Imageable embolic microsphere

Abstract

This invention concerns imageable, radiopaque embolic beads, which are particularly useful for monitoring embolization procedures. The beads comprise iodine containing compounds which are covalently incorporated into the polymer network of a preformed hydrogel bead. The beads are prepared by activating pre-formed hydrogel beads towards nucleophilic attack and then covalently attaching iodinated compounds into the polymer network. The radiopaque beads may be loaded with chemotherapeutic agents and used in methods of embolizing hyperplastic tissue or solid tumors.

Claims

1. A method of making a radiopaque hydrogel bead, comprising: forming an activated hydrogel bead by reacting a preformed hydrogel bead with a carbonyldiimidazole or a carbodiimide; and reacting functional groups on the activated hydrogel bead with an iodinated compound such at the iodinated compound is covalently coupled to the activated hydrogel bead, wherein the iodinated compound is an aromatic iodine containing compound.

2. The method according to claim 1, wherein the aromatic iodine containing compound is an iodinated benzyl or phenyl alcohol or is an iodinated benzoic acid.

3. The method according to claim 1, wherein the aromatic iodine containing compound is 2,3,5-triiodobenzoic acid.

4. The method according to claim 1, comprising preforming the preformed hydrogel bead from a polymer which has alcoholic hydroxyl substituents or acylated derivatives thereof.

5. The method according to claim 1, wherein the preformed hydrogel bead comprises a cross-linked polyhydroxy polymer selected from polyvinyl alcohol (PVA) or a copolymer of vinyl alcohol.

6. The method according to claim 1, wherein the preformed hydrogel bead comprises a polymer backbone with a 1,2-diol and/or a 1,3-diol structure to enable crosslinking with an acrylic monomer.

7. The method according to claim 6, wherein the acrylic monomer is 2-acrylamido-2-methylpropane sulfonic acid (AMPS).

8. The method according to claim 1, wherein hydroxyl moieties on and/or within the preformed hydrogel bead are activated by reaction with the carbonyldiimidazole.

9. The method according to claim 1, wherein the iodinated compound is an iodinated alcohol, an iodinated amine, or an iodinated carboxylic acid.

10. The method according to claim 1, wherein the iodinated compound is an iodinated alcohol.

11. The method according to claim 10, wherein the iodinated alcohol is a triiodobenzyl alcohol or a triiodophenyl alcohol.

12. The method according to claim 1, having coupled to its imide or imidazole function, a terminally bifunctional linker which comprises an aliphatic carbon chain with at least 2 carbons.

13. The method according to claim 12, wherein both functionalities of the terminally bifunctional linker are reactive with imide or imidazole.

14. The method according to claim 12, wherein the functionality or reactive moieties of the bifunctional linker comprise one or more of an amine, carboxylic acid and alcohol.

15. The method according to claim 12, wherein the bifunctional linker is a diamino alkane, optionally wherein bifunctional linker has general formula H.sub.2N(CH.sub.2).sub.nNH.sub.2wherein n includes any number between 2 and 20, 2 and 10, or 2 and 4.

16. The method according to claim 12 wherein the iodinated compound is covalently coupled through the bifunctional linker.

17. The method according to claim 1, further comprising absorbing a pharmacologically active agent within the radiopaque hydrogel bead.

18. The method according to claim 1, comprising: adding the carbonyldiimidazole or the carbodiimide to a suspension that contains the preformed hydrogel bead, wherein the preformed hydrogel bead is swollen with an organic solvent, in the presence of a catalytic amount of a base and under anhydrous conditions to react with the preformed hydrogel bead such that it is activated towards nucleophilic substitution; and covalently coupling the activated hydrogel bead with the iodinated compound which is reactive towards the imidazole or diimide functionality of the activated hydrogel bead.

19. The method according to claim 18, wherein the preformed hydrogel bead comprises a cross-linked polyhydroxy polymer selected from polyvinyl alcohol (PVA) or a copolymer of vinyl alcohol.

Description

(1) The invention will now be described by way of example with reference to the following figures, in which:

(2) FIG. 1 shows the size and appearance (A) pre-formed hydrogel bead, prior to activation and (B) imageable bead prepared according to the reaction described in Example 2.

(3) FIG. 2 shows Clinical CT. (A) and Micro CT images (B) of imageable bead prepared according to the reaction of Example 1.

(4) FIG. 3A shows light microscopy image from imageable beads prepared according to Example 5. FIG. 3B shows the same beading after loading with doxorubicin as described in Example 6.

(5) FIG. 4A shows Clinical CT micrographs of radiopaque beads prepared according to example 5 (A) at 3.1%, 6.2% and 12.5% packed volume of beads. FIG. 4B shoes Micro CT images of 3.1% packed bead volume in agarose phantom. FIG. 4C shows the MicroCT images of the same bead volume in which the bead had been loaded with doxorubicin hydrochloride.

(6) FIG. 5 shows Micrograph of 100-300 μm beads sieved after iodination in accordance with the reaction described in Example 8.

(7) FIG. 6 shows MicroCT images of iodinated beads with size range (A) 70-150 μm, (B) 100-300 μm, (C) 300-500 um, and (D) 500-700 μm, prepared according to Example 8.

MATERIALS AND METHODS USED IN THE EXAMPLES

Materials

(8) Sulphonate modified polyvinyl alcohol AMPS microspheres (LC/DC-Bead™, Biocomaptibles UK Ltd) were prepared as described in Example 1 of WO 2004/071495. Anhydrous dimethyl sulfoxide (DMSO), 1,1′-Carbonyldiimidazol (CDI), 2,3,5-triiodobenzyl alcohol, 2,3,5-Triiodobenzoic acid, N,N′-Diisopropylcarbodiimide (DIC), 1-Hydroxybenzotriazole hydrate (HOBt), 4-(Dimethylamino)pyridine (DMAP), 1,3-Diaminopropane, triethylamine (Et3N), and anhydrous dichloromethane (DCM) were purchased from Sigma Aldrich. Doxorubicin (Dox) was obtained from Bedford Laboratories. De-ionized water (DI water) obtained from Millipore purification system.

General Methods

PVA-AMPS Hydrogel Bead Formation

(9) The first stage of microspheresynthesis involves the preparation of Nelfilcon B—a polymerisable macromer from the widely used water soluble polymer PVA. Mowiol 8-88 poly(vinyl alcohol) (PVA) powder (88% hydrolised, 12% acetate content, average molecular weight about 67,000 D) (150 g) (Clariant, Charlotte, N.C. USA) is added to a 2 litre glass reaction vessel. With gentle stirring, 1000 ml water is added and the stirring. increased to 400 rpm. To ensure complete dissolution of the PVA, the temperature is raised to 99±9° C. for 2-3 hours. On cooling to room temperature N-acryloylaminoacetaldehyde (NAAADA) (Ciba Vision, Germany) (2.49 g or 0.104 mmol/g of PVA) is mixed in to the PVA solution followed by the addition of concentrated hydrochloric acid (100 ml) which catalyzes the addition of the NAAADA to the PVA by transesterification. The reaction proceeds at room temperature for 6-7 hours then stopped by neutralisation to pH 7.4 using 2.5M sodium hydroxide solution. The resulting sodium chloride plus any unreacted NAAADA is removed by diafiltration using a stainless steel Pellicon 2 Mini holder stacked with 0.1 m.sup.2 cellulose membranes having a pore size with a molecular weight cut off of 3000 (Millipore Corporation, Bedford, Mass. USA). Upon completion, the macromere solution is concentrated to 20-23% solids with a viscosity of 1700-3400 cP at 25° C.

(10) Hydrogel microspheres are synthesized by suspension polymerization in which an aqueous phase comprising the modified PVA macromer is added to an immiscible organic phase comprising 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and rapidly mixed such that the aqueous phase is dispersed to form droplets, the size and stability of which can be controlled by stirring rates, viscosity, ratio of aqueous/organic phase and the use of stabilizers and surfactants which influence the interfacial energy between the phases. The resulting hydrogel microspheres are recovered by filtration and washing and may be sieved to provide particular size ranges. Unless otherwise stated preformed hydrogel beads were 300-500 μm diameter.

Evaluation of Imageable Beads with Microscopy

(11) The size and appearance of beads during various steps of synthesis and doxorubicin loading were examined and imaged in a chamber slide (Electron Microscopy Sciences; ˜150 μl bead and DI water suspension). Bright field images were acquired with a 5× objective on an upright microscope (Zeiss, Axio Imager.M1, Thornwood, N.Y.) equipped with a color CCD camera (Axiovision, Zeiss).

Phantom Preparation

(12) In order to assess radiopacity, beads were suspended in an agarose matrix at bead concentrations (bead volume percent) that is relevant for in vivo applications. Bead containing agarose phantoms (0.5% w/v) with various concentrations (bead volume percents ranging from 0, 3.1, 6.2 and 12.5%) were prepared by adding a 1% agarose mixture to an equal volume of bead suspension in deionsed water. The solutions were mixed while allowing the agarose to slowly gel (over ice), resulting in a homogeneous distribution of beads. The bead volume percent is packed bead volume due to gravity alone and does not account for aqueous solution between the packed beads or altered bead packing efficiency.

In Vitro Evaluation of Imageable Beads with Clinical CT

(13) The distribution of conjugated iodine contrast agent within the radiopaque microspheres was imaged on a clinical 256 Slice CT (Philips, Andover, Mass.) to determine the overall attenuation with the following settings: 465 mAs tube current, 80 keV tube voltage, 1 mm thickness, 0.5 mm overlap. The average attenuation of an 80 mm2 rectangular region in the middle slice of a given phantom was measured using OsiriX.

In Vitro Evaluation of Imageable Beads with Micro-CT

(14) Micro-CT imaging and analysis of imageable bead containing phantoms was performed with a SkyScan 1172 high-resolution micro-CT (Skyscan, Konitch, BE) to evaluate the radiopacity of each individual bead, as well as, intra-bead distribution of iodine. The radiopaque microspheres were imaged at 5 μm resolution, 78 kV, 127 micro-Amps, using a 0.5 mm Aluminum filter. The average attenuation of individual beads was measured and reported as the mean and standard error (n=10).

Example 1: Preparation of Imageable Embolic Beads by Conjugation of 2,3,5-Triiodobenzyl Alcohol on to Preformed PVA-AMPS Hydrogel Embolic Beads

(15) ##STR00001##

(16) Pre-formed PVA based hydrogel embolic Beads [depicted by Scheme 1, 1] were washed (200 mg) with DMSO (3×5 ml) and the beads were allowed to swell in DMSO (20 ml) for 30 minutes at 50° C. The beads were activated by stirring the suspended beads with carbonyldiimidazole (CDI) (800 mg) (CDI:OH ratio of approximately 1.2:1) in the presence of catalytic amount of triethylamine (0.12 equivalent) at 50° C. for 24 hours to form activated beads (Scheme II). The reaction mixture was cooled to room temperature and washed quickly with a cocktail of DMSO and DCM (1:1) and finally with DMSO alone to provide activated beads [2]. The beads were successfully activated with CDI under very mild conditions. The beads were stored in DMSO for further use.

Example 2: Preparation of Imageable Embolic Beads by Direct Conjugation of 2,3,5-Triiodobenzyl Alcohol to Activated PVA-AMPS Hydrogel Embolic Beads

(17) ##STR00002##

(18) Activated beads were prepared according to Example 1. Activated beads were immediately transferred into a reaction flask containing a solution of 2,3,5-Triiodobenzyl alcohol (971.7 mg) in DMSO (10 ml) and stirred for 24 hours at 50° C. (Scheme II). The resulting product cooled to room temperature and was washed thoroughly with DMSO:DCM (1:1), followed by DMSO alone. Finally, the DMSO was exchanged with DI water (under continuous agitation) and the image-able beads thoroughly washed with saline and DI water consecutively. The clean imageable beads were suspended in DI water until further analysis. The beads were successfully conjugated with 2,3,5-triiodobenzyl alcohol (as depicted in Scheme II). Microscopic image comparison of pre-formed hydrogel bead (prior to activation) and 2,3,5-triiodobenzyl alcohol conjugated beads (imageable beads) revealed that the imageable beads size is slightly reduced, as shown in FIG. 1, and the beads may be more easily viewed under light microscopy

Example 3—Assessment of the Radiopacity of Imageable Hydrogel Beads

(19) The radiopacity of imageable beads prepared according to Example 2 was assessed both in clinical and micro CT and they are visualized in both radiographic techniques. In the clinical CT, visualization is based on radiopacity of the imageable beads in an agarose phantom per a given volume. A 3.1% imageable bead packed volume showed a mean attenuation of 26±15 HU (Hounsfield Units) and increased to 41±16 HU and 74±25 HU as the imageable bead packed volume increased to 6.2 and 12.5%, respectively. In micro CT, a single particle showed a mean attenuation of 952.3±93.9 HU (n=10). Images are shown in FIG. 2.

Example 4: Preparation of an Amino-Reactive Activated Hydrogel Bead

(20) ##STR00003##

(21) Activated beads, prepared according to Example 1, were reacted with the diamino alkane linker, 1,3-diaminopropane, by mixing at 50° C. for 24 hours (Scheme III). After the reaction was complete, the reaction mixture was cooled and washed thoroughly with DMSO:DCM (1:1), followed by DMSO to yield the amino-reactive hydrogel bead (depicted in Scheme III as [4]). The amino-reactive bead gave a positive ninhydrin response confirming the presence of the terminal primary amine group.

Example 5: Preparation of an Imageable Hydrogel Bead from a Reactive Hydrogel Bead

(22) ##STR00004##

(23) A solution of 2,3,5-triiodobenzoic acid (2.4 g) in DMSO (20 ml) was activated with N,N′-Diisopropylcarbodiimide (DIC) (604.5 mg), HOBt (634.2 mg) and DMAP (586.4 mg) for 30 minutes at room temperature and the amino-reactive beads prepared in Example 4 [4] were added (Scheme IV). The resulting reaction mixture was stirred at 40° C. for 3 days. After cooling, the imageable beads [5] were thoroughly washed with DMSO/DCM and DMSO and then finally the solvent was exchanged with deionized water. A light microscopy image of the radiopaque beads in shown in FIG. 3.

Example 6: Doxorubicin Loading into Imageable Beads

(24) Imageable beads, prepared according to Example 5, were loaded with Doxorubicin according to previously reported method (Lewis, A. L. et al. Journal of Materials Science-Materials in Medicine 2007, 18, 1691). Briefly, 250 μl of thoroughly washed beads with DI water was immersed into 0.5 ml of Dox (20 mg/ml) solution and shaken for 3 hrs at room temperature. As can be seen from the light microscopy image in FIG. 3B, the doxorubicin loaded radiopaque beads have taken on the characteristic red appearance of doxorubicin loaded hydrogels and appear to have decreased slightly in size.

Example 7: Radiopacity of Imageable and Drug-Loaded Imageable Beads

(25) The radiopacity, or radiodensity, of the beads prepared in Examples 6 and 7 were assessed in clinical and micro CT with both techniques confirming that both sets of beads are radiopaque and easily visualized in both radiographic techniques. In the clinical CT, visualization is based on radiopacity of the beads per given volume. A 3.1% packed bead volume showed a mean radiopacity of 129±33 HU and increased to 269±53 HU and 444±83 HU as the bead packed volume increased to 6.2 and 12.5%, respectively. This is shown in FIG. 4A. In micro CT, individual “bland” and Doxorubicin loaded beads showed a mean attenuation of 7903.99±804 HU (n=10) and 11873.96±706.12, respectively. These are shown in FIGS. 4B and 4C, respectively.

Example 8: Preparation of Radiopaque Hydrogel Beads by Activation of Iodinated Benzoic Acid

(26) In a reaction vessel, 10 g of acetone-dried PVA beads were mixed with 200 mL of anhydrous DMSO by stirring for 30 min at 50° C. Subsequently 41.6 g of 1,1′-carbonyldiimidazole (CDI) and 4.1 mL of triethylamine were added into the bead suspension under a nitrogen blanket. After the reaction was complete, the temperature was reduced to room temperature, approximately 22° C. Then 200 ml of anhydrous diethyl ether was added into the reaction mixture and stirred over 10 min, followed by removal of the solvents. The activated beads were then washed with mixed solvent of DMSO and diethyl ether (1:1, v/v) three times. Elemental analysis of the activated beads confirmed that conversion of OH by CDI activation was approximately 30%.

(27) A bifunctional linker was then grafted on to the activated beads. Activated beads were suspended in 200 ml of anhydrous DMSO at 50° C., and 18.5 g of 1,3-diaminopropane was added into the bead suspension. After the reaction was complete, the reaction vessel was cooled down to room temperature and the resulting beads were washed by diethyl ether and DMSO mixture three times, followed by the removal of solvents.

(28) In a final step, triiodobenzoic acid was activated towards reaction with the activated bead; In a round bottom flask 40 g of 2,3,5-triiodobenzoic acid (TIBA), (a concentration equivalent to ⅓ of initial OH on beads), was dissolved in 100 ml of anhydrous DMSO. The compound was then activated by adding 13 g of CDI powder, resulting in a steady release of CO.sub.2 (at room temperature), the solution becoming turbid and viscous after stirring for about 30 to 60 min. The mixture was then added into a suspension of activated beads in 100 ml of DMSO. At 50° C., the suspension was stirred for over 24 hr whilst protected from light. Finally, the beads were washed with diethyl ether and DMSO mixture and deionized water.

(29) A light micrograph image of the resulting beads, after sieving, is shown in FIG. 5. MicroCT image of bead phantoms is shown in FIG. 6. The iodine content of vacuum-dried beads was 42-45% by elemental analysis. Table 1 shows the measured bead solid content, iodine content and parameters of characterization.

Example 9: Effect of Temperature on Activation Chemistry

(30) As a comparison, this example conforms in all respects to Example 8, except that a reaction temperature of 70° C. was used in all three steps and ⅔ of TIBA equivalent to initial OH on beads are used in the third step. The beads appeared more brown color under these higher temperature conditions. The iodine content of dried beads is listed in Table 1, indicating slightly less conjugation efficiency. The beads were further autoclaved under 121° C. for 20 min, and no damage/degradation was observed post-sterilisation.

Example 10: Drug-Loading Efficiency of Radiopaque Beads

(31) Drug loading capacity of the beads prepared according to Example 9 was tested by adding 2.87 ml of doxorubicin solution (24.4 mg/ml) to 1 ml of beads (sieved, 100-300 μm) with occasional agitation. After 24 hr, the residual doxorubicin in solution was measured by UV at 483 nm, and the loading yield was calculated as 99.6%.

Example 11: Carbodiimide Coupling of Beads

(32) This example is the same in all respects as Example 9, other than that in the third step, an alternative coupling agent, N,N′-diisopropylcarbodiimide (DIC) was used in the reaction of TIBA and reactive (amino-linked) beads. Equivalent DIC and TIBA to initial OH on beads were used in this case. The iodine content of final dried beads was confirmed by elemental analysis to be 17.7%, illustrating a lower conjugation efficiency that that observed in Examples 8 & 9.

(33) TABLE-US-00001 TABLE 1 Iodine Content and Radiodensity of Radiopaque beads prepared according to the Examples 8 and 9 Solid Iodine MicroCT Example No./ content content Attenuation bead size (μm) (%) (%) (HU) 4 70-150 μm 27.2 41.9 9758 ± 1476 100-300 μm 24.8 44.3 8037 ± 1142 300-500 μm 23.7 45.1 8243 ± 1240 500-700 μm 23.0 45.6 7326 ± 773  5 70-150 μm 37.8 32.4 — 100-300 μm 34.1 38.5 — 300-500 μm 32.0 40.1 —