 30% 35.3 99.6

[0322] The efficiency of loading is calculated from the following equation:

[00004]$LC\left(mgofdrug/mLofMS\right)=\frac{m_{Druginitial}-C_{Drug\_sup}\ast V_{sup}}{V_{MS}}$

[00005]$LE\left(\%\right)=\frac{LC}{m_{Druginitial}}$

[0323] LC: Loading capacity [0324] LE: Loading efficiency [0325] M.sub.Drug.sub.initial: Amount of drug dissolved [0326] C.sub.Drug_sup: Concentration of the drug in the supernatant after loading [0327] V.sub.sup: Volume of the supernatant [0328] V.sub.MS: Volume of microspheres

[0329] The efficiency of loading without methacrylic acid is 82.6%, compared to 99.6% in the presence of 30% of methacrylic acid. The capacity of the microspheres without ionizable monomer for loading of doxorubicin can be explained by the establishment of hydrophobic or van der Waals bonds. In the presence of ionizable monomer, besides these bonds, doxorubicin is loaded by electrostatic bonds. The kinetics and the loading capacities are improved thereby.

Example 9: Synthesis by Direct Suspension Polymerization of Polymers Containing 5% of Maoetib According to the Invention in the Form of Microspheres of Size 700-900 μm

[0330] An aqueous solution of hydrolyzed polyvinyl alcohol and sodium chloride is poured into a reactor and heated to 50° C. The organic phase containing poly(ethylene glycol) methyl ether methacrylate (m-PEGMA) (hydrophilic monomer), poly(ethylene glycol) dimethacrylate (PEGDMA) (crosslinking agent), methacrylic acid (MA) (ionizable monomer), MAOETIB (halogenated monomer), hexanethiol (transfer agent), violet dye (1-(4-((2-methacryloxyethyl)oxy)phenylamino)anthraquinone) and AIBN (initiator) dissolved in toluene is then fed into the reactor. Stirring is applied with a stirrer of the propeller type at a suitable speed for obtaining droplets of the desired diameter. The temperature is then increased to 80° C. and stirring is continued for 12 hours. The mixture is then filtered and the microspheres are washed with acetone and then with water before being sieved and then autoclaved.

[0331] Table 5 below presents the main parameters and the composition of the organic phase.

TABLE-US-00007 700-900 .Math.m Process parameters O/W (oil/water volume ratio) ⅙ Volume of organic phase 140 mL Stirring speed 105 RPM PVA (30-70 kDa) 0.25% (by weight of the aqueous phase) NaCl 7% (by weight of the aqueous phase) Organic phase Ratio: weight of monomers/weight of organic phase 50% Hexanethiol 3 mol%/moles of mPEGma AIBN 1 mol%/moles of methacrylate function Monomer phase m-PEGMA 79.96 mol%/total moles of monomers PEGDMA 5 mol%/total moles of monomers Methacrylic acid 10 mol%/total moles of monomers MAOETIB 5 mol%/total moles of monomers Dye 0.04 mol%/total moles of monomers

Example 10: Suspending the Microspheres From Example 9 in a 50/50 Mixture of Contrast Agent and Normal Saline Solution, and Comparison With Equivalent Microspheres Without Maoetib

[0332] 2 mL of sediment of beads is added to a mixture of 10 mL of 50/50 normal saline solution/contrast agent (5 mL of Optiray® 300 mgl/mL and 5 mL of normal saline solution).

[0333] The mixture is passed 5 times through a three-way tap using 20-mL syringes.

[0334] The syringe containing the mixture is then positioned vertically and the destabilization of the mixture is observed; the microspheres then rise to the surface. The time corresponding to a destabilization interface arriving at mid-height of the syringe is measured.

[0335] For the particles of size 700-900 .Math.m not containing MAOETIB, this time is 20 seconds, whereas it is 120 seconds for the particles containing 5% of MAOETIB. This destabilization time even increases to 220 seconds if 7% of MAOETIB is added.

[0336] The presence of MAOETIB makes it possible to stabilize the suspension.

Example 11: Synthesis by Direct Suspension Polymerization of Polymers of Various Kinds in the Form of Microspheres of Size 700-900 μm

Synthesis

[0337] An aqueous solution of hydrolyzed polyvinyl alcohol and sodium chloride is poured into a reactor and heated to 50° C. The organic phase containing hydrophilic monomer, crosslinking agent, coloring monomer, transfer agent, the halogenated monomer if applicable, the ionizable monomer if applicable and AIBN (initiator) dissolved in toluene is then fed into the reactor. Stirring is applied with a stirrer of the propeller type at a suitable speed for obtaining droplets of the desired diameter. The temperature is then increased to 80° C. and stirring is continued for 12 hours. The mixture is then filtered on a 40 .Math.m sieve in order to collect the microspheres. The microspheres retained by said sieve are then washed 3 times with acetone and then 3 times with water. These washed microspheres are then sieved between a 900 .Math.m sieve and a 710 .Math.m sieve. The microspheres collected between these two sieves are then sterilized in an autoclave at 121° C. for 20 minutes, which will have the effect of swelling the microspheres and obtaining microspheres of the desired size, i.e. in this case 700 to 900 .Math.m. Table 6 below presents the main parameters and the composition of the organic phase.

TABLE-US-00008 batch L10 700-900 .Math.m batch L12 700-900 .Math.m Process parameters O/W (oil/water) volume ratio ⅙ ⅙ Total volume 980 mL 980 mL Volume of organic phase 140 mL 140 mL Stirring speed 100 rev/min 100 rev/min PVA (30-70 kDa) (by weight relative to the aqueous phase) 0.25% 0.25% NaCl (by weight relative to the aqueous phase) 7% 7% Organic phase Weight of monomer/weight of the organic phase 35% 35% Transfer agent (moles/moles of monomer) Hexanethiol 3% Hexanethiol 3% AIBN (moles/moles of methacrylate function) 1% Monomer phase (mol%/total moles of monomers) Hydrophilic monomer N-vinylpyrrolidone 74.96% mPEGMA 79.96% Crosslinking agent PEG-diacrylate 5% PEGDMA 5% Ionizable monomer Methacrylic acid 20% Methacrylic acid 10% Halogenated monomer / 2-(2-(2-(2,3,5-triiodobenz-amido)ethoxy)ethyl methacrylate 5% Dye 0.04% 0.04%

Characterization and Results

[0338] Characterization is carried out in the same way as in example 8bis and the results are presented in Table 7.

TABLE-US-00009 Batches Dry weight per ml of wet sediment (mg/mL) Average diameter (.Math.m) Young’s modulus (method 2) Injectability in GlideCath® 4Fr microcatheter (ID(1) = 1030 .Math.m) Batch L10 60.4 834 1.01 ± 0.25 No blockage, low resistance Batch L12 80.4 837 7.59 ± 0.75 No blockage, low resistance

Example 12: Synthesis by Direct Suspension Polymerization of Polymers Containing Different Amounts of Maoetib According to the Invention in the Form of Microspheres of Size 100-300 μm

Synthesis

[0339] An aqueous solution of hydrolyzed polyvinyl alcohol and sodium chloride is poured into a reactor and heated to 50° C. The organic phase containing poly(ethylene glycol) methyl ether methacrylate (m-PEGMA) (hydrophilic monomer), poly(ethylene glycol) dimethacrylate (PEGDMA) (crosslinking agent), methacrylic acid (MA) (ionizable monomer), MAOETIB (halogenated monomer), hexanethiol (transfer agent), violet dye (1-(4-((2-methacryloxyethyl)oxy)phenylamino)anthraquinone) and AIBN (initiator) dissolved in toluene is then fed into the reactor. Stirring is applied with a stirrer of the propeller type at a suitable speed for obtaining droplets of the desired diameter. The temperature is then increased to 80° C. and stirring is continued for 12 hours. The mixture is then filtered and the microspheres are washed with acetone and then with water before being sieved and then autoclaved.

[0340] Table 8 below presents the main parameters and the composition of the organic phase.

TABLE-US-00010 Microspheres 100-300 .Math.m L13 L14 L15 Process parameters O/W (oil/water) volume ratio ⅙ Total volume 1060 mL 1060 mL 1200 mL Volume of organic phase 88 mL 88 mL 130 mL Stirring speed 150 rpm 150 rpm 150 rpm PVA (13-23 kDa) 0.25% (by weight relative to the aqueous phase) NaCl 7% (by weight relative to the aqueous phase) Organic phase Weight of monomer/weight of the organic phase (%) 56% 50% 50% Hexanethiol 3 mol%/moles of m-PEGMA AIBN 1 mol%/moles of methacrylate function Monomer phase (in moles/total moles of monomers) m-PEGMA 84.96% 79.96% 64.96% PEGDMA 5% MA 10% MAOETIB 0% 5% 20% Dye 0.04%

Example 13: Suspending Microspheres From Example 12 With Different Levels of Maoetib in a 50/50 Mixture of Contrast Agent and Normal Saline Solution

[0341] 2 mL of sediment of beads is added to a mixture of 10 mL of 50/50 normal saline solution/contrast agent (5 mL of Xenetix® 350 mgl/mL and 5 mL of normal saline solution). The mixture is passed 5 times through a three-way tap using 20-mL syringes. The syringe containing the mixture is then positioned vertically and destabilization of the mixture is observed, either through frothing or through sedimentation depending on the concentration of MAOETIB. The time corresponding to a destabilization interface arriving at mid-height of the syringe is measured. The results are presented in Table 9.

TABLE-US-00011 Microsphere MAOETIB % Mid-height destabilization time Type of destabilization L13 0 6 min frothing L14 5 12 min frothing L15 20 2.5 min sedimentation

[0342] Adding a small amount of MAOETIB makes it possible to delay frothing by bringing the density of the microspheres closer to that of the suspension medium. However, beyond a certain concentration, the density of the microspheres becomes too high and they sediment rapidly.