Hydrogels and Compositions Containing Hydrogels

Abstract

To provide a drug delivery system that can be imaged during an embolization procedure. [Solution] The present invention relates to imageable polymers, particularly imageable polymers comprising polyvinyl alcohol, methods for making the same, and embolic microspheres comprising the polymers, which can be imaged during an embolization procedure and can be loaded with drugs or other therapeutic agents to provide an imageable drug delivery system.

Description

Technical Field

[0001] The present invention relates to a radiopaque polymer and a method for producing the same. The present invention provides a radiopaque hydrogel that can be imaged during embolization procedures, particularly radiopaque hydrogel microspheres. These microspheres can be loaded with therapeutic agents such as drugs to form an imaged drug delivery system.

Background Art

[0002] Radiopacity refers to the property of interfering with or attenuating the transmission of electromagnetic radiation, particularly X-rays. Thus, radiopaque substances can be visualized in X-ray images or during X-ray imaging and fluoroscopy. For this reason, radiopaque substances have numerous applications in radiology and medical imaging techniques such as computed tomography (CT) and fluoroscopy.

[0003] Angioembolization (blocking blood flow) is a medical procedure in which an embolism or occlusion is introduced into a blood vessel to reduce blood flow and atrophy tumors and malformations, and is an important procedure for the treatment of tumors, fibromas, and vascular malformations. There are various embolizing substances for clinical applications that require transcatheter delivery to the embolization site. Microspheres (also referred to herein as "beads") can have their shape and size controlled and are currently commonly used as embolizing substances for injection because they are more predictable in use than previous particulate substances.

[0004] Imaging embolization procedures is important because it allows clinicians to monitor the exact location of the embolizing substance and ensure that the embolizing substance is accurately administered and retained in the correct location in the vascular system, thereby improving the outcome of the procedure and reducing the risk of the procedure. Currently, imaging is possible only when using inherently radiopaque embolizing substances or when mixing non-radiopaque embolizing particles with a radiopaque substance.

[0005] Iodinated polyvinyl alcohol (I-PVA) is a radiopaque embolic substance in the form of a viscous liquid that precipitates under the aqueous conditions encountered in vivo. However, the exact location where embolization occurs can lack consistency, presenting a risk of precipitation at off-target locations.

[0006] However, contrast agents such as Ethiodol® and Isovue® are routinely mixed with embolic particles to render the injection composition radiopaque. While such compositions are useful, differences in physical properties between the aqueous suspension of embolic particles and the contrast agent can result in differential localization in vivo. After administration, it is the contrast agent rather than the embolic particles that may be visible, and the contrast agent and embolic particles may not remain in the same location within the tissue.

[0007] Therefore, it is necessary to combine the predictability and reproducibility benefits of embolic microspheres with the radiopacity of contrast agents.

[0008] European Patent No. 1810698 describes a process for forming stable radiopaque embolic beads (also referred to herein as RO beads or RO microspheres), in which PVA hydrogel embolic beads are loaded with iodinated oil to render them radiopaque. The mechanism by which the oil is retained within the beads is not elucidated. Furthermore, since this oil is a mixture of iodinated fatty acid ethyl esters, the final product is not precisely defined, and with this method, it is not possible to control the elution of the contrast agent from the beads, nor is the impact of the contrast agent on drug loading and elution considered.

[0009] International Publication No. 2011 / 110589 describes the synthesis of iodinated poly(vinyl alcohol) by grafting iodobenzoyl chloride onto poly(vinyl alcohol) via an ester bond. This polymer has been shown to be radiopaque, but in this process, an insoluble polymer is produced, and later it cannot be made into microspheres using the water-in-oil polymerization process commonly used for the production of hydrogel microspheres having desirable embolization properties. Although microspheres are mentioned in this published patent, no disclosure regarding a method for obtaining them is included.

[0010] Mawad et al. (Biomacromolecules 2008, 9, 263 - 268) describe the chemical modification of PVA-based degradable hydrogels to make the polymer radiopaque by introducing covalently bound iodine into the polymer backbone. Iodine is introduced by reacting 0.5% of the pendant alcohol groups on PVA with 4-iodobenzoyl chloride. The resulting polymer is biodegradable and forms emboli by precipitation and does not form microspheres.

[0011] Therefore, it is clear that there is a need for a single product radiopaque embolic material that combines the embolization efficiency and reproducibility of embolic beads with the radiopacity of contrast agents such as iodinated poppy seed oil ethyl ester. An ideal embolic particle is essentially radiopaque and has stability and reproducibility in size and physical properties so that a clinician can perform and image embolization procedures with greater confidence that the visible contrast is due to the embolic particles. The injection and deposition of such beads into the vascular site could be monitored, and it is thought that it would also be possible to monitor during clinical follow-up to monitor the effectiveness of embolization and confirm that the embolic material remains in the desired location and to identify areas at risk for further procedures. The time frame for obtaining follow-up imaging would be significantly increased compared to existing methods.

[0012] Radiopacity (the ability to attenuate X-rays) can be quantified according to the Hounsfield scale. The Hounsfield unit is a unit of measurement of radiopacity per unit volume (voxel). A typical voxel in computed tomography (CT) is about 1 mm 3 in size, so individual microspheres with a diameter of about 100 μm must have a high radiopacity so that they or their aggregates can increase the radiopacity of the above voxels and be visualized, for example, intravascularly. A radiopacity of greater than 100 HU, preferably greater than 500 HU, is considered appropriate.

[0013] In addition to excellent radiopacity, ideal embolization beads have properties that allow efficient drug loading and elution so that chemical embolization procedures can be monitored with confidence. SUMMARY OF THE INVENTION

[0014] The inventors have confirmed that it is possible to modify a polymer to be radiopaque by using a relatively simple chemical reaction. A low molecular weight aldehyde containing one or more covalently bonded radiopaque halogens (such as bromine or iodine) is reacted with the 1,3-diol groups of the polymer to bind to the polymer. Reaction with 1,2-glycol is also possible. This forms a cyclic acetal (a dioxane ring when reacted with 1,3-diol) to which the halogenated group is covalently bonded. This halogenated group has a molecular weight of less than 1000 daltons, usually less than 750 daltons. The minimum value is 156 daltons.

[0015] The halogenated group usually has 6 to 18, preferably 6 to 10 carbons; and optionally has one oxygen atom; and contains an aromatic ring containing one or two covalently bonded radiopaque halogens. The aromatic ring is preferably a phenyl group.

[0016] This chemical reaction results in a polymer having defined radiopaque groups covalently bonded to the polymer in a predictable and controllable manner. This reaction may be carried out with any diol-containing polymer and is particularly suitable for hydrogel polymers and pre-formed microspheres, so that non-radiopaque microspheres can be made essentially and permanently radiopaque without adversely affecting the physical properties of the microspheres (i.e., size, sphericity, high water content, swellability, and compressibility). The radiopaque microspheres have equivalent and / or better drug loading capacity and / or elution characteristics compared to the original non-radiopaque beads from which they are formed. The radiopacity of these microspheres is permanent or has a long enough lifespan to be monitored during the clinical follow-up period.

[0017] The ability to post-treat pre-formed beads allows the same manufacturing process to be used for both radiopaque and non-radiopaque beads, and size selection or sieving can be carried out to determine the size, taking into account the bead diameter variation resulting from the process of imparting radiopacity, so that only beads of a specific size or size range become radiopaque, either before post-treatment or, if necessary, after post-treatment. This provides a certain degree of flexibility in manufacturing.

[0018] Accordingly, in a first aspect, the present invention provides a polymer comprising an acetalized 1,2-diol group or 1,3-diol group with a radiopaque chemical species. By acetalizing with a radiopaque chemical species, the radiopaque chemical species is bonded to the polymer via a cyclic acetal group (dioxane in the case of a 1,3-diol polymer). Thus, the radiopacity of the polymer is due to having a radiopaque substance incorporated into the polymer by covalent bonding via a cyclic acetal linkage.

[0019] As used herein, the terms "radiopaque chemical species" and "radiopaque substance" refer to a chemical substance that is visible in an X-ray image and can be resolved from the solvent surrounding the radiopaque substance or chemical species using routine techniques such as computed tomography, or a substance modified by such a chemical substance.

[0020] The terms "microspheres" or "beads" refer to micron-sized spherical or nearly spherical occluding substances. The terms "particles" or "fine particles" refer to occluding particles having an irregular shape, generally, for example, occluding particles resulting from the fragmentation of a large single mass.

[0021] When the text refers to "halogen" or "halogenated", iodine is preferred unless otherwise specified.

[0022] When referring to the level of radiopacity in HU, it refers to the result of measurements performed by X-ray micro-computed tomography, preferably using the equipment and conditions described in this specification (Example 12), preferably in an agarose phantom as described in this specification (Example 12), preferably the measurement result when measured using a 0.5 mm aluminum filter and a power supply voltage of 65 kV. When referring to radiopacity in gray scale units, it also refers to the result of measurements performed by X-ray micro-computed tomography under the above conditions.

[0023] When referring to "wet beads" or "fully hydrated beads", it means beads that are fully hydrated in normal physiological saline (1 mM phosphate buffer (pH 7.2 - 7.4) of 0.9% NaCl) as the packed volume (e.g., quantified in a graduated cylinder).

[0024] In this and other embodiments, the polymer may be any polymer containing 1,2-diol groups or 1,3-diol groups or mixtures thereof. Preferably, the polymer, such as a polyhydroxy polymer, contains a high proportion of diol groups throughout the polymer backbone. Suitably, the polymer is a hydrogel or other crosslinked polymer network structure. Particularly suitable polymers are polymers including polyvinyl alcohol (PVA) or copolymers of PVA. PVA-based hydrogels are particularly preferred because they are well-known in the art and widely used in embolization procedures.

[0025] In certain embodiments, the polymer comprises a PVA backbone having pendant chains with crosslinkable groups that are crosslinked to form a hydrogel. The PVA backbone has at least two pendant chains containing groups such as acetates and acrylates that can be crosslinked. The crosslinking agent is desirably present in an amount of about 0.01 to 10 milliequivalents per gram of backbone (meq / g), more desirably about 0.05 to 1.5 meq / g. The PVA polymer may contain more than one type of crosslinkable group. Advantageously, the pendant chains are attached to the 1,3-diol hydroxyl groups of PVA via the hydroxyl groups of the polymer backbone attached through cyclic acetal linkages.

[0026] Crosslinking of the modified PVA may occur via any of a number of means such as physical crosslinking or chemical crosslinking. Physical crosslinking includes, but is not particularly limited to, complex formation, hydrogen bonding, desolvation, van der Waals interactions, and ionic bonding. Chemical crosslinking can be carried out by a number of means including, but not particularly limited to, methods routine to polymer chemists such as chain reaction (addition) polymerization and stepwise reaction (condensation) polymerization.

[0027] The group crosslinked on the PVA polymer main chain is preferably an ethylenically unsaturated functional group such as acetate, which can be crosslinked via polymerization initiated by free radicals without the need for the addition of an aldehyde crosslinking agent. Preferably, the PVA polymer contains pendant actetate groups formed from the acetalization of PVA with N-acryloyl-aminoacetaldehyde dimethyl acetal (NAADA). Such modification of PVA is described in U.S. Patent No. 5,583,163. One example of this type of modified PVA is Nelfilcon A.

[0028] The crosslinkable PVA is preferably crosslinked with additional vinyl comonomers, suitably hydrophilic vinyl comonomers such as hydroxy-substituted lower alkyl acrylates and methacrylates, acrylamide and methacrylamide. In certain embodiments, the above-described modified PVA is crosslinked with 2-acrylamido-2-methylpropane sulfonic acid (AMPS® monomer from Lubrizol) to obtain an acrylamide polyvinyl alcohol-co-acrylamide-2-methylpropanesulfonate hydrogel. In a preferred embodiment, the crosslinking reaction is carried out as an inverse phase emulsion polymerization reaction to obtain an acrylamide polyvinyl alcohol-co-acrylamide-2-methylpropanesulfonate hydrogel in the form of microspheres.

[0029] The polymer or hydrogel of the present invention is radiopaque due to a radiopaque substance covalently bonded throughout the polymer in the form of a cyclic acetal. Reactions for forming cyclic acetals are well known in the field of organic chemistry, and thus any radiopaque chemical species capable of forming a cyclic acetal is considered to be within the scope of the present invention. Substances known to be radiopaque include many, such as iodine, bismuth, tantalum, gadolinium, gold, barium, and iron. Elements with high electron density, such as halogens, are particularly useful. Bromine, chlorine, fluorine, and iodine can be easily incorporated into organic molecules capable of forming cyclic acetal bonds, resulting in high radiopacity. Thus, in certain embodiments, the radiopaque polymer comprises a covalently bonded halogen, preferably iodine. The radiopaque halogen covalently bonds to an aromatic group to form a radiopaque chemical species bonded to the polymer via a cyclic acetal. The aromatic group may contain one, two, three, or four radiopaque halogens such as covalently bonded bromine or iodine. This group preferably comprises a phenyl group to which one, two, three, or four such radiopaque halogens are covalently bonded. Thus, the polymer advantageously comprises a halogenated group (X in the following formula) containing a radiopaque halogen such as covalently bonded iodine that binds to the polymer via a cyclic acetal.

[0030] By acetalizing with a radiopaque chemical species, the radiopaque chemical species binds to the polymer via a cyclic acetal group, as shown below. The radiopaque polymer has, or comprises, a structure described by General Formula I (where J is -CH2- in PVA) or II (representing other polymers having 1,2-diols or 1,3-diols). By controlling the number (n) of such acetalized groups in the polymer, the amount of iodine present, and thus the radiopacity, is controlled. The number of diols per milligram of the substance will be considered later.

[0031]

Chemical formula

[0032] X is preferably of formula III

Chemical formula

[0033] Preferably, when Z is present, Z is a methylene or ethylene group or the group -(CH2) p -O-(CH2) q -, where q is 0, 1 or 2 and p is 1 or 2; more preferably, a group selected from -CH2O-, -CH2OCH2- and -(CH2)2O- In particular, Z is -CH2OCH2- or -CH2O- or does not exist, Hal is in particular three or four bromine or iodine, preferably iodine, such as 2,3,5 or 2,4,6 triiodine or 2,3,4,6 tetraiodine, J is preferably -CH2-.

[0034] Thus, preferably, radiopaque iodine is incorporated into the polymer in the form of an iodinated phenyl group. As described above, the iodinated phenyl group is incorporated into the polymer via a cyclic acetal bond.

[0035] Groups such as those described above, particularly halogenated (e.g., iodinated) phenyl groups, are useful because they can be mono-substituted, di-substituted, tri-substituted or in some cases tetra-substituted to control the amount of halogen, such as iodine, incorporated into the radiopaque polymer and thus the level of radiopacity.

[0036] The possible level of halogenation is also affected by the level of 1,3-diol or 1,2-diol groups in the polymer starting material. This level can be estimated based on the polymer structure and the presence of any substitution of -OH groups, for example, by a crosslinking agent or other pendant groups. Polymers having a level of -OH groups of at least 0.1 mmol / g dry polymer are preferred. Polymers having a level of at least 1 mmol / g are more preferred. Excellent radiopacity levels have been obtained with polymers having -OH groups in excess of 5 mmol / g (2.5 mmol / g diol).

[0037] One skilled in the art will understand that the amount of iodine or other radiopaque halogens in the polymer can also be controlled by controlling the degree of acetalization of the polymer. In the present invention, the polymer contains up to 50% of acetalized diol groups. Preferably, at least 10% of the diol groups in the polymer are acetalized, and more preferably, at least 20% of the diol groups are acetalized. Regardless of whether the amount of halogen (e.g., iodine) in the polymer is controlled by increasing substitution on the phenyl ring, for example, or by controlling the degree of acetalization of the polymer, the resulting polymer contains at least 10% halogen (halogen weight / total weight) by dry weight. Preferably, the polymer contains at least 20% halogen by dry weight, preferably more than 30%, 40%, 50% or 60%. Useful contrast is obtained with polymers having 30 - 50% halogen by dry weight.

[0038] The halogen content can also be expressed as the amount of halogen (mg) per mL of beads. This refers to the amount of halogen per mL of beads fully hydrated in physiological saline as the packed volume (quantified, for example, in a graduated cylinder). The present invention provides beads in which the level of halogen (especially iodine) is above 15 mg per mL of wet beads, for example. A halogen (especially iodine) content above 25 mg or 50 mg per mL of beads, preferably above 100 mg, has given good results.

[0039] The present invention is particularly suitable for hydrogels, especially hydrogels in the form of microparticles or microspheres. Microspheres are particularly useful for embolization because their size can be controlled, for example, by sieving, and because they are spherical and can avoid unnecessary aggregation of the embolic material. Microspheres can be produced by a number of techniques known to those skilled in the art, such as single - and two - phase emulsions, suspension polymerization, solvent evaporation, spray drying, and solvent extraction.

[0040] For example, Thanoo et al., Journal of Applied Biomaterials Vol.2, 67-72 (1991); International Publication Nos. 0168720, 03084582; 06119968 and 04071495 (the above documents are incorporated herein by reference) describe microspheres containing polyvinyl alcohol or vinyl alcohol copolymers. In certain embodiments, hydrogel microspheres are prepared from PVA modified with N-acryloyl-aminoacetaldehyde dimethyl acetal (NAADA) as described above (and as disclosed in U.S. Patent No. 5,583,163) and crosslinked with 2-acrylamido-2-methylpropane sulfonic acid as described above. This type of hydrogel microspheres is described in U.S. Patent Nos. 6,676,971 and 7,070,809.

[0041] Microspheres can be made in a size range of about 10 μm (microns) to 2000 μm. If smaller, they may pass through the capillary system and get caught elsewhere. In most applications, microspheres in a small size range are preferred to suppress aggregation and make embolization predictable. By controlling the process used to make the microspheres, microspheres in a specific desired size range can be obtained. Other methods such as sieving can be used to more precisely control the size range of the microspheres.

[0042] In certain embodiments, the hydrogel or non-hydrogel microspheres according to the present invention have an average diameter size range of 10 to 2000 μm, more preferably 20 to 1500 μm, and even more preferably 40 to 900 μm. In the preparation of microspheres, particles in a size range suitable for the planned treatment are usually obtained, for example, 100 to 300 microns, 300 to 500 microns, 500 to 700 microns or 700 to 900 microns. Since smaller particles tend to penetrate deeper into the vascular bed, for certain procedures, particles in the ranges of 40 to 75 microns, 40 to 90 microns and 70 to 150 microns are particularly useful.

[0043] In certain embodiments, the polymer is a hydrogel microsphere having a net negative charge at physiological pH (7.4).

[0044] Radiopacity can be quantified according to the Hounsfield scale, in which distilled water has a value of 0 Hounsfield units (HU) and air has a value of -1000 HU. Preferably, the embolizing microspheres have a radiopacity greater than 100 HU, and even more preferably a radiopacity greater than 500 HU. Using the methods described herein, radiopaque microspheres having a radiopacity greater than 10,000 HU have been prepared. Preferred microspheres have a radiopacity greater than 2000 HU, 3000 HU, 4000 HU, or 5000 HU. These levels of radiopacity enable the microspheres to be distinguishable from, for example, blood (30 - 45 HU), liver (40 - 60 HU), brain (20 - 45 HU), and soft tissue (100 - 300 HU).

[0045] Radiopacity can also be expressed in 0 - 255 gray scale units after subtracting the background, according to American Society for Testing and Materials (ASTM) F - 640.

[0046] Accordingly, a further aspect of the invention provides a radiopaque microsphere having a radiopacity of at least 500 HU, as described in the first aspect herein.

[0047] The hydrogel microspheres of this embodiment are used in a composition containing a suitable excipient or diluent such as water for injection and can be used directly for embolization of blood vessels. Accordingly, a further aspect of the invention provides a pharmaceutical composition comprising the hydrogel microspheres described herein and a pharmaceutically acceptable carrier or diluent.

[0048] Accordingly, a pharmaceutical composition comprising radiopaque hydrogel microspheres formed from a polymer comprising a 1,2-diol group or a 1,3-diol group acetalized with a radiopaque chemical species as described herein forms a further aspect of the present invention. The polymer preferably comprises an iodinated aromatic group covalently bonded to the polymer via a cyclic acetal linkage as described above.

[0049] A pharmaceutical composition comprising radiopaque microspheres may also contain additional radiopaque substances such as contrast agents (either ionic or non-ionic contrast agents including oily contrast agents such as iodized poppy seed oil ethyl ester (Lipiodol®)). Suitable non-ionic contrast agents include iopamidol, iodixanol, iohexol, iopromide, iobitridol, iomeprol, iopentol, iopamiron, ioxilan, iotrolan, iotrol and ioversol.

[0050] Ionic contrast agents may also be used, but since a high ion concentration favors the dissociation of ionic drugs from the matrix, it is not preferred to combine them with ion exchange microspheres loaded with drugs. Examples of ionic contrast agents include diatrizoate, metrizoate and ioxaglate.

[0051] The microspheres can be dried by any process recognized in the art, but since the microspheres can be stored in a dried state under reduced pressure, it is advantageous to dry them under vacuum, such as by freeze-drying. Using this method, rehydration is improved as discussed in International Publication No. 07147902, which is incorporated herein by reference. Typically, the pressure for storing the dried microspheres is less than 1 mBar (gauge pressure).

[0052] Alternatively or additionally, an effective amount of one or more biologically active agents may be included in the embolization composition. It may be desirable to deliver the active agent from the formed radiopaque hydrogel or microspheres. Biologically active agents that may be desirable to deliver include prophylactic, therapeutic and diagnostic agents (collectively referred to herein as "active agents", "therapeutic agents" or "drugs") including organic and inorganic molecules and cells. A wide variety of active agents can be incorporated into radiopaque hydrogels and microspheres. Release of the incorporated active agent from the hydrogel is achieved by diffusion of the agent from the hydrogel, degradation of the hydrogel, and / or degradation of the chemical bonds linking the agent to the polymer when in contact with an aqueous medium such as body fluid. In this context, "effective amount" refers to the amount of the active agent necessary to obtain the desired effect.

[0053] Accordingly, in a further aspect, the present invention provides a pharmaceutical composition comprising a radiopaque hydrogel microsphere as described above and a therapeutic agent, wherein the therapeutic agent is absorbed in the hydrogel matrix. A further aspect of the present invention provides a composition comprising one or more radiopaque hydrogel microspheres described herein, wherein the microspheres further comprise a therapeutic agent such as one or more pharmaceutically active agents. Examples of active agents or pharmaceutically active agents that can be incorporated include, but are not limited to, anti-angiogenic agents, cytotoxic agents and chemotherapeutic agents that make the microspheres particularly useful for chemoembolization procedures.

[0054] In a particularly advantageous embodiment, the radiopaque hydrogel microspheres of the present invention have a net negative charge such that a charged drug is loaded into the microspheres, for example by an ion exchange mechanism. As a result, the therapeutic agent is electrostatically retained within the hydrogel and elutes from the hydrogel in an electrolyte medium such as physiological saline or in vivo, for example in blood or tissue, releasing the drug over several hours, days or even weeks. In this embodiment, the radiopaque hydrogel microspheres of the present invention are particularly useful if they have a net negative charge over a variety of pH values, including physiological conditions (7.4), such that a positively charged drug is loaded into the microspheres in a controllable and reproducible manner and is electrostatically retained therein for subsequent sustained elution from the hydrogel in vivo. Such charges can be derived from ion exchange groups such as carboxyl or sulfonic acid groups attached to the polymer matrix. Even drugs that are uncharged at physiological pH can still be loaded into the microspheres of the present invention, which may be particularly advantageous, for example, immediately after embolization or when embolization is not required or essential, or when the low solubility of the drug under physiological conditions rather than ionic interactions dictates the release profile, or when a rapid elution or "burst effect" is desired simply to deliver the drug rapidly to the tissue.

[0055] Particularly preferred examples of drugs that can be loaded by this method include, but are not particularly limited to, camptothecin (such as irinotecan and topotecan) and anthracyclines (such as doxorubicin, daunorubicin, idarubicin, and epirubicin), anti-angiogenic agents (such as vascular endothelial growth factor receptor (VEGFR) inhibitors, for example axitinib, bortezomib, bosutinib, canertinib, dovitinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, masutinib, mubitinib, pazopanib, pazopanib semaxanib, sorafenib, tandutinib, vandetanib, batatinib, and visimodegib), microtubule assembly inhibitors (such as vinblastine, vinorelbine, and vincristine), aromatase inhibitors (such as anastrozole), platinum-based drugs (such as cisplatin, oxaliplatin, carboplatin, and miloplatin), nucleoside analogs (such as 5-FU, cytarabine, fludarabine, and gemcitabine). Other preferred drugs include paclitaxel, docetaxel, mitomycin, mitoxantrone, bleomycin, pingyangmycin, abiraterone, amifostine, buserelin, degarelix, folic acid, goserelin, lanreotide, lenalidomide, letrozole, leuprorelin, octreotide, tamoxifen, triptorelin, bendamustine, chlorambucil, dacarbazine, melphalan, procarbazine, temozolomide, rapamycin (and analogs such as zotarolimus, everolimus, umirolimus, and sirolimus), methotrexate, pemetrexed, and raltitrexed.

[0056] The radiopaque hydrogel microspheres are preferably water-swellable but water-insoluble.

[0057] In one embodiment, the beads are water-swellable but have some solubility in water. In this embodiment, the degree of swelling can be controlled by the use of an aqueous salt solution or a suitable solvent and can be determined by routine experimentation. This can be applied in particular to PVA polymers crosslinked by non-covalent bonds.

[0058] In another embodiment, the beads are swellable in water and a solvent, but are also biodegradable. In this embodiment, the beads biodegrade in vivo over a period ranging from 4 weeks to 24 months. Biodegradable polymers containing PVA are disclosed, for example, in WO 2004 / 071495, WO 2012 / 101455 and Frauke-Pistel et al., J. Control Release 2001 May 18; 73(1): 7-20.

[0059] As described above, the radiopaque polymers of the present invention can be prepared using a simple chemical reaction to directly modify pre-formed microspheres to render them essentially radiopaque. Thus, in a further aspect, the present invention provides a method of making a radiopaque polymer, comprising reacting a polymer containing a 1,2-diol group or a 1,3-diol group, preferably under acidic conditions, with a radiopaque chemical species capable of forming a cyclic acetal with the 1,2-diol or 1,3-diol.

[0060] Specifically, the radiopaque chemical species capable of forming a cyclic acetal includes a covalently bonded radiopaque halogen such as iodine, as described herein. Specifically, the halogen is covalently bonded to an aromatic group such as a phenyl group.

[0061] This chemical reaction is particularly suitable for polymers having a main chain of units with a 1,2-diol or 1,3-diol structure, such as polyhydroxy polymers. For example, polyvinyl alcohol (PVA) or a copolymer of vinyl alcohol containing a 1,3-diol backbone. The main chain may also include hydroxyl groups in the form of 1,2-glycols such as 1,2-dihydroxyethylene. These can be obtained, for example, by alkaline hydrolysis of a vinyl acetate-vinylene carbonate copolymer.

[0062] Other polymeric diols such as saccharides may also be used. In certain embodiments, the polymer is crosslinked, such as crosslinked PVA or a copolymer of PVA.

[0063] The polyvinyl alcohol that can be derivatized as described herein preferably has a molecular weight of at least about 2,000. As an upper limit, PVA can have a molecular weight of up to 1,000,000. Preferably, PVA has a molecular weight of up to 300,000, particularly up to about 130,000, and particularly preferably up to about 60,000.

[0064] In a preferred embodiment, PVA is a crosslinked PVA hydrogel, and as described above, PVA modified with N-acryloyl-aminoacetal dimethyl acetal (NAADA) is crosslinked with 2-acrylamido-2-methylpropane sulfonic acid, preferably in the form of microspheres as described in U.S. Patent Nos. 6,676,971 and 7,070,809.

[0065] Radiopaque species are acetalized and covalently attached to the polymer via a diol group. Preferred radiopaque species are chemical moieties with a high electron density, such as simple organic molecules or organometallic complexes that provide a radiopacity greater than +1 HU, and include reactive moieties that allow for the formation of cyclic acetals with the diol group on the polymer. Specific reactive moieties include aldehydes, acetals, hemiacetals, thioacetals, and dithioacetals.

[0066] In certain embodiments, the radiopaque species includes bromine or iodine. This is advantageous because organic small molecules substituted with bromine or iodine are commercially available or can be prepared using chemical reactions well-known in the art. For example, iodinated or brominated aldehydes are radiopaque and can be easily incorporated into diol-containing polymers using the methods of the present invention. Particularly useful radiopaque species include iodinated or brominated benzyl aldehyde, iodinated phenyl aldehyde, and iodinated phenoxy aldehyde.

[0067] For example, using a hydrogel polymer that has been pre-formed into microspheres (although other pre-formed hydrogel structures such as coatings are contemplated), the reaction of the radiopaque aldehyde with the diol-containing polymer proceeds surprisingly well. Thus, in another aspect, the present invention provides a method of making radiopaque hydrogel microspheres, the method comprising the steps set forth below: (a) swelling a pre-formed hydrogel microsphere comprising a polymer having a 1,2-diol group or a 1,3-diol group in a solvent capable of swelling the microsphere; (b) mixing or contacting the swollen microsphere with a solution of a radiopaque species capable of forming a cyclic acetal with the 1,2-diol or 1,3-diol under acidic conditions; and (c) extracting or isolating the microsphere.

[0068] The extracted or isolated microspheres may then be used directly, formulated into a pharmaceutical composition as described above, or dried for long-term storage.

[0069] In a preferred embodiment, the reaction is carried out on acrylamide polyvinyl alcohol-co-acrylamide-2-methylpropanesulfonate hydrogel microspheres. Examples of such microspheres are described in U.S. Patent Nos. 6,676,971 and 7,070,809.

[0070] The reaction is preferably carried out in a polar organic solvent, more specifically, in an aprotic polar solvent such as tetrahydrofuran (THF), ethyl acetate, acetone, dimethylformamide (DMF), acetonitrile (MeCN) and dimethyl sulfoxide (DMSO). Suitable solvents can be determined by those skilled in the art through routine experiments and / or by considering the properties of the solvents such as boiling point and density.

[0071] The reaction is rapid and can be carried out at room temperature or at an elevated temperature to improve the yield and shorten the reaction time. In a preferred embodiment, the reaction is carried out at a temperature above 25°C, suitably above 40°C but below 135°C, preferably below 80°C. A reaction temperature of 50 - 75°C is particularly useful. At elevated temperatures, the conversion of hydrogel beads to radiation-opaque hydrogel beads can be accomplished in just 2 - 3 hours.

[0072] As described above, the radiation-opaque species contains a functional group selected from the group consisting of aldehydes, acetals, hemiacetals, thioacetals and dithioacetals and contains iodine or other radiation-opaque halogens. In this context, groups such as acetals and thioacetals can be considered as protected aldehydes. Iodinated aldehydes such as iodinated benzaldehyde, iodinated phenylaldehyde or iodinated phenoxyaldehyde are particularly useful as they are widely available and give high reaction yields.

[0073] Thus, preferably, the radiation-opaque species is of formula IV:

Chemical formula

[0074] Preferably, A is an aldehyde, acetal, hemiacetal, thioacetal or dithioacetal group; Preferably, A is -CHO, -CHOR 1 OR 2 -CHOR 1 OH, -CHSR 1 OH or -CHSR 1 SR 2 wherein R 1 and R 2 are independently selected from C 1~4 alkyl, preferably methyl or ethyl.

[0075] Specific examples of radiopaque species that have been shown to produce radiopaque PVA hydrogel microspheres include 2,3,5-triiodobenzaldehyde, 2,3,4,6-tetraiodobenzaldehyde and 2-(2,4,6-triiodophenoxy)acetaldehyde.

[0076] In a further aspect, the present invention provides radiopaque hydrogel microspheres obtainable or obtained by reaction of a polymer containing a 1,2-diol group or a 1,3-diol group with a halogenated aldehyde, halogenated acetal, halogenated hemiacetal, halogenated thioacetal or halogenated dithioacetal.

[0077] In a preferred embodiment of this aspect, radiopaque hydrogel microspheres are obtained by the reaction of acrylamide polyvinyl alcohol-co-acrylamide-2-methylpropanesulfonate hydrogel microspheres. Examples of such microspheres are disclosed in U.S. Patent No. 6,676,971, U.S. Patent No. 7,070,809, and International Publication No. 2004 / 071495, and they have been found to react rapidly with iodinated aldehydes, iodinated acetals, and iodinated thioacetals to produce radiopaque microspheres with a high iodine content, providing good contrast in vivo. Physical properties (such as size, shape, charge, drug loading capacity, etc.) are not adversely affected by iodination and may in some cases be improved. Handling also appears to be little affected. Mechanical robustness is retained, the beads do not aggregate, and they suspend well in contrast agents and other delivery media, so delivery by catheter can be achieved relatively easily. Delivery is smooth, and it has been observed that there is no catheter occlusion. Furthermore, the beads are stable to steam sterilization and autoclaving.

[0078] Particularly suitable iodinated aldehydes include, but are not particularly limited to, 2,3,5-triiodobenzaldehyde, 2,3,4,6-tetraiodobenzaldehyde, and 2-(2,4,6-triiodophenoxy)acetaldehyde.

[0079] The above radiopaque microspheres and compositions can be used in a treatment method for occluding a patient's blood vessels by administering the microspheres described herein or a composition containing the same into the patient's blood vessels. The blood vessels can be suitable blood vessels associated with solid tumors, such as hypervascular liver tumors including hepatocellular carcinoma (HCC), as well as some other liver metastases including metastatic colorectal cancer (mCRC) and neuroendocrine tumors (NET). This treatment method is imageable and provides the clinician with sufficient visual feedback regarding the procedure in real-time or near real-time. Such a method is particularly useful when a pharmaceutically active agent is loaded into the microspheres and a therapeutically effective amount of the agent is delivered to a patient in need thereof by the treatment.

[0080] The radiopaque microspheres can also be used in procedures where the microspheres are delivered to the site of action by injection. One method for doing this is to directly deliver microspheres containing a pharmaceutically active agent to a tumor by injection.

[0081] The present invention also provides the compositions and microspheres of the present invention for use in the above treatment methods.

[0082] The microspheres described herein are surprisingly efficient in drug loading and elution. These microspheres readily load positively charged drugs, especially doxorubicin, epirubicin, daunorubicin, idarubicin, and irinotecan. Experimental studies have shown that the ability of the microspheres to load and elute drugs is the same before and after making the beads radiopaque using the chemical reaction of the present invention. In some cases, the drug loading efficiency or capacity is surprisingly improved by more than 50%. In some cases, a 100% increase in drug loading has been measured. In many cases, the degree of drug elution is not affected compared to non-radiopaque type beads, and in some cases, substantially the entire amount of drug elutes from the beads over a long period of time. In many cases, the drug elution profile is improved in that the time it takes for the drug to elute from the radiopaque microspheres is increased compared to equivalent non-radiopaque microspheres. Thus, the microspheres of the present invention surprisingly result in increased drug loading efficiency and improved, i.e., extended, drug elution compared to non-radiopaque equivalents.

[0083] Any of the polymers or microspheres of the above-described aspects and embodiments of the present invention can be used in another aspect of the present invention where an imaging method for embolization procedures is provided. In a further aspect, a method for monitoring embolization after the completion of the procedure is provided. Depending on the durability and biodegradation rate of the radiopaque polymer of the present invention, the post-treatment time frame during which embolization can be monitored can range from several days, several weeks, or even several months.

[0084] Hereinafter, the present invention will be further described by the following non-limiting examples with reference to the drawings. These are provided for illustrative purposes only, and based on these, those skilled in the art will be able to come up with other examples that fall within the scope of the claims. All references cited in this specification are incorporated by reference.

Brief Description of the Drawings

[0085]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0086] Throughout these examples, the structure of the polymer containing a 1,2-diol group or a 1,3-diol group is represented by the following structure:

Chemical Formula

Examples

[0087] Example 1: Preparation of 2,3,5-triiodobenzaldehyde from 2,3,5-triiodobenzyl alcohol [Chemical formula] In a 50 ml three-necked round-bottom flask equipped with a thermometer, a nitrogen bubbler and an airtight seal, 10.2 g of alcohol was dissolved in 100 ml of anhydrous DMSO under a nitrogen blanket and stirring conditions. Then, 1.0 molar equivalent of propane sulfonic anhydride (T3P) (50% solution in ethyl acetate) was added dropwise at 22 °C to 25 °C over 5 minutes. The reaction solution was stirred at room temperature and monitored by high performance liquid chromatography (column: Phenominex Lunar 3um C 18 : Mobile phase gradient: Phase A, water / 0.05% TFA; Phase B, ACN / 0.05% TFA; Linear gradient from A to B over 10 minutes: Column temperature 40 °C: Flow rate 1 ml / min: UV detection at 240 nm). The conversion was completed after 240 minutes. The yellow solution was poured into 100 ml of deionized water with stirring, and the resulting white precipitate was filtered and washed with the mother liquor and 50 ml of deionized water. The filter cake was slurried in 50 ml of ethyl acetate, filtered, washed again with 50 ml of water, and dried under vacuum at 40 °C for 20 hours to obtain 7.7 g of a white solid. The structure and purity were confirmed by NMR analysis and high performance liquid chromatography.

[0088] Example 2: Preparation of 2-(2,3,5-triiodophenoxy)acetaldehyde [Chemical formula] (a) Synthesis of 2-(2,4,6-triiodophenoxy)ethanol from 2,4,6-triiodophenol In a 500 ml three-necked flat-bottomed flask equipped with a thermometer, a nitrogen bubbler, and an overhead stirrer, 10 g of phenol was dissolved in 100 ml of ethanol at room temperature under a nitrogen blanket and vigorous stirring conditions. 1.25 molar equivalents of sodium hydroxide pellets were added, and the slurry was stirred for 30 minutes under a nitrogen blanket until the pellets were completely dissolved. Then, 1.1 molar equivalents of 2-iodoethanol were added while maintaining the temperature at 25 °C, and the mixture was stirred for 15 minutes. The solution was heated to reflux in ethanol. The consumption of phenol and the formation of 2-(2,4,6-triiodophenoxy)ethanol were monitored by HPLC (conditions of Example 1). After 25 hours, 0.27 molar equivalents of 2-iodoethanol were added, and the solution was stirred for an additional 2 hours while refluxing. After the solution was cooled to room temperature, 150 ml of deionized water was rapidly added under vigorous stirring conditions. The resulting slurry was filtered under vacuum and washed with the mother liquor, then three times with 30 ml of deionized water, and finally with 5 ml of ethanol. The resulting pink filter cake was dissolved in 100 ml of ethyl acetate, the organic layer was extracted with a large amount of sodium hydroxide solution (pH 14), dried over magnesium sulfate, and concentrated on a rotary evaporator to obtain 5.9 g of an off-pink solid, which was identified as 2-(2,4,6-triiodophenoxy)ethanol by comparative analysis with an analytical standard commercially available from Sigma-Aldrich.

[0089] (b) Oxidation of 2-(2,4,6-triiodophenoxy)ethanol to 2-(2,3,5-triiodophenoxy)acetaldehyde: In a 500 ml three-necked flat-bottom flask equipped with a thermometer, a nitrogen bubbler, and an overhead stirrer, 5.9 g of alcohol was dissolved in 150 ml of anhydrous DMSO under a nitrogen blanket. The solution was stirred and heated to 40 °C, and 1.6 molar equivalents of T3P (a 50% w / w solution dissolved in EtOAc) was gradually added while maintaining the temperature at 40 °C - 41 °C. The consumption of alcohol and the formation of aldehyde were monitored over time by high-performance liquid chromatography (conditions of Example 1). After 24 hours, 150 ml of water was gradually added to the reaction mixture over 2 hours using a syringe pump. An off-pink solid precipitated from the solution and was filtered under vacuum to obtain a pink filter cake, which was washed with water. The resulting agglomerates containing impurities were dissolved in ethyl acetate / hexane and then dried under vacuum at 40 °C to obtain an oil, which was identified as 2-(2,3,5-triiodophenoxy)acetaldehyde by 1H NMR analysis.

[0090] Example 3: Preparation of 1-(2,2-dimethoxyethoxymethyl)-2,3,5-triiodo-benzene from 2,3,5-triiodobenzyl alcohol and 2-bromo-1,1-dimethoxy-ethane (example of a radiopaque acetal / protected aldehyde)

Chemical formula

[0091] In a 50 ml three-necked flat-bottom flask equipped with an overhead stirrer, a thermometer, a nitrogen bubbler, and an airtight septum, 5.07 g of alcohol was dissolved in 55 ml of anhydrous 2-methyltetrahydrofuran under a nitrogen blanket and stirring conditions. Then, 2.11 g of acetal and then 0.540 g of sodium hydride (60% dispersion in mineral oil) were added. The slurry was heated to reflux under a nitrogen blanket for 1010 minutes and monitored by high-performance liquid chromatography (conditions of Example 1). The reaction mixture was dissolved in 50 ml of dichloromethane and washed 4 times with 25 ml of water. The organic layer was concentrated under vacuum to obtain a brown oil, which was identified as 1-(2,2-dimethoxyethoxymethyl)-2,3,5-triiodo-benzene by 1H NMR.

[0092] Example 4: Preparation of crosslinked hydrogel microspheres. Crosslinked hydrogel microspheres were prepared according to Example 1 of International Publication No. WO 2004 / 071495. This process was terminated after the step of vacuum drying the product to remove residual solvent. Polymers in both the high AMPS form and the low AMPS form were prepared and the beads were sieved to the appropriate size range. The beads were stored either in the dry state or in physiological saline and autoclaved. Good radiopacity results were obtained using either the high AMPS form or the low AMPS form of the polymer.

[0093] Example 5: General preparation of radiopaque microspheres from 2,3,5-triiodobenzaldehyde and preformed crosslinked PVA hydrogel microspheres [Chemical formula] In a 50 ml three-necked round-bottom flask equipped with an overhead stirrer, thermometer and nitrogen bubbler, 1.0 g of dry PVA-based beads (see Example 4 - high AMPS type) were swollen in an appropriate solvent (e.g., DMSO) under a nitrogen blanket and stirring conditions. Then, 0.20 - 1.5 molar equivalents of aldehyde (prepared according to Example 1) were added to the slurry, followed immediately by 1.0 - 10 molar equivalents of acid (e.g., sulfuric acid, hydrochloric acid, methanesulfonic acid or trifluoroacetic acid - usually methanesulfonic acid is used). Based on the properties of the PVA used and the degree of crosslinking, the theoretical level of available -OH groups was estimated (a typical value for high AMPS beads is 0.0125 mol / gm dry beads). The reaction slurry was stirred at 50 °C to 130 °C for 12 hours to 48 hours, during which the consumption of aldehyde was monitored by high performance liquid chromatography (HPLC). If necessary, a drying agent such as magnesium sulfate acid or sodium sulfate was added to further promote the reaction. In this way, batches of radiopaque microspheres with iodine incorporated at various levels could be obtained. When sufficient aldehyde had reacted on the 1,3-diol of the PVA-based hydrogel to make it sufficiently radiopaque (see below), the reaction slurry was cooled to room temperature and filtered. The cake of beads was washed with large amounts of DMSO and water until no unreacted aldehyde was detected by HPLC measurement.

[0094] Example 6: Preparation of Radiopaque Microspheres from 2,3,5-Triiodobenzaldehyde and Crosslinked PVA Hydrogel Microspheres 5.0 g of dry PVA-based beads (refer to Example 4 - high AMPS type, 105 - 150 μm) and 0.26 equivalents of aldehyde (7.27 g) (prepared according to Example 1) were placed in a 500 ml container purged with nitrogen. 175 ml of anhydrous DMSO was added under a nitrogen blanket, and the mixture was stirred to keep the beads in a suspended state. The suspension was warmed to 50 °C, and 11 ml of methanesulfonic acid was gradually added. The reaction slurry was stirred at 50 °C for 27 hours, during which the consumption of aldehyde was monitored by HPLC. Then, the reaction slurry was washed with a large amount of DMSO / 1% NaCl and then with physiological saline. The resulting beads had an iodine concentration of 141 mg I / ml of wet beads and a radiopacity of 4908 HU.

[0095] Example 7: Preparation of radiopaque PVA hydrogel beads using 2-(2,4,6-triiodophenoxy)acetaldehyde. 2-(2,4,6-Triiodophenoxy)acetaldehyde was prepared according to Example 2 and reacted with PVA-based hydrogel beads (refer to the high AMPS type of Example 4) in the same manner as in Example 5, except that the reaction temperature was maintained at 20 °C to 50 °C. The reaction time was also shortened to less than 1 hour. The iodine content was measured to be 18 mg I / ml of wet beads.

[0096] Example 8: Preparation of radiopaque PVA hydrogel microspheres using 1-(2,2-dimethoxyethoxymethyl)-2,3,5-triiodo-benzene

Chemical formula

[0097] Example 9: Preparation of Radiopaque PVA Hydrogel Microspheres from 2,3,4,6-Tetraiodobenzaldehyde 2,3,4,6-Tetraiodobenzyl alcohol (ACES Pharma; USA) was converted to 2,3,4,6-tetraiodobenzaldehyde using T3P and DMSO as described in Example 1. Then, 0.6 molar equivalent of 2,3,4,6-tetraiodobenzaldehyde (8.8 g) was added to 2.05 g of PVA hydrogel microspheres (see Example 4 - high AMPS type with size 150 - 250 μm) together with DMSO under a nitrogen blanket. The reaction mixture was heated to 50 °C and stirred for several hours. The reaction was monitored by HPLC, and when the reaction was complete, the beads were filtered and washed with DMSO, water, and then 0.9% saline. The radiopaque beads were then stored in 0.9% saline solution for analysis. The iodine content was measured to be 30 mg / ml wet beads.

[0098] Example 10: Characterization of Radiopaque Beads Optical microscope images of typical beads prepared in Examples (5 and 6) are shown in Figure 1. The dry weight of the beads was measured by removing the filled physiological saline and sucking out the remaining physiological saline with tissue paper. Then, the beads were vacuum-dried at 50 °C overnight to remove moisture, from which the dry bead weight of the polymer and the solid content (w / w%) were obtained.

[0099] According to the Schoniger flask method, the iodine content (w / w%) of the dry beads was measured by elemental analysis. The calculation of the iodine content of the wet beads is as follows: Solid content of beads (%) × Iodine content of dry beads (%).

[0100] Depending on the chemical reactions and reaction conditions used, the solid content of the radiopaque hydrogel beads in 0.9% physiological saline prepared according to Example 5 was measured to be 5% - 16% w / w, and the weight / weight dry iodine content was measured to be 5% - 56%.

[0101] Another way to represent the iodine content is mg I / mL wet beads (wet filled bead volume), which is the same unit used for contrast agents. Using the protocol according to Example 5, iodine contents in the range of 26 mg I / ml beads to 214 mg I / ml beads were achieved.

[0102] Higher iodine contents (up to 250 mg I / ml beads) could be achieved using a similar protocol except using microspheres based on low AMPS polymers (Example 4).

[0103] Example 11 - Micro-CT Analysis of Radiopaque Beads Micro-CT was used to evaluate the radiopacity of a sample of radiopaque embolization beads prepared according to Example 5 above.

[0104] The samples were prepared in Nunc cryotube vials (product code V7634, 48 mm × 12.5 mm from Sigma-Aldrich). The beads were suspended in a 0.5% agarose gel (prepared using Sigma-Aldrich product code A9539). The resulting suspension is generally referred to as a "bead phantom". To prepare these bead phantoms, first, a solution of agarose (1%) is raised to a temperature of about 50 °C. Then, beads of a known concentration are added and the two are gently mixed until the solution begins to solidify or gel. The solution gels upon cooling, and the beads are maintained in a uniformly dispersed and suspended state within the agarose gel.

[0105] At RSSL Laboratories (Reading, Berkshire, UK), micro-computed tomography (μCT) using a Bruker Skyscan 1172 μCT scanner fitted with a tungsten anode was used to test the radiopacity of the bead phantoms. Each phantom was analysed using the same instrument settings with the tungsten anode operating at a voltage of 64 kV and a current of 155 μA. An aluminium filter (500 μm) was used.

[0106] Acquisition parameters: Software: SkyScan1172 version 1.5 (build 14), NRecon version 1.6.9.6, CT Analyser version 1.13.1.1 Light source type: 10Mp Hamamatsu 100 / 250 Camera resolution (pixels): 4000 × 2096 Camera binning: 1 × 1 Power supply voltage kV: 65 Power supply current μA: 153 Image pixel size (μm): 3.96 Filter: Al 0.5 mm Rotation step (degrees): 0.280 Output format: 8-bit BMP Dynamic range: 0.000~0.140 Smoothing: 0 Beam hardening: 0 Post-alignment: Correction Ring artefact: 16

[0107] A small amount of purified MilliQ water was carefully poured into each sample tube. Each sample was then analyzed by X-ray micro-computed tomography using a single scan to include the water control and beads. The samples were then reconstructed using NRecon and calibrated against the purified water control of the volume of interest (VOI). After calibration, air and water in the region of interest (ROI) were analyzed to verify the Hounsfield calibration.

[0108] Radiopacity was reported in both gray scale units and Hounsfield units from line scan projections across the beads. Values used for the dynamic range of all samples in NRecon (thresholding): -0.005, 0.13 (minimum and maximum attenuation coefficients). Representative images and line scans are shown in Figure 2.

[0109] Table 1 shows the radiopacity of microspheres prepared under various time and aldehyde equivalent conditions according to Example 4 in both gray scale units and Hounsfield units. The radiopacity data are the average values of 10 line scans of beads of approximately 150 microns.

[0110]

Table 1

[0111] Figure 10 shows a sample of cross-sectional images of 10 beads having an average dimension of 153 μm and an average radiopacity of 4908 HU.

[0112] Example 12. Drug Loading of Radiopaque Beads: Example 12(a) Doxorubicin 1 mL of the RO bead slurry prepared according to Example 5 (size 100 - 300 μm, 47 mg I / ml wet beads) was weighed using a graduated cylinder and the liquid was removed. While continuously infiltrating at ambient temperature, 4 mL of doxorubicin solution (25 mg / mL) was mixed with the radiopaque beads. After loading for 20 hours, the consumed solution was removed and the drug-loaded beads were washed 4 - 5 times with deionized water (10 mL). By measuring the doxorubicin concentration of the combined consumed loading solution and washing solution with a UV spectrophotometer at 483 nm, it was calculated that the loaded doxorubicin was 80 mg / mL beads. The doxorubicin hydrochloride drug loading capacity of the radiopaque beads was determined to be a non-linear function of the iodine content in the beads.

[0113] In another experiment, 1.5 ml of RO beads of 70 - 150 μm with an iodine content of 158 mg / ml wet beads were loaded as above using 3 ml of doxorubicin solution (25 mg / ml). Non-RO beads of the same size as a control were also loaded in the same manner. The RO beads were loaded with 50 mg / ml of doxorubicin and the control beads were loaded with 37.5 mg / ml.

[0114] In another experiment, the loading of RO beads (size 70 - 150 μm; iodine content 150 mg I / ml) was substantially completed after 3 hours.

[0115] The radiopaque beads prepared according to Example 5 above were loaded with a 37.5 mg / ml doxorubicin solution according to the method above. Figure 3A shows the radiopaque beads before loading, and Figure 3B shows the beads loaded with the drug. Before drug loading, the beads were observed as spherical microspheres with a light brown to dark brown color. When doxorubicin was loaded into the beads, the beads turned dark red. In this example, the beads were autoclaved to show the stability of the beads during sterilization. During autoclaving, the integrity of the beads was maintained, and the average bead diameter decreased from 177 μm to 130 μm during autoclaving. When the beads were loaded with doxorubicin, a further shift was observed in the bead size distribution, which was consistent with the drug loading observed in non-radiopaque beads. In a further example, when the drug was loaded at 51 mg / ml, the average bead diameter decreased from 130 μm to 102 μm. The resulting beads remained within a clinically useful range even after modification, sterilization, and drug loading.

[0116] Example 12(b) Epirubicin In the same manner as for doxorubicin, epirubicin was loaded into RO beads (prepared according to Example 5) and non-RO beads (high AMPS sized 70 - 150 μm prepared according to Example 4). 1 ml of beads was loaded using 1.5 ml of the loading solution (25 mg / ml epirubicin). The final loading amount in the radiopaque beads after 90 minutes was 37.49 mg (loading efficiency 99.97%), and in the non-RO beads it was 36.43 (loading efficiency 97.43%).

[0117] Example 12(c) Sunitinib A sunitinib DMSO solution was prepared by dissolving 400 mg of sunitinib powder in anhydrous DMSO in a 10 mL volumetric flask. 1 mL of RO bead slurry (70 - 150 μm, 134.4 mg I / ml wet beads, prepared according to Example 5) was pre-washed three times with 10 mL of DMSO to remove residual water. 2.5 mL of sunitinib-DMSO solution (40 mg / mL) was mixed with the RO bead slurry and mixed for 1 - 2 hours. Then, after removing the loading solution, 10 mL of physiological saline was added to the bead slurry to precipitate sunitinib inside the beads. The washing solution and drug particles were filtered through a cell strainer, and the washing was repeated 3 - 4 times. Non-RO beads (100 - 300 μm, prepared according to Example 4) were treated in the same manner.

[0118] Example 12(d) Sorafinib 1 mL of RO PVA microspheres (size 70 - 150 μm, iodine content 134 mg iodine / ml beads, prepared according to Example 5) or non-RO PVA microspheres (DC Bead™ 100 - 300, Biocompatibles; UK) was pre-washed three times with 10 mL of DMSO to remove residual water. A sorafenib / DMSO solution (39.8 mg / mL in anhydrous DMSO) was mixed with 1 mL of the bead slurry for 1 hour (2.5 mL for radiopaque beads and 2 mL for non-radiopaque beads). After removing the loading solution, 20 mL of physiological saline was added to the bead slurry. The bead suspension was filtered through a cell strainer, and the washing was repeated 3 - 4 times. The final loading level was determined by DMSO extraction of a few hydrated beads and determination of the drug concentration by HPLC (column: Kinetex 2.6u XB-C18 100A 75×4.60 mm; mobile phase, water:acetonitrile:methanol:trifluoroacetic acid = 290:340:370:2 (v / v); detection at 254 nm; column temperature 40 °C; flow rate: 1 mL / min).

[0119] 49.9 mg of sorafinib was loaded in 1 mL of RO beads, and 34.7 mg was loaded in 1 mL of non-RO (DC Bead™) beads.

[0120] Example 12(e) Vandetinib. Using sonication in a 25 ml amber volumetric flask, 500 mg of vandetanib was dissolved in 14 ml of 0.1 M HCl and deionized water was added to make up to 25 ml to prepare a 20 mg / ml vandetanib solution. Subsequently, vandetanib was loaded into both RO PVA hydrogel microspheres (prepared according to Example 5: size 70 - 150 μm; iodine content 147 mg / ml beads) and non-RO microspheres (DC Bead 100 - 300; Biocompatibles UK) according to the following protocol:

[0121] 1 ml of microspheres containing the loading solution was aliquoted with a graduated cylinder and transferred to a 10 mL vial. Subsequently, the loading solution was removed using a pipette. Then, 3 ml of the 20 mg / ml drug solution was added to the non-RO beads or 1.5 mL of the same drug solution was added to the RO beads. In the radiopaque bead loading experiment, the pH of the solution was set to 4.6 - 4.8, and in the DC bead loading experiment, the pH was set to approximately 4.2. Two hours after loading, the residual solution was removed and the beads were washed three times with 5 mL of deionized water. The combined spent loading solution and wash solution was analyzed by C 18 reverse-phase HPLC to determine the loading yield. For sterilization, if necessary, the loaded beads were placed in 1 ml of deionized water and autoclaved at 121 °C for 30 minutes, or lyophilized for 24 hours and then gamma-sterilized at 25 kGy.

[0122] The radiopaque beads were loaded with vandetanib up to a level of 29.98 mg / ml of wet beads.

[0123] The non-radiopaque beads were loaded with vandetanib up to a level of 26.4 mg / ml.

[0124] Example 12(f) Milteplatin 1 mL of each vial of hydrated RO microspheres (size 70 - 150 μm, iodine content 134 mg iodine / ml beads, prepared according to Example 5) and non-RO PVA microspheres (DC Bead 100 - 300, Biocompatibles; UK) were washed 4 times with 5 mL of 1-methyl-2-pyrrolidinone. Then, the solvent was removed. 0.147 g of miliplatine was mixed with 25 mL of 1-methyl-2-pyrrolidinone, and the suspension was heated to 75 °C in a water bath to dissolve the miliplatine. 2 mL of the drug solution was added to the washed beads, and the mixture was placed in a 75 °C water bath for 1 hour. After filtering the bead suspension through a cell strainer to remove the loading solution, it was washed with approximately 100 mL of physiological saline.

[0125] Beads of known volume were washed with deionized water and lyophilized. The total amount of platinum was determined by elemental analysis using ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometry) and converted to the miliplatine level.

[0126] This experiment was repeated in the same manner for the loading of lyophilized beads. Table 2 shows the results of loading miliplatine into wet RO beads and lyophilized RO beads.

[0127]

Table 2

[0128] Example 12 (g) Irinotecan 2 mL of each bead sample of non-RO beads (100 - 300 μm - prepared according to the high AMPS type of Example 4) and RO beads (100 - 300 μm, 163 mg I / mL, prepared according to Example 5) was mixed with 10 ml of an irinotecan aqueous solution (10 mg / mL). The loading amount was measured by determining the level of irinotecan in the consumed loading solution by UV spectrophotometry at 384 nm. For both non-RO and RO beads, almost 100% of the drug was loaded within 90 minutes.

[0129] Example 12 (h) Topotecan 1 mL of each bead sample of non-RO beads (70 - 150 μm, prepared according to the high-AMPS type of Example 4) and RO beads (70 - 150 μm, 146 mg I / mL, prepared according to Example 5) was mixed with an aqueous topotecan solution (15.08 mg / mL), and a dose of 40 mg (2.5 ml) or 80 mg (5 ml) was loaded under stirring. After about 1.5 hours, the loading amount of topotecan was measured by determining the level of topotecan in the depleted loading solution as described above by UV spectrophotometry at 384 nm. Table 3 shows that up to 80 mg of topotecan was loaded into the RO bead sample. Both non-RO beads and RO beads had more than 98% of 40 mg of topotecan loaded.

[0130]

Table 3

[0131] Example 13. Drug Elution from Radiopaque Beads Example 13(a) Doxorubicin. Doxorubicin-loaded beads (70 - 150 μm, 158 mg I / ml, 50 mg / ml doxorubicin) prepared according to Example 12(a) were added to 1000 ml of PBS in a brown bottle at room temperature. The bead suspension was stirred at a low speed with a magnetic stirrer. At the time of sampling, 1 mL of the elution medium was taken out with a 5-μm filter needle and analyzed by UV against a standard substance at 483 nm. The elution profile is shown in Figure 4.

[0132] Example 13(b) Sunitinib 400 ml of PBS in a brown bottle, 0.5 g / L Tween 80, and the sunitinib-loaded beads prepared according to Example 12(c) were added in a water bath at 37°C. The bead suspension was stirred at a low speed with a magnetic stirrer. At the sampling time points of 1 hour, 2 hours, 3 hours, and 4 hours, 10 mL of the elution medium was taken out with a 5-μm filter needle for HPLC analysis (under the conditions of Example 13(c)), and 10 mL of fresh PBS solution was added to make up the volume. At the sampling time points of 5 hours, 25 hours, 48 hours, and 73 hours, 100 mL of the elution medium was exchanged with an equal volume of fresh PBS solution. The samples were analyzed by HPLC. The elution profile is shown in Figure 6.

[0133] Example 13(c) Sorafenib 400 mL of PBS containing 0.5 g / L Tween 80 in a brown bottle, and the sorafenib-loaded beads prepared according to Example 12(d) were added in a water bath at 37°C. The bead suspension was stirred at a low speed with a magnetic stirrer. At the sampling time points of 1 hour, 2 hours, 4 hours, and 6 hours, 10 mL of the elution medium was taken out with a 5-μm filter needle for HPLC analysis, and 10 mL of fresh PBS solution was added to make the volume 400 mL. At the sampling time points of 8 hours, 24.5 hours, and 31 hours, 100 mL of the elution medium was exchanged with an equal volume of fresh PBS solution. Two repeated tests were carried out for each type of bead. The elution profiles of sorafenib from RO beads and non-RO beads are shown in Figure 6.

[0134] Example 13(d) Vandetinib Vandetinib-loaded RO beads and non-RO beads (2 ml of beads at vandetinib / ml beads, 70 - 150 μm beads and 141 mg I / ml wet beads for RO beads) prepared according to Example 12(e) were placed at ambient temperature in an amber bottle containing 500 mL of PBS with a magnetic flea. At each sample collection time point, all of the PBS elution medium was removed from the bottle through a cannula filter by a peristaltic pump and replaced with the same volume of fresh PBS. 5 μl of the elution medium was analyzed by reverse phase HPLC with detection at 254 nm. The elution profile is shown in Figure 7. 18 The elution profile is shown in Figure 7.

[0135] Example 13(e) Miliplatine Miliplatine-loaded beads prepared according to Example 12(f) were added to 50 mL of PBS containing 1% Tween 80 in a 100 mL Duran® bottle. The bottle was floated in a 37 °C water bath and rotated at 75 rpm to agitate the beads. At sample collection time points of 1 day, 5 days, 11 days, 15 days and 22 days, 20 mL of the elution medium was taken out for ICP analysis and 20 mL of fresh PBS / Tween solution was added to make up a volume of 50 mL. The elution profiles of miliplatine from RO beads and non-RO beads are shown in Figure 8.

[0136] Example 13(f) Irinotecan 163 mI / ml of the sample prepared in Example 12(g) was added at 37 °C to 500 ml of PBS in a brown bottle and stirred at low speed by a magnetic stirrer. At the sample collection time point, 1 ml of the elution medium was taken out with a 5 μm filter needle and analyzed by UV against a standard at 369 nm. The elution profile is shown in Figure 9.

[0137] Example 14. Radiopacity of Drug-Loaded Radiopaque Beads A portion of the doxorubicin-loaded beads prepared according to Example 13(a) was subjected to micro-CT analysis as described in Example 11. The drug-loaded beads were found to be radiopaque. The average bead radiopacity (gray scale) was determined to be 139 (n = 3).

[0138] Example 15. Freeze-drying protocol. The microspheres of the present invention, with or without drug loading, can be freeze-dried according to the protocol described in WO 07 / 147902 (page 15) using an Epsilon 1-6D freeze-dryer (Martin Christ Gefriertrocknungsanlagen, Osterode am Harz, Germany) equipped with a Lyo Screen Control (LSC) panel and a Pfeiffer DUO 10 Rotary Vane Vacuum pump and controlled by Lyolog LL-1 documentation software, as briefly described below.

[0139] The microspheres are freeze-dried at about -30°C for at least 1 hour without using vacuum, and then the temperature is raised to about -20°C while gradually reducing the pressure over about 30 minutes until the pressure ranges from 0.35 to 0.40 mbar. After maintaining these temperature and pressure conditions overnight, the temperature is raised to room temperature over about 1 to 2 hours at the same pressure, and then the pressure is reduced to about 0.05 mbar at room temperature for a certain period until the total cycle time reaches 24 hours.

[0140] If it is necessary to maintain the preparation under reduced pressure, the vials are sealed under vacuum by preventing substantial air entry at the end of the cycle and operating the vial sealing mechanism that lowers the shelf position to seal the vials on the lower shelf. Then, air is introduced into the chamber until atmospheric pressure is reached. Then, the shelf is returned to its original position and the chamber is opened. If the sample is not maintained under reduced pressure, the pressure is gradually returned to atmospheric pressure before sealing.

[0141] Example 16: In vivo embolization test Male Yorkshire crossbred domestic pigs (about 14 weeks old) were used in this study.

[0142] After induction of anesthesia, a sheath was placed in the femoral artery, and under fluoroscopy, a guide wire was passed through the introducer and advanced to the aorta. Subsequently, a guiding catheter was placed at the entrance of the celiac artery through the guide wire. The guide wire was removed, and the branches of the celiac artery were visualized using a contrast agent.

[0143] A combination of a micro wire and a micro catheter was passed through the guiding catheter and used for the selection of the common hepatic artery, and 25 - 50% of the liver volume was isolated. The micro catheter was inserted into the liver lobe through the guide wire, the guide wire was removed, and angiograms of the liver lobe were captured using a contrast agent. Digital subtraction angiography was performed to confirm the position of the catheter.

[0144] 2 ml of RO beads (size 75 - 150 μm, iodine content 141 mg I / ml) prepared according to Example 5 were transferred to a 20 - 30 mL syringe, and the filling solution was discarded. A smaller syringe containing 5 mL of non - ionic contrast agent (Visipaque® 320) was connected to the larger syringe via a three - way stopcock, and the beads were mixed with the contrast agent by passing through the stopcock. The total volume was adjusted to 20 mL by adding the contrast agent. This suspension was gradually administered under fluoroscopy until a nearly stagnant state was achieved. The volume of the suspension delivered to achieve the stagnant state was 2 - 6 ml.

[0145] Abdominal CT images were taken before administration, 1 hour and 24 hours after administration, and on the 7th and 14th days. On the 14th day, baseline CT images were taken and 75 cc of contrast material was injected. After injection of the contrast material, a second CT image was taken. The images were analyzed for the visibility of the beads in the liver.

[0146] The RO beads were visible on both X-rays and CT during the procedure. This was best demonstrated on the CT scans obtained on days 7 and 14 without the use of intravenous contrast agent (see Figure 11). The beads were easily visible in multiple branches of the hepatic artery. The beads had greater attenuation than the intravenous contrast agent and could be distinguished from it.

Claims

**Claim 1**: A hydrogel having a crosslinked polymer network structure comprising a polymer having a polyvinyl alcohol (PVA) main chain and a plurality of pendant groups having crosslinkable ethylenically unsaturated functional groups, and a radiopaque chemical species covalently bonded to the polymer by a cyclic acetal group. The radiopaque chemical species has an iodinated aromatic group comprising one or more covalently bonded radiopaque iodine. The hydrogel has iodine of 10% or more by dry weight, the hydrogel. **Claim 2**: The hydrogel according to claim 1, wherein the plurality of pendant groups are formed from acetalization of PVA with N-acryloyl-aminoacetaldehyde dimethyl acetal, and the PVA is crosslinked by 2-acrylamido-2-methylpropanesulfonic acid (PVA-AMPS). **Claim 3** The hydrogel according to claim 1, wherein the radiopaque chemical species comprises an iodinated phenyl group. **Claim 4**: The hydrogel according to claim 1, wherein the hydrogel is in the form of microparticles or microspheres. **Claim 5**: The hydrogel according to claim 4, wherein the hydrogel is in the form of microspheres and has an average diameter in the range of 10 to 2000 μm. **Claim 6**: The hydrogel according to claim 1, wherein the hydrogel is in the form of microspheres or microparticles having an average radiopacity of 500 HU or more. **Claim 7**: The hydrogel according to claim 1, wherein the hydrogel has a net charge at physiological pH.

8. 【Fig. 1】 The hydrogel has a structure of general formula I or II, wherein X is a group substituted by one or more iodine, and J is a group or bond of formula -CH2-. The hydrogel according to claim 1.

9. 【Fig. 2】 X is a group of formula III, wherein Z is a linking group bonded to the cyclic acetal, or the phenyl group does not exist so as to be bonded to the cyclic acetal. When Z is present, Z is C1-6 alkylene, C1-6 alkoxylene or C1-6 alkoxyalkylene. Hal is one, two, three or four covalently bonded radiopaque halogens. The hydrogel according to claim 8. **Claim 10**: A composition comprising the hydrogel according to claim 1 and a therapeutic agent absorbed in the matrix of the hydrogel.

11. The composition according to claim 10, wherein the therapeutic agent is electrostatically retained within the hydrogel and elutes from the hydrogel in an electrolyte medium.

12. The hydrogel according to claim 1, which is used for embolization of blood vessels.

13. The composition according to claim 10, which is used for embolization of blood vessels.

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