Radiopaque polymers

Abstract

A hydrophilic polymer comprising pendent groups of the formula I: Wherein: W is independently selected from —OH, —COOH, —SO.sub.3H, —OPO.sub.3H, —O—(C.sub.1-4alkyl), —O—(C.sub.1-4alkyl)OH, —O—(C.sub.1-4alkyl)R.sup.2, —O—(C.sub.2H.sub.5O).sub.qR.sup.1—(C═O)—O—C.sub.1-4alkyl and —O—(C═O)C.sub.1-4alkyl; or a group —BZ; wherein —OH, COOH, O—PO.sub.3H and SO.sub.3H maybe in the form of a pharmaceutically acceptable salt; wherein: B is a bond, or a straight branched alkanediyl, oxyalkylene, alkylene oxaalkylene, or alkylene (oligooxalkylene) group, optionally containing one or more fluorine substituents; and Z is an ammonium, phosphonium, or sulphonium phosphate or phosphonate ester zwitterionic group; X is either a bond or a linking group having 1 to 8 carbons and optionally 1 to 4 heteroatoms selected from O, N and S; G is a coupling group through which the group of the formula I is coupled to the polymer and is selected from ether, ester, amide, carbonate, carbamate, 1,3 dioxolone, and 1,3 dioxane; R.sup.1 is H or C.sub.1-4 alkyl; R.sup.2 is —COOH, —SO.sub.3H, or —OPO.sub.3H.sub.2 q is an integer from 1 to 4; n is an integer from 1 to 4; p is an integer from 1 to 3; and n+p is from 2 to 5; and wherein —COOH, —OPO.sub.3H.sub.2 and —SO.sub.3H as well as phenolic —OH maybe in the form of a pharmaceutically acceptable salt.

Claims

1. A radiopaque microsphere comprising a hydrophilic polymer comprising pendent groups of the formula I: ##STR00040## wherein: W is independently selected from —OH, —COOH, —SO.sub.3H, —OPO.sub.3H, —O—(C.sub.1-4 alkyl), —O—(C.sub.1-4 alkyl)OH, —O—(C.sub.1-4 alkyl)R.sup.2, —O—(C.sub.2H.sub.5O).sub.qR.sup.1—(C═O)—O—C.sub.1-4 alkyl and —O—(C═O)C.sub.1-4 alkyl; or a group —BZ; wherein —OH, COOH, O—PO.sub.3H and SO.sub.3H maybe in the form of a pharmaceutically acceptable salt; wherein: B is a bond, or a straight branched alkanediyl, oxyalkylene, alkylene oxaalkylene, or alkylene group, optionally containing one or more fluorine substituents; Z is an ammonium, phosphonium, or sulphonium phosphate or phosphonate ester zwitterionic group; X is either a bond or a linking group having 1 to 8 carbons and optionally 1 to 4 heteroatoms selected from O, N and S; G is a coupling group through which the group of the formula I is coupled to the polymer and is selected from 1,3 dioxolane and 1,3 dioxane; R.sup.1 is H or C.sub.1-4 alkyl; R.sup.2 is —COOH, —SO.sub.3H, or —OPO.sub.3H.sub.2 q is an integer from 1 to 4; n is an integer from 1 to 4; p is an integer from 1 to 3; and n+p is from 2 to 5; and wherein —COOH, —OPO.sub.3H.sub.2 and —SO.sub.3H as well as phenolic —OH may be in the form of a pharmaceutically acceptable salt.

2. The microsphere of claim 1, wherein the polymer is a polyhydroxylated polymer.

3. The microsphere of claim 1, which is a polymer or co-polymer of polyvinyl alcohol and wherein the groups of the formula I are coupled through hydroxyl groups of the polyvinyl alcohol.

4. The microsphere of claim 1, in which n is two or three.

5. The microsphere of claim 1, in which the phenyl ring of the group of the formula I is 3,5 diiodinated, 3,4,5 tri iodinated or 2,4,6 triiodinated.

6. The microsphere of claim 1, in which W is independently selected from —OH, —COOH, —SO.sub.3H, —OPO.sub.3H.sub.2, —O—(C.sub.1-4 alkyl), —O—(C.sub.1-4 alkyl)OH, —O—(C.sub.1-4 alkyl)R.sup.2, —O—(C.sub.2H.sub.5O).sub.qR.sup.1—(C═O)—O—C.sub.1-4 alkyl and —O—(C═O)C.sub.1-4 alkyl or a group —BZ, wherein B is a bond, a C.sub.1 to 6 branched or non branched alkanediyl group or a branched or non branched C.sub.1-6 oxyalkylene group; and Z is a group of formula II ##STR00041## wherein A.sup.3 and A.sup.4, are the same or different and are selected from —O, —S, and —NH—; and W.sup.1+ is —W.sup.2—N.sup.+R.sup.4.sub.3, in which W.sup.2 is C.sub.1-6 alkanediyl and R.sup.4 are the same or different and each is hydrogen or C.sub.1-4 alkyl; and wherein —OH, COOH, —OPO.sub.3H.sub.2 and —SO.sub.3H may be in the form of a pharmaceutically acceptable salt.

7. The microsphere of claim 1, in which W is selected from —OH, —COOH, —SO.sub.3H, —OPO.sub.3H.sub.2, —O—(C.sub.1-4 alkyl)R.sup.2 and —O—(C.sub.2H.sub.5O).sub.qR.sup.1; wherein —OH, —COOH, —OPO.sub.3H.sub.2 and —SO.sub.3H may be in the form of a pharmaceutically acceptable salt.

8. The microsphere of claim 1, in which the phenyl ring of the group of the formula I is substituted in one of the following patterns: ##STR00042##

9. The microsphere of claim 1, in which p is two or three.

10. The microsphere of claim 1, which is cross-linked.

11. The microsphere of claim 1, wherein the microsphere is in dried form.

12. The microsphere of claim 1, wherein the microsphere is in the form of a hydrogel comprising greater than 50% water wt/wt.

13. The microsphere of claim 1, having an iodine content of greater than 10 mg iodine per mg dried polymer.

14. The microsphere of claim 1, which has a radiodensity of greater than 500 HU.

15. The microsphere of claim 1, which is substituted by groups, other than those in W, which are charged at pH7.4.

16. The microsphere of claim 1, which additionally comprises a pharmaceutical active ingredient.

17. The microsphere of claim 16, wherein the polymer is charged and the pharmaceutical active ingredient is reversibly bound within the polymer by ionic interaction.

Description

FIGURES

(1) FIG. 1 illustrates a selection of microspheres of the invention prepared according to the examples below.

EXAMPLES

Example 1: Synthesis of 3,5-Diiodo-2-(2-(2-methoxyethoxy)ethoxy)benzaldehyde

(2) ##STR00024##

(3) To an HEL PolyBlock8 parallel synthesis 125 ml reactor fitted with a reflux condenser and suspended magnetic stirrer, was added 3,5-diiodosalicylaldehyde (13.9011 g, 37.72 mmol, 1.0 eq) and TBAI (2.7481 mg, 0.802 mmol, 0.2 eq). To this was added water and the pH adjusted to 9.5 with 1M NaOH (total aqueous volume 97 ml). The reactor was set to 500 rpm stirring until full dissolution to give a bright yellow solution and 1-bromo-2-(2-methoxyethoxy)ethane (5.00 ml, 37.17 mmol, 1.0 eq) was added. The reactor zone was set to heat to 120° C. The reaction was monitored by Thin Layer Chromatography (TLC) (30% EA in i-hex) and after 2 hours additional bromide was added (2.50 ml, 18.59 mmol, 0.5 eq). After a further 0.5 hours, the pH was readjusted to 9.5 due to consumption of the bromide. After a further 2 hours additional bromide (1.25 ml, 9.29 mmol, 0.25 eq) were added and the reactor turned down to 50° C. and left to stir overnight. After 19 hours, the resulting suspension was reheated to reflux for 1 hour, cooled to room temperature and transferred to a separating funnel in ethyl acetate (400 ml). The organics were washed twice with saturated sodium bicarbonate, dried with magnesium sulfate, hot filtered from toluene, and recrystallised from toluene/isohexane to give, after filtration and hi-vacuum drying, the desired product as a yellow powder (15.2909 g, 86.4% yield); δ.sub.H (CDCl.sub.3, 500.1 MHz)/ppm; 10.31 (1H, s), 8.31 (1H, d, 2.2 Hz), 8.09 (1H, d, 2.2 Hz), 4.26 (2H, app. t, 4.5 Hz), 3.89 (2H, app. t, 4.5 Hz), 3.67 (2H, app. t, 4.6 Hz), 3.55 (2H, app. t, 4.6 Hz), 3.38 (3H, s); δc NMR (CDCl.sub.3, 125.8 MHz)/ppm; 188.71 (CH), 161.55 (q), 152.43 (CH), 137.57 (CH), 131.75 (q), 94.07 (q), 89.19 (q), 75.56 (CH.sub.2), 71.90 (CH.sub.2), 70.79 (CH.sub.2), 70.06 (CH.sub.2), 59.13 (CH.sub.3).

Example 2: Synthesis of 3-Hydroxy-2,4,6-triiodobenzaldehyde

(4) ##STR00025##

(5) To a 2 L 3-necked round bottomed flask with large oval stirrer bar was added 3-hydroxybenzaldehyde (10.007 g, 81.89 mmol), sodium iodide (0.614 g, 4.09 mmol, 0.05 eq) and sodium carbonate (93.028 g, 877.44 mmol, 10.7 eq), rinsed in with a total of 750 ml of deionised water. When the benzaldehyde had dissolved to give a bright yellow stirred solution, iodine balls (70.008 g, 275.80 mmol, 3.37 eq) was added in 2 portions over 30 minutes and rinsed in with 225 ml of water each time. The reaction is followed by TLC (60% DCM in i-hex) and over 3 hours, the iodine almost completely dissolves resulting in a dark yellow/orange precipitate. The solid was isolated by Buchner filtration and washed with i-hexane to remove any residual iodine. The isolated solid was re-dissolved in warm water (2 L, 45° C.) to give a clear brown solution to which 100 ml of sat. sodium thiosulfate solution were added to reduce any remaining iodine. The pH of the solution was cautiously reduced from 10.2 to 3.26 using 1M HCl (care due to evolution of CO.sub.2). The solid was isolated by filtration, washed with water (2×500 ml) and dried in a high vacuum oven at 30° C. to give the desired compound as a yellow solid (37.002 g, 90.3% yield, 97.2% HPLC purity); δ.sub.H (CDCl.sub.3, 500.1 MHz)/ppm; 9.65 (1H, s), 8.35 (1H, s), 6.42 (1H, s); δc NMR (CDCl.sub.3, 125.8 MHz)/ppm; 194.90 (CH), 155.12 (q), 149.77 (CH), 135.69 (q), 88.78 (q), 87.66 (q), 85.70 (q).

Example 3: Synthesis of 2,4,6-triiodo-3-(2-(2-methoxyethoxy)ethoxy)benz aldehyde

(6) ##STR00026##

(7) To a flame dried 250 ml 3-necked round bottomed flask under a nitrogen atmosphere containing a stir bar and fitted with a reflux condenser, were added 3-hydroxy-2,4,6-triiodobenzaldehyde (15.627 g, 31.3 mmol, 1.0 eq), sodium iodide (469 mg, 3.13 mmol, 0.1 eq), anhydrous sodium carbonate (3.981 g, 37.6 mmol, 1.2 eq) and anhydrous dimethylformamide (DMF) (160 ml). The suspension was stirred until the aldehyde had completely dissolved, then 1-bromo-2-(2-methoxyethoxy)ethane (6.87 g, 37.5 mmol, 1.2 eq) was added by syringe and the reaction heated to reflux. After 2 hours, TLC analysis (10% EA in i-hex) indicated the start material was consumed and the reaction was cooled to room temperature, transferred to a 250 ml round bottomed flask and evaporated to dryness under high vacuum. The resulting suspension was diluted with 500 ml of ethyl acetate, washed with 3×100 ml 1M NaOH, 2×100 ml sat. brine, decolourised with activated charcoal and dried with magnesium sulfate. The resulting solution was concentrated to dryness, and purified by silica column chromatography (2-20% ethyl acetate in i-hexane) and dried under high vacuum to give the desired compound as a yellow powder (7.556 g, 40.1%); δ.sub.H (CDCl.sub.3, 500.1 MHz)/ppm; 9.65 (1H, s), 8.44 (1H, s), 4.20 (2H, t, 6.4 Hz), 4.01 (2H, t, 6.4 Hz), 3.79 (2H, app. t, 5.8 Hz), 3.60 (2H, app. t, 5.8H), 3.41 (3H, s); Sc NMR (CDCl.sub.3, 125.8 MHz)/ppm; 194.97 (CH), 159.10 (q), 150.83 (CH), 138.27 (q), 97.06 (q), 95.70 (q), 90.40 (q), 72.47 (CH.sub.2), 72.04 (CH.sub.2), 70.89 (CH.sub.2), 68.89 (CH.sub.2), 59.19 (CH.sub.3).

Example 4: Synthesis of 2,4,6-Triiodo-3-(2-(2-(2-methoxyethoxy)ethoxy) ethoxy)benz aldehyde

(8) ##STR00027##

(9) To a flame dried 100 ml 3-necked round bottomed flask containing a stirrer under a nitrogen blanket, was added triphenylphosphine (1.7216 g, 6.502 mmol, 1.3 eq) and anhydrous tetrahydrofuran (THF) (35 ml). The stirring was started and, after full dissolution of the Triphenylphosphine (PPh.sub.3), the reactor was cooled to ca 0° C. in an ice-bath. To the colourless solution was added to diisopropyl azodicarboxylate (DIAD) (1.28 ml, 6.502 mmol, 1.3 eq) dropwise via syringe resulting in a persistent yellow solution. After stirring for 5 minutes, triethylene glycol monomethyl ether (1.04 ml, 6.502 mmol, 1.3 eq) was added dropwise by syringe. After stirring for a further 5 minutes, the 3-hydroxy-2,4,6-triiodobenzaldehyde (2.5077 g, 5.002 mmol, 1.0 eq) was added in one portion resulting in an immediate colour change. The reaction was monitored by TLC (5% Et.sub.2O in toluene) and left to stir overnight. The solution was diluted with ether to precipitate triphenylphosphine oxide and then concentrated to dryness. The resulting thick oil was purified by column chromatography (2-10% Et.sub.2O in toluene) to give, after concentration and high vacuum drying, the desired product as a yellow powder (3.2077 g, 99% yield, 94.4% HPLC purity); δ.sub.H (DMSO-D.sub.6, 500.1 MHz)/ppm; 9.58 (1H, s), 8.47 (1H, s), 4.08 (2H, t, 4.9 Hz), 3.57-3.53 (4H, m), 3.44 (2H, app. t, 4.8 Hz), 3.24 (3H, s).

Example 5: Synthesis of 3,4,5-Triiodosalicylaldehyde

(10) ##STR00028##

(11) To a 3-necked 2 L round bottomed flask containing a large oval stirrer was added 4-iodo-salicilaldehyde (25.01 g, 100.86 mmol, 1.0 eq) and acetic acid (300 ml). After stirring for 5 mins to allow the solid to dissolve, pre-warmed liquid iodine monochloride (39.11 g, 2.4 eq) was diluted with AcOH (100 ml) and transferred to a dropping funnel on the round bottomed flask. This solution was added over 10 mins. The reactor was then placed in a large silicone oil batch a fitted with a 1 L dropping funnel, thermometer and condenser and set to heat to 80° C. During the heat up, water (700 ml) was slowly added to the solution causing a yellow/orange precipitation. After 20 mins at 80° C., the heating was turned off. After a further 30 minutes the heating bath was removed and the black solution/yellow suspension allowed to cool to room temperature and stirred for 65 hours; the reaction was analysed by TLC (20% EA in iHex). The solid was isolated by Buchner filtration and washed with water (2×500 ml). To remove residual iodine crystals, the solid was repeatedly re-slurried with i-hexane (200 ml) until the i-hexane supernatant was no longer purple. The isolated solid was dried in a hi-vac oven overnight to give the desired product as a yellow crystalline solid (40.84 g, 81% yield, 93.2% pure by HPLC analysis). The product could be further recrystallised to higher purity from acetone:water (9:1); δ.sub.H (CDCl.sub.3, 500.1 MHz)/ppm; 12.15 (1H, s), 9.67 (1H, s), 8.09 (1H, s); 6c NMR (CDCl.sub.3, 125.8 MHz)/ppm; 194.53 (CH), 159.58 (C), 142.24 (CH), 133.39 (C), 120.87 (C), 101.68 (C), 94.02 (C).

Example 6: Synthesis of 3,4,5-Triiodo-2-(2-(2-methoxyethoxy)ethoxy) benzaldehyde

(12) ##STR00029##

(13) (5 g scale): To a flame dried 3-necked 250 ml round bottomed flask containing a small octagonal stirrer bar under a positive pressure of nitrogen, was added triphenylphosphine (2.76 g, 10.5 mmol, 1.05 eq) and dry THE (70 ml) by syringe. The round bottomed flask was placed in a Dewer bath fitted with a low temperature thermometer and cooled to −68° C. with an ethanol/liquid nitrogen bath. Diethyl azodicarboxylate (1.65 ml, 10.5 mmol, 1.05 eq) was added dropwise by syringe over 1 min and left to stir for 5 mins to give a yellow suspension. Diethyleneglycol mono-methyl ether (1.77 ml, 15 mmol, 1.5 eq) was then added dropwise and left to stir for 5 mins. To this was added solid 3,4,5-triiodosalicylaldehyde (5.00 g, 10.0 mmol, 1.0 eq) in one portion. The initial dark orange/red suspension lightened to give a pale yellow solution which was allowed to stir for 2 hours, monitored by TLC analysis (20% ether in toluene) and left to warm up to room temperature overnight. TLC indicated complete consumption of aldehyde starting material with a clean reaction profile. The resulting solution was transferred to a 500 ml round bottomed flask, diluted with ether (200 ml) and cooled in the freezer. The resulting suspension was filtered through a short silica plug to remove triphenylphosphine oxide and flushed with further ether (200 ml). The resulting solution was concentrated to dryness, and purified by column chromatography eluting with ether in toluene (2-20%) with product fractions concentrated to dryness and dried under high vacuum to give the desired product as a yellow amorphous solid (4.91 g, 82% yield, 96% HPLC purity); δ.sub.H (CDCl.sub.3, 500.1 MHz)/ppm; 10.26 (1H, s), 8.34 (1H, s), 4.22 (2H, t, 4.5 Hz), 3.90 (2H, t, 4.5 Hz), 3.90 (2H, t, 4.6 Hz), 3.55 (2H, t, 4.6 Hz), 3.38 (3H, s); δc NMR (CDCl.sub.3, 125.8 MHz)/ppm;

Example 7: Synthesis of 5-((2,2-Dimethoxyethyl)amino)-2,4,6-triiodoisophthalic acid

(14) ##STR00030##

(15) To a flame dried 500 ml round bottomed flask under nitrogen, was added solid 5-amino-2,4,6-triiodoisophthalic acid (46.95 g, 84.03 mmol, 1.0 eq), sodium bicarbonate (28.21 g, 335.8 mmol, 4.0 eq) and DMF (ca 400 ml) via cannula. To the resulting brown solution was added 2-bromo-1,1-dimethoxyethane (13 ml, 110.0 mmol, 1.3 eq) dropwise and the resulting solution heated to reflux for 18 hours. After cooling to room temperature, the majority of DMF was removed by rotary evaporation under vacuum (9 mBar, 55° C.) and the resulting orange solid extracted with ethyl acetate (1 L). This suspension was washed with saturated lithium chloride solution (7×400 ml) to remove residual DMF and salts, dried over magnesium sulfate, filtered and evaporated to dryness. The resulting solid was recrystallised from ethyl acetate, washed with i-hexane and filtered. This process was repeated a total of 3 times and the resulting orange solid dried under high vacuum to give the title compound (33.04 g, 61%, 91.7% HPLC purity). The product could be further purified via silica gel column chromatography (MeOH in DCM, 0-15%) (4.91 g, 82% yield, 96% HPLC purity); δ.sub.H (CDCl.sub.3, 500.1 MHz)/ppm; 8.01 (1H, s), 4.86 (2H, br s), 4.76 (1H, t, 5.5 Hz), 4.37 (2H, d, 5.5 Hz), 3.44 (δ.sub.H, s); δc NMR (CDCl.sub.3, 125.8 MHz)/ppm;

Example 8: Synthesis of Potassium 3-(3-formyl-2,4,6-triiodophenoxy)propane-1-sulfonate and 3-(1-formyl-3,4,5-triiodophenoxy)propane-1-sulfonate, sodium salt

(16) ##STR00031##

(17) In a 150 mL three-neck round bottom flask, 3-hydroxy-2,4,6-triiodobenzaldehyde (10 g, 20 mmol, 1.0 eq) was dissolved in anhydrous THF (50 ml) by magnetic stirrer. Potassium t-butoxide (2.47 g 22 mmol, 1.1 eq) was mixed with 20 mL of THF and the suspension was added slowly into the flask under nitrogen atmosphere at room temperature, followed by increasing temperature to 40° C. to allow a full dissolution of product. Then sultone (15 g, 120 mmol, 6.0 eq) of was dissolved in 15 mL of THF and the mixture was added slowly to the reaction flask. A precipitation appeared almost immediately. After 3 hours reaction at 40° C., the reaction mixture were poured into 500 mL of ethyl acetate to receive solid raw product. The filtered solid was washed with 100 mL of ethyl acetate, and recrystallized from ethanol. After vacuum drying over 24 hours, the desired product (10.7 g, 80% yield) was isolated; δ.sub.H (D.sub.2O, 500.1 MHz)/ppm; 2.24-2.34 (m, 2H), 3.12-3.25 (t, 2H), 3.88-4.02 (t, 2H), 8.18-8.25 (s, 1H), 9.42-9.50 (s, 1H) δ.sub.C NMR (CDCl.sub.3, 125.8 MHz)/ppm; Element analysis result: C 18.56, H 2.22, S 5.66, 152.31, K 6.27. Cal: C 18.20, H 1.22, S 4.85, 157.68, K 5.92.

(18) 3-(1-formyl-3,4,5-triiodophenoxy)propane-1-sulfonate, sodium salt was synthesized analogously from 3,4,5-triiodosalicylaldehyde (Example 6).

Example 9: Preparation of Microspheres

(19) Microspheres were prepared according to Example 1 of WO2004/071495 (high AMPS method). The process was terminated after the step in which the product was vacuum dried to remove residual solvents. Beads were then sieved to provide appropriate size ranges. Beads were either stored dry or in physiological saline and autoclaved. Unless otherwise stated coupling was carried out on batches of microspheres having diameters between 70 and 170 μm and reactions were carried out on dried beads that were swollen in the appropriate solvent prior to use.

Example 10: General Microsphere Coupling Method

(20) To a pre-dried reactor under a nitrogen blanket was added the desired chemical substrate (typically 0.6 eq w.r.t. PVA diol functionalities), anhydrous solvent (typically dimethyl sulfoxide (DMSO) or N-Methyl-2-pyrrolidone (NMP), 30 vol w.r.t. particle mass) and catalyst (typically 2.2 vol w.r.t. particle mass). With stirring, the solution was warmed up to reaction temperature (40-80° C.). Bead micro-particles were then added, rinsed in to the reactor with further anhydrous solvent (typically 5 vol w.r.t. particle mass). The reaction was then stirred under an N.sub.2 blanket and the reaction conversion was monitored by High Performance Liquid Chromatography (HPLC) for consumption of the chemical substrate. At a pre-determined time (typically when bead uptake of chemical had ceased), the stirring was switched off and the beads allowed to settle. The supernatant fluid was removed by aspiration through a filter membrane and solvent (typically 35 vol of either 0.5% w/w NaCl in DMSO or NMP) was charged and stirred for up to 10 minutes. The solvent washing was repeated for a total of 5 solvent washes and a further 5 washes with 0.9% saline (typically 50 vol w.r.t. particle mass). The resulting particle suspension was transferred to a 10 ml Schott vial in PBS and autoclaved at 121° C. for 30 mins then cooled to room temperature.

Example 11: Characterization of Radiopaque Microspheres

(21) The dry weight of beads was measured by removing the packing saline and wicking away remaining saline with a tissue. The beads were then vacuum dried at 50° C. overnight to remove water, and the dry bead weight and solid content (w/w %) of polymer were obtained from this. To determine iodine levels per unit volume, settled volume of fully hydrated beads is determined, for example by measuring cylinder, and the beads are then dried and iodine content is determined. The iodine content in dry, beads were measured by elemental analysis according to the Schöniger Flask method.

Example 12: X-Ray Analysis of Individual Radiopaque Beads and Liquid Embolic Polymers

(22) Micro-CT was used to evaluate the radiopacity of samples of radiopaque embolic beads prepared according to general example 10 above. The samples were prepared in Nunc cryotube vials (Sigma-Aldrich product code V7634, 48 mm×12.5 mm). The beads were suspended in 0.5% agarose gel (prepared with Sigma-Aldrich product code A9539). The resulting suspension is generally referred to as a “Bead Phantom”. To prepare these bead phantoms, a solution of agarose (1%) is first raised to a temperature of approximately 50° C. A known amount of the beads is then added, and the two gently mixed together until the solution starts to solidify or gel. As the solution cools it gels and the beads remain evenly dispersed and suspended within the agarose gel.

(23) Bead phantoms were tested for radiopacity using micro-Computer Tomography (Micro-CT) using a Bruker Skyscan 1172 Micro-CT scanner at the RSSL Laboratories, Reading, Berkshire, UK, fitted with a tungsten anode. Each phantom was analysed using the same instrument configuration with a tungsten anode operating at a voltage of 64 kV and a current of 155 μA. An aluminium filter (500 μm) was used.

(24) For liquid embolic samples, a two part analysis method is used. Initially an interpolated region of interest is created coving the inner tube diameter to include the plug and any void structures then the image is segmented to isolate the polymer from the void structures so as to report polymer radiodensity. The radiodensity in HU was then calculated using the water standard acquired on the same day. Table 1 gives the acquisition parameters.

(25) TABLE-US-00001 TABLE 1 SkyScan1172 Version 1.5 (build 14) NRecon version Software: 1.6.9.6 CT Analyser version 1.13.1.1 Source Type: 10 Mp Hamamatsu 100/250 Camera Resolution 4000 × 2096 (pixel): Camera Binning:   1 × 1 Source Voltage 65 kV Source Current uA 153 Image Pixel Size (um):  3.96 Filter Al 0.5 mm Rotation Step (deg)  0.280 Output Format  8 bit BMP Dynamic Range 0.000-0.140 Smoothing  0 Beam Hardening  0 Post Alignment corrected Ring Artefacts  16

(26) A small amount of purified MilliQ® water was carefully decanted into each sample tube. Each sample was then analysed by X-Ray micro-computer tomography using a single scan, to include the water reference and the beads. The samples were then reconstructed using NRecon and calibrated against a volume of interest (VOI) of the purified water reference. A region of interest (ROI) of air and water was analysed after calibration to verify the Hounsfield calibration.

(27) Radiodensity was reported in Hounsfield units from line scan projections across the bead. Values used for dynamic range for all samples in NRecon (thresholding): −0.005, 0.13 (minimum and maximum attenuation coefficient).

(28) Table 2 gives the radiodensity, iodine and solid content of microspheres prepared according to general example 10. Radiodensity data are the mean of ten line scans of each individual microsphere. Multiple microspheres were analysed for each preparation.

(29) TABLE-US-00002 TABLE 2 Solid Iodine Iodine Radio doxorubicin Microsphere content (% wt/wt (mg/cm.sup.3 density loading prototype Product (mg/ml) Dry) wet) (HU) time (min) 1 268.99 37.4 100.7 10 2 304.8 36.4 111.0 3668 5 3 329.9 41.4 136.6 60 4 368.9 40.8 150.3 4643 20 5 151.9 33.37 50.7 956 <10 6 245.6 46.3 113.7 3860 <5 7 397.9 43.6 173.4 5389 15 8 329.1 43.8 144.2 5368 30

Example 13: Drug Loading of Microsphere Prototypes

(30) 1 mL of microspheres (70-150 μm) were suspended in 1.5 mL of doxorubicin solution (concentration 25 mg/mL) under constant agitation. At predetermined time points the supernatant solution was sampled and doxorubicin concentration determined at UV at 483 nm against a known reference. Table 2 (above) shows time to greater than 95% loading for microsphere prototypes. Non-radiopaque microspheres (DC Bead M1 (70-150 μm: Biocompatibles UK Ltd. UK) were loaded to greater than 95% in less than 10 mins. Commercial radiopaque microspheres carrying a tri iodophenyl group coupled to the microsphere through a 1,3 dioxane group (DC Bead LUMI Biocompatibles UK Ltd. UK) were loaded to greater than 95% in 30 mins.

Example 14: General Liquid Embolic Synthesis Conditions

(31) To a pre-dried reactor under a nitrogen blanket is added PVA (typically 5-10 g) and anhydrous solvent (typically DMSO or NMP, 40 vol w.r.t. PVA mass) and catalyst (e.g. methanesulfonic acid typically 2.2 vol w.r.t. PVA mass). The stirred suspension is heated to elevated temperature (ca 90° C.) to dissolve the PVA. When a homogeneous solution had been obtained, the mixture is cooled to the desired reaction temperature (typically 50-80° C.) the desired chemical substrate (typically 0.1 to 0.6 eq w.r.t. PVA diol functionalities) is added. The reaction is then stirred under an N.sub.2 blanket and the reaction conversion is monitored by HPLC for consumption of the chemical substrate. At a pre-determined time (typically when consumption of the chemical substrate had ceased) an anti-solvent is added (typically, acetone, DCM, MeCN or TBME, ca 40 vol) dropwise from a dropping funnel. The supernatant fluid is removed by aspiration through a filter membrane and further reaction solvent (typically 40 vol) is charged and stirred until the solids had fully dissolved. This solvent washing stage is repeated up to 3 times. Then the solid is re-dissolved in reaction solvent, and precipitated by the slow addition of water (typically up to 100 vol). The resulting aggregated solid is removed from the supernatant and homogenised in a blender in water (ca 11). The suspension is filtered and re-suspended in water (typically 100 vol) and slurried for up to 30 minutes and filtered. The water slurrying is repeated until pH neutral is obtained, then the damp solids are slurried in acetone (100 vol, 30 mins stir, 2 repetitions), filtered and dried in a high vacuum oven at 30° C. for up to 24 hours.

Example 15: Preparation of Liquid Embolic Prototypes

(32) A sample prototype is prepared in the following fashion: iodinated PVA prepared according to general example 12, is weighed into a 10 ml vial, to which was added the desired solvent (typically DMSO or NMP) such that the overall concentration was in the range 4-20% w/w with a total volume being less than 10 ml. To this, if desired to create ionic liquid embolic species, sodium hydroxide (4M) is added at tis time. The vial containing the thick suspension is then sealed and placed in a sonicator, and sonicated until complete dissolution had occurred (typically ca 4 hours).

Example 16. Preparation of 3,4,5-Triiodosalicylaldehyde (TISA)-PVA

(33) To a dry 600 ml HEL (ltd) PolyBLOCK® vessel under a nitrogen blanket, was added DMSO (200 ml, 67 vol) and the stirring initiated at 500 rpm. To this was charged PVA (85-124 kDa, 100% hydrolysed, 3.0051 g) which was rinsed into the reactor with DMSO (10 ml) and the suspension heated to 80° C. (internal probe) until all the solids had dissolved. The solution was then cooled to 60° C. internal and 3,4,5-triiodosalicylaldehyde (3,4,5-TISA, 6.8140 g, 13.6 mmol, 0.25 eq w.r.t. PVA-1,3-diol units) was charged and rinsed in with DMSO (10 ml). After full dissolution, methanesulfonic acid (6 ml, 2 vol) was added in one portion and the reaction was stirred at 60° C. until HPLC analysis showed consumption of 3,4,5-TISA had halted. The solution was cooled to room temperature and transferred to 2 L glass breaker containing a large stirrer bar to which was added from a dropping funnel, dichloromethane (DCM) (250 ml) then toluene (500 ml). The yellow supernatant was decanted and the resulting solid slowly re-dissolved in DMSO (150 ml) at 50° C. for 1.5 hours. The polymer was precipitated by the slow addition of toluene (500 ml) and the coloured supernatant removed by in-situ filtration. The polymer was re-dissolved in DMSO (150 ml) overnight, then precipitated by the dropwise addition of water (500 ml). The resulting solid was removed, was blended in water to achieve a homogeneous suspension. The pH of the solution was confirmed a pH7, and the solids were isolated by filtration on a Buchner funnel, washed with water (250 ml) and acetone (250 ml) and dried in a hi-vacuum oven at 30° C. overnight to give the desired product as a yellow/white solid (9.1517 g, 93.2% w/w yield).

(34) Table 3 shows yield and iodine content (w/w) for sample liquid embolic preparations prepared according to this general protocol, with varying molecular weight samples of PVA and TISA/PVA ratios.

(35) TABLE-US-00003 TABLE 3 Yield % I.sub.2 Prep. MW PVA Eq. TISA Conversion (% w/w) (w/w) 1 85-124 kDa  0.1 eq  100% 88.8% 28.1% 100% hydrolysed 2 85-124 kDa 0.25 eq 99.4% 97.3% 44.3% 100% hydrolysed 3 85-124 kDa  0.4 eq   97% 93.2% 51.9% 100% hydrolysed 4 85-124 kDa 0.6 eq   90% 90.2% 55.4% 100% hydrolysed 5 67 kDa, 88%  0.6 eq   57% 67.4% 51.8% hydrolysed

(36) In an analogous way the following commercially available aldehydes may be also be coupled to PVA:

(37) (a) 2-sulfobenzaldehyde sodium salt, (Sigma Aldrich UK)

(38) (b) 4-formylbenzene 1,3 disulfonic acid disodium-salt, (Sigma Aldrich UK)

(39) (c) 4-formylbenzoic acid (Sigma Aldrich UK).

Example 17: Precipitation of Liquid Embolic Under Flow Conditions

(40) A clear detachable tube was attached to a flow system through which PBS was pumped through the detachable tubing using a peristaltic pump to mimic blood flow conditions. A 2.4 Fr catheter was used to deliver the liquid embolic preparation into the detachable tube. As the liquid embolic left the catheter and came into contact with PBS, it precipitated inside the detachable tubing. The length of any precipitate was then measured from the end of the catheter tip. Flow rate and rate reduction were also recorded. The “longest length of advancement” was recorded. If reflux had occurred, its length was also recorded as the “longest length of reflux” (cm). Table 4 records precipitation properties of liquid embolic preparations

(41) TABLE-US-00004 TABLE 4 Longest eq length of Longest base advance- length of Flow rate eq (per wt/wt ment reflux reduction TISA TISA) Solvent polymer (cm) (cm) (%) 1 0.1 NMP 8 3.5 1 99.8 2 0.25 NMP 8 4 0.5 99.7 1 0.1 DMSO 8 4 1 99.8 2 0.25 DMSO 8 5 1 99.8 3 0.4 DMSO 8 2.4 1.9 95.0 DMSO 12 3.7 1 85.5 NMP 8 3.5 1 97.2 NMP 12 6 0 86.5 4 0.6 DMSO 8 4.7 1.2 90.0 DMSO 12 5.5 2 65.5 NMP 8 3.5 1.5 96.9 NMP 12 3.5 1.5 100.0 5 0.6 0.33 NMP 12 2.5 cm   1 cm 100.0 0.66 12 — — — 0.22 12 2.5 cm 1.5 cm 100.0 0.11 12 3.5 0.5 98.0

Example 18: X-Ray Analysis of Precipitated Liquid Embolic Samples

(42) In order to obtain radiopacity measurements for the material, 1 cm sections of precipitated formulations are cut and embedded in warm (55° C.) 100 agarose in a polypropylene capped tube, (such as a Nunc tube) and scanned using Micro-CT according to Example 12. Table 5 illustrates radiopacities of prepared formulations of Example 13

(43) TABLE-US-00005 TABLE 5 TISA Original Added Radiopacity Eq Plug Solvent Concentration (NaOH) of polymer 0.6 NMP 12% (w/w) 0.11 eq 4414 HU 0.4 NMP 12% (w/w)   0 eq 3815 HU 0.6 NMP 12% (w/w)   0 eq 4809 HU