NZ714077B2 - Gel formulations for guiding radiotherapy - Google Patents

Abstract

The present invention describes an X-ray contrast composition for local administration, wherein the X-ray contrast composition exhibits contrast properties and wherein at least 60% of an administrated amount of said X-ray contrast composition remains more than 24 hours within 10 cm from an injection point when the X-ray contrast composition is administrated to a human or animal body. point when the X-ray contrast composition is administrated to a human or animal body.

Description

GEL FORMULATIONS FOR GUIDING RADIOTHERAPY Field of the invention The t invention relates to improved formulations for guiding radiotherapy.

Technical Background Every year more than 12 million people are diagnosed with cancer worldwide and over 7.5 million people die from cancer each year. These numbers are ed to increase e of population growth and due to the lifestyle in the Western world. Radiotherapy is an important part of modern cancer ent and more than 50% of cancer patients receive radiotherapy at least once. Modern radiotherapy relies on advanced high precision planning, ent equipment and imaging techniques (such as, e.g., computed tomography (CT), positron-emission tomography (PET) and magnetic imaging resonance (M Rl)) in order to deliver high radiation doses to a precisely defined target in patients.

One ofthe main difficulties in external beam radiotherapy is that both tumors and the surrounding tissue move significantly and unpredictably during radiotherapy; both within each single treatment, and during the whole course of radiotherapy, lasting usually 5—7 weeks. These movements can be dramatic (e.g. several cm within seconds) and may be caused by various factors such as respiration, bladder- and bowel filling, air passing colon, tumor shrinkage and set-up variation of the patient. One way of minimizing this problem is the implantation of markers in or adjacent to the tumor allowing frequent imaging and treatment adaptation. So far, markers have been inserted using long and thick needles, a complicated procedure with a significant risk of complications, which is ng the practical usefulness of markers in radiotherapy.

Ideally, a tissue marker should enable tracking of tumor movement; be visible on several image ties; be visible for an extended period (e.g., at least 4 ; be non-toxic; and be easy to insert.

Various attempts have been made for improvements within the field of radiotherapy. 935 describes a composition for lled release of a W0 2014!187962 substance WO9403155 describes a hydrogel composition prepared from a ne bonded to a cross—linking agent. The hydrogels may be loaded with therapeutic drugs and diagnostic labels, including X-ray contrast imaging agents for disease diagnostics and treatment. U520120065614 discloses a hybrid system for bio imaging. Gold is bound into a matrix comprising a hydrogel or polymer or similar. In U520100297007 a substantially bi concave shaped nanoparticle is disclosed, the nanoparticle comprising an aqueous inner core and a hydrophilic outer shell comprising an hilic polymer.

Furthermore, U52009110644 discloses a nanoparticle consisting of a polymer which is a metal chelating agent coated with a magnetic metal oxide, wherein at least one active agent is covalently bound to the polymer. In the documents U520100290995 and U52005036946, radio-opaque biodegradable compositions are disclosed by ing terminal groups of synthetic and natural biodegradable polymers such as polylactones with iodinated moieties and in SE403255 a contrast agent is disclosed that comprises a polymer comprising hydroxy— and/or carboxy- and/or amino groups further comprising X-ray contrast giving iodo-substituted aromatic groups. Further yet, the document W09519184 discloses air ulating micro particles formed by ionotropically gelling synthetic polyelectrolytes such as poly(carboxylato—phenoxy)phosphazene, poly(acrylic acid), ethacrylic acid) and rylic acid copolymers (Eudragit's) by contact with multivalent ions such as m ions.

There are several drawbacks to the current al practice using solid markers and the methods described in the documents above. Installation of solid markers is invasive due to the large ion ofthe solid implant which may cause severe complications limiting is usefulness in radiotherapy. By combining gel- g, scosity solutions with solid particles and/or organic X-ray contrast agents (or other imaging modalities) injectable gels can be ated with fine— tuned properties as these can be modified by multiply parameters with t to the gel forming solution and the contrast agents used. The solid particles can, besides contributing to the overall contrast of the system, also carry pharmaceutical substances and control their release in a controlled manner.

W0 2014!187962 2014/060673 One aim of the present ion is to provide new formulations comprising rming, low-viscosity systems that are easy to administer parenterally, and wherein the present invention provides good visualization by one or multiple imaging modalities, including X—ray imaging.

Summary ofthe invention X-ray imaging of a locally administered reference marker is achieved by use of an X-ray contrast composition, wherein the X—ray contrast composition exhibits contrast properties and n at least 60% of an administrated amount of said X- ray contrast composition remains more than 24 hours within 10 cm from an injection point when the X-ray contrast composition is administrated to a human or animal body.

Detailed description of the invention The formulation is preferably in the form adapted for parenteral administration, and should preferably t of pharmaceutically acceptable constituents. The formulation which as such has a comparable low viscosity is intended for injection in the body of a human or animal, where after the formulation becomes more viscous, e.g. it goes through a sol—gel transition (liquid to gel) or forms a amorphous glass , due to the presence ofthe gel-forming system. It is red that the viscosity of the formulation after injection in the body of a human or animal increases by at least 50 %, such as at least 80 %, such as at least 100 %, or at least 150 %, or at least 200 %, or at least 300 %, or at least 500 %, or at least 750 %, or at least 1000 %, or at least 10,000%, or that the formulation s essentially solid (non-viscous).

The formulation is ably adapted for injection via a thin needle used for injection into a body or surgical related procedures, such as but not limited to biopsy. The viscosity of the hydrogel or gel-forming formulation before ion can be any suitable viscosity such that the formulation can be parenterally administered to a patient.

Exemplary formulations include, but are not limited to, those having a viscosity (prior to administration/injection) lower than 10,000 centipoise (cP), e.g. lower than 2,000 cP, such as 10 to 2,000 cP, such as 20 to 1,000 cP, such as 150 to W0 2014!187962 350 cP, such as 400 to 600 cP, such as 600 to 1,200 cP or such as 1,000 to 2,000 cP, or 10 to 600 cP, or 20 to 350 cP, at 20 °C.

Alternative formulations include, but are not limited to, those having a viscosity (prior to administration/injection) lower than 10,000 centipoise (cP), e.g. lower than 2,000 cP, such as 10 to 2,000 cP, such as 20 to 1,000 cP, such as 150 to 350 cP, such as 400 to 600 cP, such as 600 to 1,200 cP or such as 1,000 to 2,000 cP, or 10 to 600 cP, or 20 to 350 cP, at 5 °C.

When referred to herein, the (dynamic) viscosity is measured at the specified temperature in accordance with the method described in ASTM D7483.

Hydrogels, gels or amorphous glass matrixes may be formed either through covalent bond formation or ionic— or hydrophobic interactions. Physical (non- covalent) cross-links may result from complexation, hydration, hydrogen bonding, desolvation, Van der Waals ctions, ionic bonding, combinations thereof, and the like, and may be initiated by mixing two precursors that are physically separated until combined in situ, or as a consequence of a prevalent condition in the logical environment, including ature, pH, ionic strength, combinations thereof, and the like. al ent) cross linking may be accomplished by any of a number of mechanisms, including free radical polymerization, condensation polymerization, anionic or cationic polymerization, step growth polymerization, electrophile-nucleophile reactions, combinations f, and the like. Figures 1-6 illustrate exemplary hydrogel and/or rming and/or amorphous glass matrix systems that can be used in the present invention.

The hydrogel, gel or amorphous glass matrix forming compositions may be loaded with organic x-ray agents such as iodinated polymers or sugars and nanoparticles or submicron particles either prior to or during gel formation, such as when the formulation is in a sol-state or in transition to the gel—state, e.g., by diffusion into the el composition. These x—ray agents or les may either be entrapped in the gel matrix without any chemical cross-linking, or they may be , non—covalently or covalently, to the backbone or cross—linking agent of the hydrogel, gel or amorphous glass matrix. The organic x-ray agents may be one component in the gel and the particles r component, where the particles are W0 2014!187962 either a contrast agent for imaging by x—ray, MRI, PET, SPECT, fluorescence or ultrasound, and/or contain pharmaceutical agents. Pharmaceutical agents may be, but not limited to, radiosensitzers, chemotherapeutics or hormones. MRI agents such as gadolinium may be a component in the gel g systems. Pharmaceutical agents can furthermore be covalent or non-covalently embedded in the el, gel or amorphous glass matrix.

After ion, the formulation typically provides a well defined assembly of x-ray contrast agents which provides contrast in e.g. X-ray imaging, and which may serve as a marker, thus, enabling tracking of tumor movement during e.g. radiotherapy or al procedures.

U52001/0142936 discloses covalently linked hydrogels particles in the eter range (lOum — 500um) with/without radiopaque agents for use of conformal filling of surgical sites with optional imaging in order to ensure that the implants are oned correctly. The present invention offers several advantageous features as it exploits organic x-ray contrast agents that may be in combination with nano-sized particles combined with a gel forming able liquid. Nano—sized particles exhibit low/no sedimentation rate due to the s of Brownian motion which is problematic for micrometer sized particles. Furthermore, ng the particles and the gel forming solution into two components enables control over particle diffusion, release etc. within the gel which is advantageous for controlling the overall properties of the formulation. U52011/0142936 is built on the invention that swelling of the gel will increase the distance between normal and tumor tissue by injecting into enic (”medically produced”) spaced. The present invention aims at infiltrate tissue with minimal impact on the shape and position of the target tissue typically being a cancer. Furthermore, the ion of the present invention is to infiltrate tissue with minimal change in size and location why swelling is for this invention a antage. This in contrast to USZOOl/0142936 In the context of the present invention, a ”marker” or ”tissue marker” is a detectable agent or composition which does not move, or stays substantially in the same position, for several days or weeks once it has been administered or implanted into a specific site or tissue of a ian body. A tissue marker can, for example, WO 2014!187962 comprise one or more X—ray contrast agents, ctive compounds, paramagnetic compounds, fluorescent agents, or other detectable agents.

In the context of the present invention, a ”ge I” ‘ IS defined as a carrier matrix in which the detectable agent (contrast agent) is dispersed and/or dissolved within.

The term ”gel” includes systems such as hydrogels, gels or amorphous glass matrixes which upon injection into a human or an animal ses viscosity due to chemical and/or physical stimulus.

An ”imageable tissue marker” or able marker” comprises a detectable agent in a form and/or a sufficient amount to allow for detection of the tissue marker by an external imaging modality if administered or implanted into a mammalian body. ary external imaging modalities include, but are not limited to, X-ray imaging, CT imaging, MRI, PET imaging, single photon emission computed tomography (SPECT) imaging, nuclear scintigraphy imaging, ultrasonography imaging, onic imaging, near—infrared imaging and/or fluorescence imaging. Some examples ofthe brand names and types of different image techniques are e.g. ExacTrac® (BrainLAB), Cone Beam (e.g. Vairan) and OBI (e.g. On-Board ® Varian).

Contrast agents Contrast may be ed using organic x—ray contrast agents, such as radiopague agents such as iodinated nds, which may be combined with chelators of MRI agents such as nium, and/or combined with chelators of PET g agents such as copper-64, which may further be combined with solid inorganic particles. Chelators may be DOTA, EDTA, or DTPA and chelators will be non—covalently embedded or covalently conjugated to the gel—forming components.

The ed contrast agents should preferably be visible by at least CT imaging.

Preferred contrast agents are iodinated compounds such as rs or sugar molecules such as derivatives ofglucose or sucrose or other oligosaccharides. Solid particles may comprise, or t of, one or more X-ray contrast agents, i.e., compounds that are able to block or attenuate X—ray radiation. Such compounds include transition metals, rare earth metals, alkali metals, alkali earth metals, other metals, as defined by the periodic table. A metal or alkali metal may appear in non- W0 2014!187962 2014/060673 oxidized or any of the ng oxidation states for the metal. These oxidation states include monovalent cations, divalent cations, trivalent cations, tetravalent cations, pentavalent cations, hexavalent cations and heptavalent cations.

In one embodiment, the one or more X—ray contrast agents are selected from Iodine (I), gold (Au), bismuth (Bi), gadolinium (Gd), iron (Fe), barium (Ba), calcium (Ca) and magnesium (Mg). In a particular embodiment, the detectable compound comprises one or more compounds selected from the group of gold (Au) and bismuth (Bi). The one or more X-ray contrast agents are typically present in metal form, in alloy form, in oxide form or in salt form.

It should be understood that besides iodinated compounds which provides a useful contrast for X-ray imaging, the formulation may also include solid particles that are visible by X—ray imaging or other imaging modalities than X-ray imaging. In one embodiment, the solid-particles are furthermore visible by MR and/or PET imaging, or by other imaging ties.

In a particular ment, the gel-forming composition may further se a radioactive or paramagnetic compound for one or more g ties such as MRI, PET imaging, SPECT imaging, nuclear scintigraphy imaging, ultrasonography imaging, ultrasonic imaging, near-infrared imaging and/or fluorescence imaging.

In some interesting embodiments, the formulation according to any one of the preceding claims, contain solid particles that comprise one or more radioactive, paramagnetic or ferromagnetic particles.

Moreover, individual les may comprise two or more types of compounds which are visible in different imaging modalities.

Said radioactive compounds may se isotopes of Copper (61Cu, 64Cu, and 67Cu), Indium (lllln), Technetium (99mTc), Rhenium (186Re, 188Re), Gallium (67Ga, 68Ga), Strontium (89$r), Samarium ), Ytterbium (169Yb), Thallium (201Tl), Astatine (211At), Lutetium (177Lu), Actinium (225Ac), Yttrium (90V), Antimony (1195b), Tin (117Sn, 113Sn), Dysprosium (159Dy), Cobalt , Iron (59Fe), Ruthenium (97Ru, , Palladium (103Pd), Cadmium (115Cd), Tellurium (118Te, 123Te), Barium (1318a, 140Ba), nium (149Gd, 151Gd), m (160Tb), Gold (198Au, 199Au), num (140La), W0 2014!187962 Zirconium (892r) and Radium (223Ra, 224Ra), wherein said isotope of a metal radionuclide may appear in any of the existing oxidation states for the metal. These oxidation states include monovalent cations, divalent cations, trivalent cations, tetravalent cations, pentavalent cations, hexavalent cations and heptavalent cations.

Said paramagnetic or ferromagnetic compounds may also be selected from the group of Scandium (Sc), Yttrium (Y), num (La), Titanium (Ti), Zirconium (Zr), Hafnium (Hf), Vandium (V), Niobium (Nb), Tantalum (Ta); Chromium (Cr), Molybdenium (Mo), en (W), Manganese (Mn), Technetium (Tc), Rhenium (Re), Iron (Fe), Ruthenium (Ru), Osmium (Os), Cobalt (Co), Rhodium (Rh), Iridium (Ir), Nickel (Ni), Palladium (Pd), Platinum (Pt), Copper (Cu), Silver (Ag), Gold (Au), Zinc (Zn), Cadmium (Cd), Mercury (Hg), the lanthanides such as Lathanum (La), Cerium (Ce), Praseodymium (Pr), ium (Nd), Promethium (Pm), um (Sm), Europium (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb), um (Lu)) and the actinides such as Actinium (Ac), Thorium (Th), Protactinium (Pa), Uranium (U), Neptunium (Np), Plutonium (Pu), Americium(Am), Curium (Cm), Berkelium (Bk), Californium (Cf), Einsteinium(Es), Fermium (Fm), Mendelevium (Md), um (No) and Lawrencium (Lr), wherein said paramagnetic or ferromagnetic nds may appear in any of the existing oxidation states for the metal. These oxidation states include monovalent cations, divalent cations, ent cations, tetravalent cations, pentavalent cations, hexavalent cations and heptavalent cations.

Said one or more radioactive, paramagnetic or ferromagnetic compounds may be covalently linked to gel-forming components or the ized particles or non—covalently associated with the gel-forming components or nano-sized les.

In one embodiment, the gel-forming components or nano-sized particles further comprise one or more fluorophore nds for near infrared fluorescence imaging. Said nds may comprise a fluorescent proteins, peptides, or fluorescent dye molecules. Common classes of fluorescent dyes e xanthenes such as rhodamines, rhodols and fluoresceins, and their derivatives; bimanes; coumarins and their derivatives such as umbelliferone and aminomethyl coumarins; aromatic amines such as dansyl; squarate dyes; benzofurans; fluorescent W0 2014!187962 cyanines; carbazoles; dicyanomethylene pyranes, polymethine, oxabenzanthrane, xanthene, pyrylium, carbostyl, perylene, acridone, quinacridone, rubrene, anthracene, coronene, phenanthrecene, pyrene, butadiene, stilbene, lanthanide metal e complexes, rare-earth metal chelate complexes, and derivatives of such dyes. Typical fluorescein dyes include 5-carboxyfluorescein, fluorescein-5— isothiocyanate and oxyfluorescein; examples of other fluorescein dyes can be found, for example, in US 6,008,379, US 5,750,409, US 5,066,580, and US 4,439,356.

The species may also include a ine dye, such as, for example, ethylrhodamine-6—isothiocyanate, 5—carboxytetramethylrhodamine, 5— y rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 yl chloride (sold under the tradename of TEXAS RED), and other rhodamine dyes. The species may alternatively include a e dye, such as, for example, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy. Or IRDye 800CW, IRDye 680LT, Qdot 800 nanocrystal, Qdot 705 nanocrystal or porphyrazine compounds In another ment, the nano—sized particles further comprise or consist of one or more gasses encapsulated in lipid, polymer or inorganic based particles for ultrasonography imaging. Said gasses may comprise air, sulphur halides such as sulphur hexafluoride or disulphur uoride; fluorocarbons such as orocarbons; fluorinated (e.g. perfluorinated) ketones such as perfluoroacetone; and fluorinated (e.g. perfluorinated) ethers such as perfluorodiethyl ether. Representative perfluorocarbons, which may for example contain up to 7 carbon atoms, include perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g. perfluoro—n—butane, optionally in a mixture with other isomers such as perfluoro-iso—butane), perfluoropentanes, perfluorohexanes and perfluoroheptanes; perfluoroalkenes such as oropropene, perfluorobutenes (e.g. perfluorobut—Z-ene) and perfluorobutadiene; perfluoroalkynes such as orobut-Z-yne; perfluorocycloalkanes such as perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, orotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, W0 2014!187962 perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane; and mixtures of any of the foregoing, ing es with gases such as nitrogen, carbon dioxide, oxygen etc, but not limited to those.

In another embodiment, contrast in achieved using small organic iodine containing nds. Said small organic iodine containing compounds includes commercial available iodinated contrast agents such as diatrizoate (marketed e.g. under the trade name GastrografenTM), ionic dimers such as ioxaglate (marketed e.g. under the trade name HexabrixTM), nonionic monomers such as iohexol (marketed e.g. under the trade name Omnipaque‘”), iopamidol (marketed e.g. under the trade name IsovueT'V'), iomeprol (marketed e.g. under the trade name lomeronTM) and the non—ionic dimer iodixanol ted under the trade name and VisipaqueTM).

Additional examples of small organic iodine containing nds includes the ones disclosed in W02009/071605 , EP1186305, EP686046, EP108638, EPOO49745, 992, W02003080554, W02000026179, W01997000240, WO9208691, US3804892, US4239747, US3763226, 227 and U53678152, but not limited to those. In another interesting ment, the said small c iodine ning compounds includes iodinated derivates of sucrose acetate isobutyrate (SAIB). In contrast to what is disclosed in for example EP1006935, where a ition for controlled release of a substance is disclosed which composition ses SAIB, this ic embodiment according to the present invention aims at providing a stable contrast agent embedded in SAIB—gel. Examples of such iodinated derivates of sucrose acetate isobutyrate (SAIB) are illustrated in figure 7, but not limited to those. Such nds may be used alone or in combination with solid particles to achieve an injectable gel visible by at least CT imaging. In one specific embodiment of the invention the hydration sensitive gel forming component is sucrose acetate isobutyrate (SAIB) a hydrophobic component composed of sucrose (the scaffold) which has been acylated with isobutyrate and acetate. red scaffolds of this invention are monosaccharides, disaccharides or trisaccharides. A particularly preferred dissacharide scaffold is sucrose, however, the alcohol containing scaffold may be derived from a polyhydroxy alcohol having from about 2 to about 20 hydroxy W0 2014!187962 2014/060673 groups and may be formed by esterifying 1 to 20 polyol molecules. le alcohol moieties include those derived by removing one or more hydrogen atoms from: monofunctional C1-C20 alcohols, difunctional C1-C20 alcohols, trifunctional alcohols, hydroxy—containing carboxylic acids, hydroxy-containing amino acids, phosphate— ning alcohols, tetrafunctional alcohols, sugar alcohols, monosaccharides, and disaccharides, sugar acids, and polyether polyols. More specifically, alcohol es may include one or more of: dodecanol, hexanediol, more particularly, 1,6- hexanediol, ol, glycolic acid, lactic acid, hydroxybutyric acid, hydroxyvaleric acid, hydroxycaproic acid, , ATP, pentaerythritol, mannitol, ol, glucose, galactose, se, maltose, lactose, glucuronic acid, polyglycerol ethers containing from 1 to about 10 glycerol units, polyethylene glycols containing 1 to about 20 ethylene glycol units. Additionally, any oligosaccharide containing from 3 to about 6 monosaccharides may be used as the scaffold in the present invention. In general, the scaffold esters of the invention can be made by reacting one or more ls, in particular one or more polyols, which will form the alcohol moiety of the resulting esters with one or more carboxylic acids, es, lactams, carbonates, or anhydrides of the carboxylic acids which will form the acid moieties of the resulting esters. The esterification reaction can be conducted simply by heating, although in some instances addition of a strong acid or strong base esterification catalyst may be used. Alternatively, an esterification catalyst such as stannous 2—ethylhexanoate or activation reagents such as N-(3—Dimethylaminopropyl)—N’-ethylcarbodiimide (EDC), N,N'—Dicyclohexylcarbodiimide (DCC), O-(7—azabenzotriazol-l-yl)—N,N,N’,N’- tetramethyluronium hexafluorophosphate (HATU) and the like can be used.

The acyl groups forming the y substituents of the invention may be any moiety derived from a ylic acid. More particularly, the acyl groups of the compositions of the invention may be of the RC0—, where R is optionally oxy- substituted alkyl of 2-10 carbon atoms which may be linear or branched hydrocarbons with one or more functional groups present in the chain. Using carboxylic acids and/or polyols of ent chain length and using carboxylic acids having oxy-substitution allows control ofthe degree of hydrophilicity and of the solubility of the resulting ester. Such materials are sufficiently resistant to W0 87962 dissolution in vivo that they are able to form stabile hydrophobic gels which may encapsulate the said contrast agents of the present invention. The gels may further comprise a ceutical agent in combination with the contrast agent.

Coating of solid particles The solid particles may r comprise a variety of other components.

Useful solid particles include uncoated or coated metal particles, uncoated or coated solid metal salts, as well as liposomes, polymersomes, dendrimers, water-soluble cross-linked polymers, and micelles comprising such solid particles. As used herein, a solid particle which is "coated” ses a shell or surface coating around a solid core material. The shell or surface coating can be attached to the core material covalently, non-covalently, or by a mixture of covalent and non—covalent bonds.

Exemplary shell or surface coatings are described herein. In one embodiment, the solid particle comprises a r surface coating non-covalently or ntly attached to the particle core surface. The polymer may be a homopolymer, a copolymer, block copolymer, or a graft copolymer, or a dendrimer-type copolymer of synthetic or natural origin, but not limited to those. lly, the polymer coating ses polyethylene glycol (PEG), typically with a PEG molecular weight from 2,000 to 70,000 Daltons, such as 5,000 Daltons; dextrans, typically with a molecular weight between 2,000 and 1,000,000 Daltons; and/or hyaluronic acid, typically with a molecular weight between 2,000 and 1,000,000 Daltons. The polymers are typically combined as block copolymers in such a way that the overall polymer structure in negatively charged, allowing electrostatic ctions with a positively charged nano-sized particle surface to achieve ent coating. In a particular embodiment, the solid particles comprise ated o, PEGZOOO, PEG3000, PEG5000 or PEGloooo, i.e., PEG preparations having an average molecular weight of approximately 1,000, 2,000, 3,000, 5,000 and 10,000 Daltons, respectively, but not limited to those. In an additional embodiment, the solid particles comprise conjugated PNIPAMlOOO, ZOOO, PNIPAMgooo, PNIPAM5000 or PNIPAMlOOOO, i.e., PNIPAM preparations having an average molecular weight of approximately 1,000, 2,000, 3,000, 5,000 and 10,000 Daltons, respectively, but not limited to those. In one embodiment, the solid les comprise a shell or surface coat comprising a lipid W0 2014!187962 layer such as a lipid monolayer and/or one or more lipid bilayers, and a particle core comprising an inorganic particle. Surface—coating lipids for the purpose of the present invention, and include, for e, fatty acids, neutral fats, phosphatides, glycolipids, ceramides, sphingoglipids, aliphatic alcohols, and steroids. Specific, non- limiting examples of solid les are gold nano-sized particles synthesized with a PEG coating or PEGylated gold nanorods as described in and Kim et al 2007 [Invest. Radiol., 2007, 42, 797—806], polymer-coated bismuth sulphide nano-sized particles as described in Rabin 2006 [Nat. Mater., 2006, 5, 188-122], calcium phosphate me hell nanocomposites, dendrimers of PAMAM with entrapped gold nano-sized particles for CT imaging as described in Haba et al. 2007 [Langmuir, 2007, 23, 5243—5246] and Kojima et al 2010 [Bioconjugate Chem., 2010, 21, 1559-1564] and other solid particles comprising X-ray contrast agents known in the art. In a specific embodiment ofthe present invention, the shell of the nano—sized particle comprises stearoyl-sn—glycero—3—phosphocholine (DSPC) ”A”, cholesterol ”B”, and 1,2-distearoyl-sn-glycerophosphoethanolamine-N- [methoxy (polyethylene glycol)—2000] (DSPE-PEG—2000) ”C”, and 1,2-distearoyl—sn- glycero-3—phosphoethanolamine—N—[methoxy (polyethylene glycol)-2000]-TATE PEGRGD) ”D” with the molar ratio A:B:C:D, wherein A is ed from the interval 45 to 65, B is selected from the interval 35 to 45, C is selected from the interval 5 to 13, D is selected from the interval 0 to 3, and wherein A+B+C+D = 100. g of the solid particles can be exploited to introduce the desired chemical and/or physical properties to the colloid particles. Properties such as hydrophobicity/hydrophilicity, particle charge, hydrodynamic diameter and stability in various environments such as high/low salt concentrations, organic solvents, reductive environments and heat, among others, can be controlled by choosing the t surface coating material. These properties, uced to the solid particles by the surface coating, are ant factors to control in order to tune the overall or of the X-ray contrast composition described here.

The amount of contrast agent comprised within the gel—forming composition including an ed the nano—sized particles according to the present ion may be quantified by the weight percent of the contrast agent relative to the total W0 2014!187962 weight ofthe gel—forming system including an embedded nano—sized particle, excluding any water comprised by the nano-sized particle, by defining the weight percent of the contrast agent relative to the weight of the shell of the nano-sized le, or by quantifying the size of the contrasting agent within the prepared nano-sized particles. The latter can be measured by conventional methods in the art, such as cryo-transmission electron microscopy or dynamic light scattering.

Shape and size The nano-sized particles according to the present invention can be quasi spherical, spherical or non—spherical such as rod—shaped. Suitable nanoparticles e those having a size up to 50 um, preferably up to 5 pm.

Preferably, the nano-sized particles ing to the present ion are of a size in the range of 1 to 1000 nm, such as 2 to 10 nm, or such as 10 to 100 nm, such as 10 to 80 nm, such as 10 to 50 nm, such as 10 to 20 nm, such as 10 to 15 nm, or such as to 20 nm, or such as 20 to 50 nm, or such as 50 to 80 nm, or such as 80 to 110 nm, or such as 110 to 140 nm, or such as 140 to 170 nm, or such as 170 to 200 nm or such as 200 to 220, or such as 220 to 250 nm, or such as 250 to 280 nm, or such as 280 to 310 nm, or such as 310 to 340 nm, or such as 340 to 370 nm, or such as 370 to 400 nm, or such as 400 to 420, or such as 420 to 450 nm, or such as 450 to 480 nm, or such as 480 to 500 nm, or such as 500 to 1000 nm. The size may according to the present invention be measured in terms of the diameter, length or width, ing the number average diameter, length or width. In a preferred embodiment, the nano-sized particles in the composition of the present invention have a number average diameter in the range of 10 nm to 150 nm, such as 10 to 100 nm, such as 10 to 80 nm, such as 10 to 50 nm, such as 10 nm to 30 nm, such as 10 to 20 nm, or such as 30 nm to 40 nm, or such as 40 nm to 50 nm, or such as 50 nm to 60 nm, or such as 60 nm to 70 nm, or such as 70 nm to 80 nm, or such as 90 nm to 100 nm, or such as 100 nm to 110 nm, or such as 110 nm to 120 nm, or such as 120 nm to 130 nm, or such as 130 nm to 140 nm, or such as 140 nm to 150 nm. Controlling the shape and the size ofthe nano-sized particles may have significant nce on the stability of the nano-scale colloidal suspensions as well as the in vivo fate of the W0 2014!187962 particles. In a preferred embodiment, the nano—sized particles in the composition of the present invention have a number average diameter in the range of 10 nm to 100 nm. Such nano-sized particles exhibit low/no sedimentation rate due to the effects of an motion. In another preferred embodiment, the nano—sized particles in the composition of the present invention have a number average diameter <10 nm.

Such particles may be cleared, after degradation ofthe hydrogel, by e.g. renal filtration with uently excretion into the urine, which may prevent prolonged tissue retention and/or thus lower the risk of toxicity.

The c gel-forming system Suitable rming components include, but are not limited to, those composed of organic tuents such as derivatized saccharides such as esterified rides, derivatized polyols such as esterified polyols, polymers, lipids, peptides, proteins, low lar weight gelators and non-water soluble high-viscosity liquid carrier materials as well as ations hereof.

The saccharides and polyols gel g sysemts may be sucrose acetate isobutyrate (SAIB) a hydrophobic component composed of sucrose (the scaffold) which has been acylated with isobutyrate and acetate. Preferred scaffolds of this ion are monosaccharides, disaccharides or trisaccharides. A particularly preferred dissacharide scaffold is sucrose, however, the alcohol containing scaffold may be derived from a polyhydroxy alcohol having from about 2 to about 20 hydroxy groups and may be formed by esterifying 1 to 20 polyol molecules. le alcohol moieties include those d by removing one or more hydrogen atoms from: monofunctional C1-C20 alcohols, difunctional C1-C20 alcohols, trifunctional alcohols, hydroxy—containing carboxylic acids, hydroxy-containing amino acids, phosphate— containing ls, tetrafunctional alcohols, sugar alcohols, monosaccharides, and disaccharides, sugar acids, and polyether polyols. More specifically, alcohol moieties may include one or more of: dodecanol, diol, more particularly, 1,6- hexanediol, glycerol, ic acid, lactic acid, hydroxybutyric acid, hydroxyvaleric acid, hydroxycaproic acid, serine, ATP, pentaerythritol, mannitol, sorbitol, glucose, galactose, fructose, maltose, e, glucuronic acid, polyglycerol ethers containing from 1 to about 10 glycerol units, polyethylene glycols containing 1 to about 20 W0 2014!187962 ethylene glycol units. Additionally, any oligosaccharide containing from 3 to about 6 monosaccharides may be used as the ld in the present invention. In general, the ld esters ofthe invention can be made by reacting one or more alcohols, in ular one or more polyols, which will form the alcohol moiety of the resulting esters with one or more carboxylic acids, lactones, s, carbonates, or anhydrides of the carboxylic acids which will form the acid moieties of the resulting esters. Such systems are known to form biodegradable, amorphous carbohydrate glass matrixes upon hydration due to solvent induced phase separation.

The polymer may be a homopolymer, a copolymer, block copolymer, or a graft copolymer, or a mer-type copolymer of synthetic or natural origin.

Specific examples of suitable monomers may include: Lactide, glycolide, N-vinyl pyrrolidone, vinyl pyridine, acrylamide, methacrylamide, N-methyl acrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl te, methacrylic acid and acrylic acid having an acidic group, and salts of these acids, vinyl sulfonic acid, styrenesulfonic acid, etc., and derivatives having a basic group such as N,N—dimethylaminoethyl methacrylate, N,N- diethylaminoethyl methacrylate, N,N-dimethylaminopropyl acrylamide, salts of these derivatives, etc. Other rs may include: acrylate derivatives and methacrylate tives such as ethyl acrylate, methyl methacrylate, and glycidyl methacrylate; N-substituted alkyl methacrylamide derivatives such as N-n-butyl methacrylamide; vinyl chloride, acrylonitrile, styrene, vinyl acetate, lactones such as s-caprolactone, es such as e—caprolactame and the like. Additional examples of suitable monomers include alkylene oxides such as propylene oxide, ethylene oxide and the like, but not restricted to any of these specific examples.

On the other hand, specific examples of polymeric blocks to be combined with (or bonded to) the above-mentioned monomers may include: methyl cellulose, dextran, polyethylene oxide, polypropylene oxide, polyvinyl l, poly N-vinyl idone, polyvinyl pyridine, polyacrylamide, polymethacrylamide, poly N-methyl mide, polyhydroxymethyl te, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid, and salts of these acids; poly N,N- dimethylaminoethyl rylate, poly N,N-diethylaminoethyl methacrylate, poly W0 2014!187962 N,N-dimethylaminopropyl acrylamide, and salts of these, poly lactic-co—glycolic acid, prolactone and combinations hereof, but not limited to those.

The lipid may be any phospholipid including one or more of a sterol such as cholesterol, and tanol, a fatty acid having a saturated or unsaturated acyl group having 8 to 22 carbon atoms and an antioxidant such as tocopherol. es of the phospholipids include, for example, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, atidylinositols, phosphatidyl— glycerols, cardiolipins, sphingomyelins, ceramide phosphorylethanolamines, ceramide phosphorylglycerols, ceramide phosphorylglycerol phosphates, 1,2— dimyristoyl-1,2-deoxyphosphatidylcholines, plasmalogens, phosphatidic acids, and the like, and these may be used alone or two or more kind ofthem can be used in combination. The fatty acid residues of these phospholipids are not ularly limited, and es thereof include a saturated or rated fatty acid residue having 12 to 20 carbon atoms. Specific examples include an acyl group derived from a fatty acid such as lauric acid, myristic acid, ic acid, stearic acid, oleic acid and linoleic acid. Further, phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used. Also suitable are, for example, di— and tri-glycerides, 1,2-bis(oleoyloxy)—3-(trimethylammonio)propane (DOTAP), 1-N,N- dimethylaminodioleoylpropane (DODAP), 1—oleoyl—2-hydroxy-3—N,N-dimethylamino- propane, 1,2-diacylN,N-dimethylaminopropane, 1,2-didecanoylN,N- dimethylamino-propane, 3- beta—[n-[(N',N'-dimethylamino)ethane]—carbamoyl]— cholesterol (DC-Chol), 1,2—dimyristyloxypropyl-3—dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyloxypropyldimethylhydroxyethylammonium bromide , and the like, but not limited to those.

A ”peptide” or ”polypeptide” refers to a string of at least two a-amino acid residues linked together by chemical bonds (for example, amide bonds). Depending on the context, the term ”peptide” may refer to an individual peptide or to a collection of peptides having the same or different sequences, any of which may contain only naturally occurring a—amino acid residues, non-naturally occurring 0L— amino acid residues, or both. The peptide may exhibit self-assembling properties, for example, peptide amphiphiles, and peptides with B-sheet or cal forming W0 2014!187962 ces. The peptides may include D—amino acids, L-amino acids, or combinations thereof. Suitable, naturally—occurring hydrophobic amino acid residues which may be in the self-assembling peptides include Ala, Val, Ile, Met, Phe, Tyr, Trp, Ser, Thr and Gly. The hydrophilic amino acid residues may be basic amino acids (for example, Lys, Arg, His, Orm); acidic amino acids (for example, Glu, Asp); or amino acids that form hydrogen bonds (for example, Asn, Gln). ation of L—amino acids produces amino acids that may be reused by the host . L-configured amino acid residues occur naturally within the body, distinguishing es formed from this class of compounds from numerous other patible substances. L- configured amino acids contain biologically active sequences such as RGD adhesion sequences. The amino acid residues in the self-assembling peptides may be naturally occurring or non-naturally occuring amino acid es. Naturally occurring amino acids may include amino acid residues encoded by the standard genetic code, amino acids that may be formed by modifications of rd amino acids (for example pyrrolysine or cysteine), as well as non-standard amino acids (for example, amino acids having the D—configuration instead of the L—configuration). Although, non—naturally occurring amino acids have not been found in nature, they may be incorporated into a peptide chain. These include, for example, D-alloiso- Ieucine(2R,3S)-2—amino—3—methylpentanoic: acid, L—cyclopentyl glycine (S)—2-amino- 2-cyclopentyl acetic acid. Self-assembling peptides used in accordance with the disclosure may vary in length so long as they retain the ability to e.g. self-assemble to an extent useful for one or more ofthe purposes described herein. Peptides having as few as two OL-amino acid residues or as many as approximately 50 residues may be suitable. In embodiments, OL—amino acid analogs can be used. In particular, 0L- amino acid residues of the D-form may be used. Useful peptides may also be branched. One or more of the amino acid residues in a self-assembling peptide may be functionalized by the addition of a chemical entity such as an acyl group, a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, or a linker for conjugation. This functional group may provide for inter-peptide es, or linkages between the peptide and the hydrogel or hydrogel sor. For example, the hydrophobic portion of an amphiphilic peptide W0 2014!187962 may be onalized with acetylene groups. Alternatively, either or both ends of a given peptide may be modified. For example, the carboxyl and/or amino groups of the carboxyl- and amino-terminal residues, respectively, may be ted or not ted. Examples of self assembling peptides include the ones disclosed by Nagai, et al. [J. lled Release, 2006, 115, 18-25], der et al. [PLoS ONE, 2008, 1, 1—8] and rink et al. [PNAS, 2002, 99, 138].

The protein is not particularly limited and may have a molecular weight from -500 kDa, such as 20-200 kDa. It may be of natural origin or human engineered protein expressed in accessible biological expression systems such as e.g. yeast, ian, and bacterial expression systems. Preferably, is has a responsive domain such as o—helical coiled-coil or leucine zipper domain — but not limited to those, which upon external or internal stimuli results in hydrogel formation which structurally respond to changes in e.g. pH, temperature, and ionic strength.

Examples of such proteins include the ones disclosed by Banta et al. [Annu. Rev.

Biomed. Eng., 2010, 12, 167—86].

The low lar weight gelators include any molecule with molecular weight from 100-4,000 s, such as 250-1,000 Daltons with an amphiphilic structure capable of forming a hydrogel. Specific, miting examples of low molecular weight rs as described in A2, Chem. Rev., 2004, 104, 1201—1217 and Eur. J. Org. Chem., 2005, 3615-3631.

The non-water soluble high-viscosity liquid carrier materials include, but are not limited to, sucrose acetate isobutyrate, stearate esters such as those of propylene glycol, glyceryl, diethylaminoethyl, and glycol, stearate amides and other long—chain fatty acid amides, such as N,N'—ethy|ene distearamide, stearamide MEA and DEA, ethylene bistearamide, cocoamine oxide, long—chain fatty alcohols, such as cetyl alcohol and stearyl alcohol, long-chain esters such as myristyl ate, behenyerucate, glyceryl phosphates, acetylated sucrose distearate (Crodesta A—IO), and the like.

The gel of the present invention having biodegradability and sol—gel phase transition which depends on pH, ature, ion—concentration, enzymatic activity, electric field or hydration.

W0 2014!187962 The composition of the solvent (dispersion medium) should not be particularly limited, and examples include, for example, a buffer such as phosphate buffer, citrate buffer, and phosphate-buffered physiological saline, physiological saline, a medium for cell culture and patible organic solvent such as ethanol, ethyl lactate, propylene carbonate, glycofurol, N—methylpyrrolidone, 2-pyrrolidone, propylene glycol, e, methyl acetate, ethyl acetate, methyl ethyl ketone, benzyl l, triacetin, dimethylformamide, dimethylsulfoxide, ydrofuran, caprolactam, decylmethylsulfoxide, oleic acid, cylazacycloheptanone and the like. Although the formulation can be stably dispersed in these solvents (dispersion media), the solvents may be further added with a saccharide (aqueous on), for example, a monosaccharide such as glucose, galactose, mannose, fructose, inositol, ribose and xylose, disaccharide such as lactose, e, cellobiose, trehalose and maltose, trisaccharide such as ose and melezitose, and polysaccharide such as OL-, [3—, or y-cyclodextrin, sugar l such as erythritol, xylitol, sorbitol, mannitol, and maltitol, or a polyhydric alcohol (aqueous solution) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol mono-alkyl ether, diethylene glycol lkyl ether and 1,3-butylene glycol.

Additives may furthermore be selected from the group consisting of bioavailable materials such as amiloride, procainamide, acetyl-beta-methylcholine, ne, dine, lysozyme, fibroin, albumin, collagen, transforming growth factor-beta (TGF-beta), bone morphogenetic proteins (BM Ps), fibroblast growth factor , dexamethason, vascular elial growth factor (VEGF), fibronectin, fibrinogen, thrombin, proteins, dexrazoxane, leucovorin, ricinoleic acid, phospholipid, small intestinal submucosa, vitamin E, polyglycerol ester of fatty acid, Labrafil, Labrafil M1944CS, citric acid, glutamic acid, hydroxypropyl, isopropyl myristate, Eudragit, tego betain, dimyristoylphosphatidyl-choline, scleroglucan, and the like; organic ts such as cremophor EL, ethanol, dimethyl sulfoxide, and the like; preservatives such as methylparaben and the like; sugars such as starch and derivatives thereof, sugar—containing s such as sucrose-mannitol, glucose— mannitol, and the like; amino acids such as alanine, ne, glycine, and the like; W0 2014!187962 polymer-containing s such as trehalose-PEG; sucrose-PEG, e—dextran, and the like; sugar—containing amino acid such as sorbitol—glycine, sucrose-glycine, and the like; tants such as poloxamer of various molecular weights, Tween 20 Tween 80, Triton X—100, sodium dodecyl sulfate(SDS), Brij, and the like; sugar— ning ions such as trehalose—ZnSO4, maltose-ZnSO4, and the like; and bio- acceptable salts such as silicate, NaCI, KCI, NaBr, Nal, LiCI, n-Bu4NBr, Br, Et4NBr, Mg(OH)2, Ca(OH)2, ZnC03, Ca3(P04)2, ZnClz, (C2H302)ZZn, ZnC03, CdClz, HgClz, CaClz, (CaN03)2, BaClz, MgClz, PbClz, AICIZ, FeClz, FeCI3, NiClz, AgCl, AuCl, CuClz, sodium tetradecyl sulfate, ltrimethyl—ammonium bromide, dodecyltrimethylammonium chloride, tetradecyltrimethyl-ammonium bromide, and the like, but not limited to those.

In one ment of the present invention, the content of the additive is from 1><10'6 -30 wt%, preferably 1><10'3 to 10 wt%, based on the total weight of the gel forming component(s).

A preferred injectable medical gel-forming system can have one or more, preferably all, of the following features: (1) In order to be injectable, the system should be in a sol state before administration. The sol state should be of sufficiently low viscosity — typically lower than 10,000 cP, preferably lower than 2,000 cP, at 20 °C (or alternatively lower than lower than 10,000 cP, preferably 2,000 cP, at 5 °C) - to allow for small needle head to alleviate the patient discomfort and simplify insertion procedure. (2) Gelation via either chemical cross-linking, physical association or hydration starts to happen or is complete after injection. (3) The gels should be radable or gradually dissolvable within a controlled time period, and the products should be cleared/secreted through normal pathways. (4) The polymer itself and the degradable products should be biocompatible.

Likewise, if ves are added, such as cross-linking agents, initiators etc. these should also be biocompatible. (5) The gel could potentially have cell/tissue-adhesive properties.

W0 2014!187962 (6) The gel should not result in adverse effects such as immune response, e.g. inflammation.

It should be understood, that the gel-forming system should preferably be biocompatible, i.e. does not stimulate a severe, long-lived or escalating biological se to the formulation when injected into a mammal, in ular a human. To facilitate metabolism of the gel scaffold, degradable linkages can be included through the use of polylactide, polyglycolide, poly(lactide—co-glycolide), polyphosphazine, polyphosphate, polycarbonate, polyamino acid, polyanhydride, and polyorthoester — based building blocks, among others. Additionally, small molecule crosslinking agents containing similar hydrolyzable moieties as the polymers such as carbonates, esters, urethanes, orthoesters, amides, imides, imidoxy, hydrazides, thiocarbazides, and phosphates may be used as building .

Additionally, polyglycolide diacrylate, thoester diacrylate and acrylate- substituted polyphosphazine, acrylate-substituted polyamino acid, or acrylate— substituted polyphosphate polymers can be used as degradable building blocks. rylate or acrylamide moieties can be employed instead of acrylate es in the above examples. Similarly, small molecules containing a hydrolyzable segment and two or more acrylates, methacrylates, or acrylamides may be used. Such degradable rs and small molecule building blocks may be functionalized with acrylate, methacrylate, acrylamide or similar moieties by methods known in the art.

In order to be injectability, the system should be in a sol state before stration. The sol state should be of iently low viscosity to allow for small needle head to alleviate the t discomfort and simplify insertion procedure.

Gelation via either chemical cross linking or physical association starts to happen or is complete after injection.

Preferred properties of the rming system include one or more of the following: The gel-forming system may form a hydrogel. Hydrogels are comprised of cross—linked polymer networks that have a high number of hydrophilic groups or domains. These networks have a high affinity for water, but are prevented from dissolving due to the al or physical bonds formed between the polymer W0 2014!187962 . Water penetrates these networks causing swelling, giving the hydrogel its form. Fully n hydrogels have some physical properties common to living tissues, including a soft and y consistency, and low interfacial tension with water or biological fluids. The elastic nature of fully swollen or hydrated hydrogels can minimize irritation to the surrounding s after implantation. A low interfacial n between the el surface and body fluid minimizes protein adsorption and cell adhesion, which reduces the risk ofan adverse immune reaction.

Many polymers used in hydrogel preparations (e.g. polyacrylic acid (PAA), PHEMA, PEG, and PVA) have mucoadhesive and bioadhesive characteristics that enhance drug residence time and tissue permeability. This adhesive property is due to inter- chain bridges between the hydrogel polymer's onal groups and the mucus roteins, which can help enhance tissue specific g.

Preferably, before in vivo stration, the gel-forming system according to the invention is a flowable solution. The organic x—ray contrast agent, such as iodinated SAIB derivatives as illustrated in figure 7 or other iodinated polymers, and solid inorganic particles can, for example, be added to the gel-forming system simply by mixing before injection. Once ed, the gel—forming system rapidly gels under physiological conditions. An injectable matrix can thus be ted in the human body with minimal surgical procedure. After gelation in situ, the matrix can provide a reference marker for imaging and image—guided radiotherapy.

A number of activators or conditions can be used to trigger this transition upon injection, either externally applied or in response to the tissue micro- environment. Examples of this include gelation as a response to pH, temperature, ion—concentration, enzymatic activity, electric field and hydration (Figure 1). In relation to the invention it is relevant to be able to tune the mechanical stability within the tissue to allow for single injections.

Gel—forming system in response to temperature change In one embodiment, the gel-forming system undergoes gel-formation in response to a temperature in the range of 10-65°C, preferably in the range C.

The favored thermosensitive material might exhibit an inverse sol—gel transition. The term “inverse” here means that gelation occurs upon heating instead W0 2014l187962 2014/060673 of cooling. Exemplary biodegradable or bioabsorbable thermogelling polymers are shown in Figure 2. According to the origin of materials, gelling hydrogels can be classified into l (or seminatural) polymeric systems and synthetic polymeric systems. The polymers in the former system include cellulose, chitosan, ucan, gelatin etc. and their derivatives. The rs in the latter class include some polyethers, block copolymers of polyethers and biodegradable polyesters, synthetic polypeptides, and other polymers (Figure 2).

Other examples of such gel-forming s are those described in; i) Eur. J.

Pharm. Biopharm., 2004, 57, 53—63, ii) Chem. Soc. Rev., 2008, 37, 1473—1481, iii) Adv.

Drug Deliv. Rev., 2010, 62, 83-99, iv) Macromol. Biosci., 2010, 10, 563-579, v) J.

Controlled Release, 2005, 103, 609-624, vi) Expert Opin. Ther. Patents, 2007, 17, 965—977, vii) Appl. Microbiol. Biotechnol., 2011, 427-443, viii) Science, 1998, 281, 389-392, ix) Eur. J. Pharm. Biopharm. 2008, 68, 34-45, x) Biomacromolecules, 2002, 4, 865-868, xi) Colloids and Surfaces B: Biointerfaces, 2011, 82, 196—202, xii) Biomacromolecules, 2010, 11, 1082-1088, xiii) Adv. Eng. Mater., 2008, 10, 515—527, xiv) Eur. J. Pharm. Biopharm., 2004, 58, 409-426, xv) Adv. Drug Deliv. Rev., 2002, 54, 37—51, xvi) Biomater., 2004, 25, 3005-3012, xvii) J. Biomed. Mater. Res., 2000, 50, 171-177, xviii) xix) WO 50651, xxi) , xx) , xxii) WO 99/07416, xxiii) Park K., Shala by W.S.W., Park H., Biodegradable hydroge/s for drug ry. Basel: Technomic Publishing Co., Inc., 1993. ISBN 1004-6, Print, xxiv) Biomedical rs and polymers therapeutics, Ed. ini E., SunamotoJ., Migliaresi C., Ottenbrite R.M., Cohn D., New York, Kluwer Academic Publishers, 2002, ISBN 01, Print - and references herein, but not limited to those.

In one interesting embodiment the thermo sensitive r is poly(ethylene glycol)-b-poly(propylene glycol)-b—poly(ethylene glycol) (PEG—PPG— PEG, Pluronic® or Poloxamer) or derivates hereof. By controlling the PEG/PPG composition, the molecular weight and the tration, reversible gelation can occur at physiological temperature and pH.

In another interesting embodiment the thermo sensitive polymer is chitosan.

Chitosan can be a thermally ive, pH dependent, gel—forming system by the W0 87962 addition of polyol salts (e.g. B-glycerophosphate, GP). These formulations s a neutral pH, remain liquid at or below room ature, and form monolithic gels at body temperature. The stability of the sol at room temperature and the on time increase as the chitosan degree of ylation decreases [Int. J. Pharm., 2000, 203, 89-98]. The gelation for these chitosan-based systems occurs by the combination of charge neutralization, ionic and hydrogen bonds and, as the main driving force, hydrophobic interaction factors. Additionally, such systems are highly compatible with biological compounds and can be used to inject in vivo biologically active growth factors and cells [Biomater., 2000, 21, 161].

In one very interesting ment the thermo sensitive polymer is poly(caprolactone—b-ethylene glycol-b-caprolactone) EG—PCL), poly(ethylene glycol-b-caprolactone- ethylene glycol) (PEG-PCL—PEG) or poly(ethylene glycol-b— caprolactone) (PEG-PCL). This family of block ymers can be tuned to be free flowing solutions at room temperature and strong biodegradable gels at body temperature. Such polymers are highly biocompatible having showed very little toxicity with a maximum tolerance dose of 25g/kg body weight by subcutaneous administration [J. Pharm. Sci., 2009, 98, 4684-4694] and have been found stabile in vivo for more than 4 weeks [Tissue Eng. 2006, 12, 2863-2873].

In another interesting embodiment the thermo sensitive polymer is poly(ethylene glycol-b-[DL-lactic acid-co—glycolic acid]—b—ethylene glycol) (PEG-PLGA- PEG) triblock copolymers. PEG-PLGA-PEG (33 wt%) is a free-flowing sol at room temperature and become a gel at body temperature. The gel showed good mechanical strength and the integrity of gels ted longer than 1 month [J.

Biomed. Mater. Res., 2000, 50, 171—177]. Additional examples includes poly(N— isopropylacrylamide)-g-methylcellulose copolymer as a reversible and rapid temperature-responsive sol—gel hydrogel. By tuning the methylcellulose content gelation temperature, gelation time and mechanical strength can be controlled [Biomater., 2004, 25, 3005-3012].

W0 2014l187962 Gel—forming system in response to change in ion—strength In another embodiment, wherein the gel—forming system undergoes gel— formation in response to change in rength in the range of 1 uM-SOO mM — preferably in the range of 1—50 mM or 50—200 mM. miting examples of such gel-forming systems include those illustrated in Figure 3 and those described in i) Int. J. Pharm. 1989, 57, 163—168, ii) J. Controlled Release, 1997, 44, 201-208, iii) J. Am. Chem. Soc., 2001, 123, 9463-9464, iv) J.

Controlled Release, 2003, 86, 253-265, v) Biomater., 2001, 22, 511-521, xi) Park K., Shala by W.S.W., Park H., radable hydroge/sfor drug delivery. Basel: Technomic Publishing Co., Inc, 1993. ISBN 1004—6, Print xii) Biomedical polymers and polymers therapeutics, Ed. Chiellini E., Sunamoto J., Migliaresi C., Ottenbrite R.M., Cohn D., New York, Kluwer ic Publishers, 2002, ISBN 0— 30646472-1, Print; and references cited therein.

One intriguing example of such a gel—forming system is that of alginate.

Alginic acid is an unbranched binary copolymer of 1-4 glycosidically linked L- guluronic acid (G) and its C-5 epimer D-mannuronic acid (M). The proportion as well as the distribution of the two monomers ines to a large extent the chemical properties of alginate.

In one embodiment, the gel—forming system is based on an aqueous solution of an alginate. Alginates are a family of linear polysaccharides, which, in aqueous solutions, can gel after addition of multivalent cations. The use of alginate as an immobilizing agent in most applications rests in its ability to form heat—stable strong gels which can develop and set at room temperatures. It is the alginate gel ion with calcium ions which has been of interest in most applications. r, te forms gels with most di- and multivalent cations. Monovalent cations and Mg2+ ions do not induce gelation while ions like Ba2+ and Sr2+ will produce er alginate gels than Ca2+. The gel strength depends on the guluronic content and also ofthe average number of G-units in the G-blocks. Gelling of alginate occur when divalent cations takes part in the interchain binding between G- blocks giving rise to a three-dimensional network in the form ofa gel (Figure 1). The alginate gel as an lization matrix is sensitive to ing compounds such as W0 2014!187962 phosphate, lactate and citrate, presence of anti—gelling cations such as Na+ or Mg“.

To avoid this gel beads may be kept in a medium containing a few millimolar free calcium ions and by g the Na+/Ca2+ ratio less than 25:1 for high G alginates and 3:1 for low G alginates. An alternative is also to replace Ca2+ with other divalent cations with a higher affinity for alginate. There has been found a ation between mechanical gel th and affinity for cations. It has been found that gel strength may se in the following orders: Pb2+ > Cu2+ = Ba2+ > Sr“ > Cd2+ > Ca2+ > Zn2+ > Co2+ > Ni2+ However, in applications involving lization of living cells toxicity is a limiting factor in the use of most ions, and only Sr“, Ba“ and Ca2+ are considered as nontoxic for these purposes. Alginate gels have been found stable in a range of organic solvents.

Since the gel-inducing factor is added before injection, slow physical gelation is required in order to avoid syringe jam. To combat this, calcium ions can be slowly released from, e.g., CaSO4 powder after the powder has been added to a sodium alginate aqueous solution [J. Biomater. Sci., Polym. Ed., 1998, 9, 475-487]. In another sting embodiment co-injection of the gel-inducing factor and the s alginate solution using a double e results in rapid gelation in the tissue of st thus avoiding syringe jam. Another interesting embodiment is Gellan gum (Gelrite®, Figure 3) — a high molecular weight polysaccharide (500kDa) produced by the microbe Sphingomonas elodea. Gellan gum is consists of four linked monosaccharides, including one molecule of rhamnose, one molecule ofglucuronic acid and two les of glucose. It forms gels when positively charged ions (i.e., cations) are added. Thus, the properties ofthe gel can be lled by manipulating the concentration of potassium, magnesium, calcium, and/or sodium salts.

In another interesting embodiment the ion-strength sensitive gel-forming system is a peptide such as H-(FEFEFKFK)2-OH (FEK16) which is known to self- assemble into 6-sheet structures in an ionic-strength dependent manner [J. Am.

Chem. Soc., 2001, 123, 9463-9464]. FEK16 has been found to be highly soluble in pure H20 but form self-assembled hydrogels at concentrations >10 mg/mL in the presence of mM concentrations of NaCl, KCI, and CaClz.

W0 2014l187962 Gel—forming system in se to change in pH In still another embodiment, the gel-forming system undergoes gel- formation in response to changes in pH. Optionally, the gel-forming system undergoes gel-formation in response to a combined change in pH and ature, such as a pH in the range of 6-8 and a ature in the range of 35 to 40 °C. miting examples of such gel—forming systems are illustrated in Figure 4, and e those described in i) Macromol. Biosci., 2010, 10, 9, ii) J.

Controlled Release, 2001, 73, 205-211, iii) Topics in tissue engineering — Smart Polymers, Vol. 3, 2007, Chapter 6, iv) Adv. Drug Delivery Rev., 2010, 62, 83—99, v) J.

Controlled Release, 2003, 86, 253-265 vi) Biodegradable hydrogels for drug delivery.

Basel: Technomic hing Co., Inc., 1993. ISBN 1004-6, Print, vii) Biomedical rs and polymers therapeutics, Ed. Chiellini E., Sunamoto J., Migliaresi C., Ottenbrite R.M., Cohn D., New York, Kluwer Academic Publishers, 2002, ISBN 0—30646472—1, Print, and references cited therein.

The pH of the formulation (before injection) is preferably in the range of pH = 2—10, optionally in a range selected from 4-6, 6-8 and 8—9.

The properties of pH responsive hydrogels are highly depending on the pKa of the ionizable moiety, the hydrophobic moieties in the polymer ne, their amount and distribution. When ionizable groups become neutral — non—ionized— and electrostatic repulsion forces disappear within the r network, hydrophobic interactions dominate. The introduction of a more hobic moiety can offer a more compact conformation in the uncharged state and a more accused phase transition. The hydrophobicity ofthese rs can be controlled by the copolymerization of hydrophilic ionizable monomers with more hydrophobic monomers with or without pH-sensitive moieties, such as 2-hydroxyethyl methacrylate, methyl methacrylate and maleic anhydride.

An example of a gel—forming system responsive to pH changes is that which employs the pH-sensitive property of chitosan solutions at low pH. Once injected into the body, these polymer solutions face different environmental pH conditions and form gels. One example is mucoadhesive pH—sensitive chitosan/glyceryl monooleate (C/GMO) in situ gel system which consisted of 3% (w/v) chitosan and W0 2014l187962 3% (w/v) GMO in 0.33 M citric acid. an is normally insoluble in neutral or alkaline pH. However, in dilute acids (pHSS.0), it becomes e due to the protonation of free amino groups on the chitosan chains (RNH3+). The solubility of chitosan in acidic medium also depends on its molecular weight. Acidic solutions of chitosan when exposed to alkaline pH or body ical pH lose this charge and form viscous gels. Chitosan and GMO both own mucoadhesive property which has been applied in drug delivery system. ve charges on the chitosan backbone may give rise to a strong electrostatic interaction with mucus or a negatively charged mucosal surface.

Gel-forming system in response to enzymatic ty In still another embodiment, the gel-forming system undergoes gel- formation in se to enzymatic activity.

Non-limiting es ofsuch rming systems are illustrated in Figure 5 and include those described in i) Tissue Eng., 2006, 12, 1151—1168, ii) Biomater. 2001, 22, 453-462, iii) Biomater., 2002, 23, 2703—2710, iv) Colloids Surf., B, 2010, 79, 142—148, v) Biomacromolecules, 2011, 12, 82-87, vi) Macromolecules 1997, 30, 5255-5264, vii) Biodegradable hydrogelsfor drug delivery. Basel: Technomic Publishing Co., Inc., 1993. ISBN 1004-6, Print, viii) Biomedical polymers and polymers therapeutics, Ed. Chiellini E., Sunamoto J., Migliaresi C., Ottenbrite R.M., Cohn D., New York, Kluwer Academic hers, 2002, ISBN 0-30646472—1, Print, and nces cited n.

The enzyme or its origin is not particularly limited. I can be added prior, during or after injection of the gel forming system, thus function as a trigger molecule to induce gel formation. It may be encapsulated in an e.g. liposomes etc. which upon re to an internal or external stimuli releases the enzyme.

Additionally, the enzyme might be present in the injected tissue, either as a natural tissue component, or as an up-regulated enzyme due to the pathophysiological conditions at the site of injection.

In one embodiment, the enzyme triggered gel-forming system is based on caseins, a group of phosphoproteins with a molecular weight in the range from 20 kDa to 30 kDa. Such system can be turned into a hydrogel by addition of microbial W0 2014!187962 transglutaminase (MTGase), a natural tissue enzyme, at physiological temperature and pH ids Surf., B, 2010, 79, 142-148].

Another interesting example of a gel forming system based on enzymatic activation is based on Schiff base formation of lysine rich peptides clue to activation by either lysyl e or plasma amine oxidase [Biomacromolecules, 2011, 12, 82-87]. ion of e-amino groups of lysine by either lysyl oxidase or plasma amine oxidase results in de formation which readily forms a Schiff base with an additional e-amino group of lysine resulting in hydrogel formation.

Gel—forming system in response to an initiator In still another ment, the gel-forming system undergoes gel- formation in response to contact with an initiator, e.g. a le or irradiation which results in gel ion by cross linking the gel forming system by the means of a covalent chemical bond.

Non—limiting examples of such gel—forming systems are described in i) US 5410016, ii) J. Controlled Release, 2005, 102, 619-627, iii) Macromol. Res., 2011, 19, 294-299, iv) Polym. Bull. 2009, 62—699-711, v) J. Biomater. Sci., Polym. Ed., 2004, 15, 895—904, and references cited therein.

In one embodiment the gel forming system is cross linked by photoinitiation by free l generation, most preferably in the e or long wavelength ultraviolet radiation. The preferred polymerizable regions are acrylates, lates, crylates, methacrylates, dimethacrylates, oligomethoacrylates, or other biologically acceptable photopolymerizable groups. Useful photoinitiators for the above mentioned system which can be used to initiate by free radical generation polymerization of the macromers without cytotoxicity and within a short time frame, minutes at most and most preferably s. Preferred dyes as initiators of choice for visible light initiation are ethyl eosin, 2,2-dimethoxy—2—phenyl acetophenone, other acetophenone derivatives, and camphorquinone. In all cases, cross linking are initiated among macromers by a light activated free-radical polymerization initiator such as 2,2—dimethoxy—2—phenylacetophenone or a combination of ethyl eosin and triethanol amine, for example.

W0 87962 In another embodiment the gel forming system is cross linked by — or homo bifunctional linkers such as e.g. dithiothreitol, glutaraldehyde, diphenylmethanebismaleimide, inimidyl suberate, bis(sulfosuccinimidyl) suberate, dimethyl adipim and the like, but not limited to those. An example of such a gel forming system is multiacrylate PEG-based polymers which have been reported to form a hydrogel upon addition ofthe initiator DTT [J. Controlled Release, 2005, 102, 619-627]. The properties the gel could be fine tuned by controlling the size of the polymer and the amount of initiator added and the gel could be formed under physiological ature and pH. An additional example of such a system is hydrogel formation by chemically cross-linking an hyaluronic acid (HA) derivative with a hydrazide moiety and another HA derivative with an aldehyde, thus, forming a slowly hydrolysable hydrazone bond [Eur. J. Pharm. Biopharm., 2008, 68, 57-66].

This method has the age of allowing in situ cross-linking without the use of initiators, cross-linking chemicals, or extra equipment for cross—linking such as a light source.

Gel—forming system in response to hydration In still another embodiment, the gel-forming system undergoes gel- formation in response to hydration. Example of such gel-forming systems are those is selected from,- 1') , ii) Adv. Drug Delivery Rev., 2001, 47, 229—250, iii) US 2007/0092560 - and references herein, but not limited to those.

Formulations composed of neutral diacyllipids and/or tocopherols and/or phospholipids lized in biocompatible, oxygen containing, low viscosity organic solvent may form a liquid crystalline phase structure upon ion, e.g. contact with an s fluid such as vascular fluid, extracellular fluid, interstitial fluid or plasma, but not limited to those. Other systems include ter soluble high- viscosity liquid carrier materials such as sucrose acetate isobutyrate (SAIB). Such a system may be mixed with solid particles described in the present invention followed by parental ion, thus functioning as a injectable contrast agent which that can be visualized by one or multiple imaging modalities, including X—ray imaging.

W0 2014!187962 Gel—forming systems with cross linking groups In still another embodiment, any of the afore mentioned rming systems, are further functionalized by ucing one or more cross-linkable groups such as acrylate, methacrylate, acrylamide, methacrylamide, vinyl ether, styryl, e, maleic acid derivative, diene, substituted diene, thiol, alcohol, amine, hydroxyamine, carboxylic acid, carboxylic anhydride, carboxylic acid halide, aldehyde, ketone, isocyanate, succinimide, carboxylic acid hydrazide, glycidyl ether, siloxane, alkoxysilane, alkyne, azide, 2'-pyridyldithiol, phenylglyoxal, iodo, maleimide, imidoester, dibromopropionate, and halo acetates, such as bromoacetate, but not limited to those.

Gel—forming systems with chelating groups In an additional embodiment, the rming system is comprised of a chelating agent that is known to chelate ions. Any ion chelating agent now known or later discovered may be used in the articles of the t invention. Examples of metal ion (e.g., Gd3+ or Cu“) chelating agents include, but are not limited to, expanded porphyrins and rin-like derivatives, DOTA, DTPA, AngioMARKTM (a backbone—functionalized DTPA chelate), DTPA-BMA (a neutral bis—methyl amide derivative of DTPA), and HP-D03A (a DOTA-like macrocyclic compound wherein one chelate arm is replaced with a hydroxylpropyl group). Additional chelates include, but are not limited to, DPDP (TeslaScanT'V‘) and Deferoxamine (e.g. Fe3+ and Zr“).

Other constituents of the formulation The ation may further e other constituents, such as (1-, [3-, and/or y-cyclodextrins and any derivate hereof. Such constituents may form guest/host complexes with the gel forming system and the nano—sized particles, thus, both aiding in the gel formation and possible alter the le leakage profile [Adv. Drug Delivery Rev., 2008, 60, 017]. In one very interesting embodiment the gel forming system is based on PEG-PHB-PEG triblock copolymers, a—cyclodextrin and PEG coated solid nano sized particles. In such a formulation, d-cyclodextrin may form inclusion complexes with both the PEG blocks of the PEG—PHB-PEG triblock copolymers and the PEG coated solid nano sized les which, combined with hydrophobic interactions between the PHB middle block, forms a strong hydrogel W0 2014!187962 with enhanced retention of solid nano sized particles due a-cyclodextrin interactions which thus altering the particle leakage profile.

The formulation may further comprise compounds or polymers which are visible in imaging modalities other than X—ray imaging.

In one embodiment, the formulation further comprises an iodine-containing polymer, e.g. nylpyrrolidone-iodine (PVP-I), or one selected from i) Polym.

Chem., 2010, 1, 1467-1474, ii) US 3852341, iii) US 4406878, iv) US 5198136, v) Biomedical polymers and polymers therapeutics, Ed. Chiellini E., Sunamoto J., resi C., Ottenbrite R.M., Cohn D., New York, Kluwer Academic hers, 2002, ISBN 01, Print, and references cited n. Such polymers can be added to the gel forming components prior to gelation and on as contrast agent in vivo. Such polymers may additionally or alternatively be covalently bound to the one or more of the gel g components or adhered to the particles of the present invention.

In one specific ment, the formulation consist of SAIB/6,6'-(2,4,6— triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH. The said combination enables the formation of stabile injectable formulations with very high iodine content which may be used to provide good visualization by one or multiple imaging modalities, including X—ray imaging. High iodine contents (high HU—contrast) is especially important for less ive imagining techniques such as e.g. fluoroscopy among others. The iodine concentration of the said ation consisting of SAIB/6,6'— (2,4,6-triiodophenoxy)acetoxy-isobutyric—Sucrose (8)/EtOH can be fine tuned by varying the weight t (w%), as defined by the weight of the atom/molecule giving x-ray contast such as iodoine divided by the total weight of the material composition times 100, of 6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8) added to the . The elemental composition of 6,6'—(2,4,6-triiodophenoxy)— acetoxy-isobutyric—Sucrose (8) is; C, 34.96; H, 3.61; I, 42.62; 0, 18.81, based on this, the overall iodine content (w%) in various formulations can be calculated: SAIB/6,6'- (2,4,6-triiodophenoxy)acetoxy-isobutyric—Sucrose (8)/EtOH (75:5:20) equals 2.13w%/2.67w% iodine before/after injection sion of EtOH out of the formulation after injection causes an increases the w% of iodine); SAIB/6,6'-(2,4,6- W0 2014!187962 triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH (70:10:20) equals 4.26w%/5.33w% iodine before/after injection; SAlB/6,6'—(2,4,6- triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH (60:20:20) equals 8.52w%/10.66w% iodine before/after injection; ,6'—(2,4,6— triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH (55:25:20) equals .65w%/13.32w% iodine before/after injection; SAIB/6,6'-(2,4,6— triiodophenoxy)acetoxy-isobutyric—Sucrose (8)/EtOH (45:35:20) equals 14.92w%/18.65w% iodine before/after injection; SAIB/6,6'-(2,4,6- ophenoxy)acetoxy—isobutyric—Sucrose OH (30:50:20) equals 21.30w%/26.64w% iodine /after injection.

An increase in iodine concentration of the ation can directly be correlated to the observed contrast in Hounsfield units (HU). The following contrast (HU) was observed at ent energies; 80-, 100-, 120- and 140kV, all 200mAs, 2 mm (col 40 x 0.6mm) for the following formulations; a) SAIB/6,6'—(2,4,6— triiodophenoxy)acetoxy-isobutyric-Sucrose (8)/EtOH (70:10:20) (4.26w%/5.33w% iodine before/after injection) 2500HU (80kV), 1800HU (100kV), 1500HU ) and 1300HU (140kV); b) SAIB/6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric—Sucrose (8)/EtOH (55:25:20) (10.65w%/13.32w% iodine before/after injection) 5000HU (80kV), 4500HU (100kV), 3500HU (120kV) and 3000HU (140kV); c) SAIB/6,6'-(2,4,6- triiodophenoxy)acetoxy-isobutyric-Sucrose OH (30:50:20) (21.30w%/26.64w% iodine before/after injection) 10500HU (80kV), 8800HU (100kV), 6200HU (120kV) and 5900HU (l40kV).

The gel-forming formulation may further comprise ceutical agents including prodrugs (in short "drugs"; y interpreted as agents which are able to modulate the biological processes of a mammal). Examples of pharmaceutical active agents include small drugs, plasmid DNA (e.g. for gene therapy), mRNA, siRNA, carbohydrates, peptides and proteins. Specific examples of ceutical agents e; a) chemotherapeutic agents such as doxorubicin, cin, paclitaxel, nitrogen mustards, etoposide, camptothecin, 5—fluorouracil, etc.; b) radiation sensitizing agents such as gemcitabine and doranidazole, porphyrins for photodynamic therapy (e.g. visudyne) or 108 clusters or 157Gd for neutron capture W0 2014!187962 therapy; c) peptides or ns that modulate apoptosis, the cell cycle, or other crucial signaling es; d) Anti matory drugs, such as methylprednisolone hemisuccinate, B-methasone; e) Anti anxiety muscle relaxants such as diclofenac, pridinol; f) Local anesthetics such as ine, bupivacaine, dibucaine, aine, procaine; g) Analgesics such as opiods, non-steroidal anti-inflammatory drugs (NSAIDs); h) Antimicrobial medications such as pentamidine, azalides; i) Antipsychotics such as chlorpromazine, perphenazine; j) The antiparkinson agents such as ne, prodipine, benztropine te, trihexyphenidyl, L-DOPA, dopamine; k) Antiprotozoals such as quinacrine, chloroquine, amodiaquine, chloroguanide, primaquine, mefloquine, quinine; l) Antihistamines such as diphenhydramine, hazine; m) Antidepressants such as serotonin, imipramine, ptyline, doxepin, desipramine; n) Anti anaphylaxis agents such as epinephrine; o) Anticholinergic drugs such as atropine, decyclomine, methixene, propantheline, physostigmine; p) Antiarrhythmic agents such as quinidine, propranolol, timolol, pindolol; q) noids such as prostaglandins, thromboxane, prostacyclin, but not limited to those. These drugs can be formulated as a single drug or as a combination of two or more ofthe above mentioned drugs in its active form or as a prodrug.

Additional examples of antitumor agents include camptothecin derivatives such as irinotecan hydrochloride, can hydrochloride, exatecan, 00, lurtotecan, 50, Bay-383441, PNU—166148, IDEC-132, BN—80915, DB-38, DB- 81, DB-90, DB-91, CKD-620, T-0128, ST-1480, ST-1481, DRF-1042 and DE—310, taxane derivatives such as docetaxel hydrate, IND—5109, EMS-184476, EMS-188797, T—3782, TAX-1011, 31012, SBT-1514 and DJ-927, ifosfamide, nimustine hydrochloride, carboquone, cyclophosphamide, dacarbazine, thiotepa, busulfan, melphalan, ranimustine, estramustine phosphate sodium, 6—mercaptopurine riboside, enocitabine, gemcitabine hydrochloride, carmofur, cytarabine, cytarabine ocphosphate, tegafur, doxifluridine, ycarbamide, fluorouracil, methotrexate, topurine, fludarabine phosphate, actinomycin D, aclarubicin hydrochloride, idarubicin hydrochloride, epirubicin hloride, daunorubicin hydrochloride, pirarubicin hydrochloride, bleomycin hydrochloride, zinostatin stimalamer, neocarzinostatin, mytomycin C, bleomycin sulfate, peplomycin sulfate, vinorelbine W0 2014!187962 tartrate, vincristine sulfate, vindesine e, vinblastine sulfate, amrubicin hydrochloride, nib, exemestan, capecitabine, TNP-470, TAK—165, KW-2401, KW- 2170, 1, KT-5555, KT-8391, TZT-1027, S-3304, CS-682, YM-511, YM-598, TAT- 59, TAS-101, TAS—102, TA—106, FK—228, FK—317, E7070, E7389, KRN—700, KRN-5500, J- 107088, 4, SM-11355, ZD-0473 and the like.

Additional examples of radiation sensitizing agents include ium ,10,15,20—tetrakis(4-su|phophenyl)-porphine dodecahydrate, PYROA protein (Emericella nidulans), photosan |||, lomefloxacin, azine, tiaprofenic acid and the like, but not limited to those.

The drugs are included in the composition in an amount ient to achieve a desired effect. The amount of drug or biologically active agent incorporated into the composition depends upon the d release profile, the concentration of drug required for a biological effect, and the d period of release of the drug. The biologically active substance is typically present in the composition in the range from about 0.5 percent to about 20 percent by weight relative to the total weight ofthe composition, and more typically, between approximately 1 percent to about 15 t by weight. Another preferred range is from about 2 percent to about 10 percent by weight. For very active agents, such as growth factors, preferred ranges are less than 1 % by weight, and less than 0.0001 %.

Viscosity of the ation The viscosity of the formulation is before the injection preferably lower than ,000 cP, in particular lower than 2,000 cP, at 20 °C. Alternatively, the viscosity of the formulation is before the injection typically lower than 2,000 cP at 5 °C.

The organic gel-forming system of the formulation is preferably one which, after injection or under conditions ing those in a human body, forms a gel having a viscosity at 37 °C in the range of 2,000 to 50,000,000 cP. More particularly, the viscosity of the hydrogel can be about 2,000 cP, about 5,000 cP, about 10,000 cP, about 20,000 cP, about 30,000 cP, about 50,000 cP, about 75,000 cP, about 100,000 cP, about 125,000 cP, about 150,000 cP, about 200,000 cP, about 30,000 cP, about 800,000 cP, about 1,000,000 cP, about 2,000,000 cP, about 5,000,000 cP, about ,000,000 cP, about 20,000,000 cP, about 30,000,000 cP, about 40,000,000 cP, W0 2014!187962 about 50,000,000 cP, or ranges thereof. Preferably, the viscosity of the hydrogel after injection (i.e. when present in the d location) is above 20,000 cP, e.g. in the range of 20,000 cP to 1,000,000 cP. In particular, the formulation after injection is preferably essentially solid.

Use of the formulation The present invention also provides the formulation as defined hereinabove for use in X—ray imaging as a marker of ic tissue, such as computer tomography (CT), of the body of a mammal.

In one interesting embodiment, the formulation is parenterally administered to a predetermined location of the body of a human or animal, and wherein an X-ray image of at least a part ofthe body of the human or animal including the predetermined location is ed.

A kit comprising the formulation The present invention r comprises a kit comprising a syringe, a needle used for injection into a body or surgical related ures, such as but not limited to biopsy, adapted to the open end of said syringe, and a formulation as defined above. In one embodiment, the formulation is held in the interior or said syringe.

The gel forming system may be provided as a Iyophilized powder, a suspension or a solution. Different components may be provided in one or more dual vials or pre-mixed in the interior or said e. Exemplary different components include, but are not limited to, the gel-forming system and the solid particles, and the formulation and one or more initiators.

The syringe may consist of a single, a multiple barrel syringe (e.g. MEDMIX SYSTEMS AG) or a double champer syringe (e.g. Debiotech SA.) and the like, but not limited to those. Multiple barrel syringes and double champer syringes and the like may be useful for e.g. two components formulations were one component is a mixture of the gel forming system and the st agent(s) and the other ent is an initiator or salt sion of e.g. Ca2+ in the case there the gel forming system is based on alginate.

W0 2014!187962 The needle of the syringe can, in some embodiments, be one suitable for fine—needle biopsies. Non—limiting examples of syringes and needles for such embodiments are described in U.S. Patent No. 7,871,383, U.S. patent publication No. 20040162505, and references cited therein. Such syringes and needles can advantageously be used in procedures where a biopsy of a tissue is to be taken in conjunction with imaging of the same, using a formulation of the invention.

Preferably, the kit has a shelf-life of at least 6 months, such as at least 12 months when stored at, e.g., room temperature (typically 18 to 25 °C) or lower temperatures, such as, e.g., 2 to 10 °C, such as about 5 °C. The shelf-life can, for example, be determined as the period wherein the kit can be stored at 25 °C, at 80 % RH and 1 atm. re, and where the viscosity is kept within 1r 5 % of the initial viscosity.

A method of recording an X-ray image ofa body of animal or human The present invention also provides a method of recording an X—ray image of the body of a mammal, comprising the steps of: (a) ing a formulation sing an organic gel-forming system that is a nous liquid before injection that comprise an organic x-ray contrast agent such as an iodinated compound detectable by X-ray imaging; (b) administering the formulation to a t, and (c) recording X-ray-based images, such as Computed Tomography (CT) s or 2D X-ray images.

In one ment, the method is forjoint herapy and X—ray imaging of a target tissue in an individual, wherein the images in step (c) provides a definition of the target tissue, and further comprises the step of: (d) using the definition of the target tissue obtained in c) to direct external beam radiotherapy to the target tissue.

The target tissue is typically one that comprises undesirably growing cells. In one embodiment, the rably g cells are tumor cells, such as malignant cells, and the individual is suffering from or at risk for cancer. In a particular embodiment, the undesirable growth of cells is associated with lung cancer, prostate cancer, cervix or n cancer. Other types of conditions or diseases associated W0 87962 with undesirable cell growth include extra uterine (ectopic) pregnancy, benign tumors in brain, such as benign tumors located closely to the optical nerve, glandule with overproduction of hormone, such as for example alamus, bone and cartilage in relation with nerve compression, blood cells which may be killed prior to transplantation, conditions associated with large tonsils such as acute tonsillitis or ditis, ctive sleep apnoea, nasal airway obstruction, snoring, or peritonsillar abscess or hyperplasic or angiogenic eye disorders.

In embodiments where the gel-forming system is one that gels upon the addition of an initiator, the stration step (a) or (b) may further comprise mixing with an initiator.

The formulation according to the present invention may be stered parenterally, such as by intravenous, intramuscular, intraspinal, subcutaneous, intraarterial, intracardiac, intraosseous, intradermal, intracisternal, intrathecal, intracerebral, transdermal, transmucosal, inhalational, epidural, sublingual, intravitreal, intranasal, intrarectal, intravaginal or intraperitoneal administration.

The parental administration may be performed by, e.g., infusion or injection.

Typically, the formulation is administered into, or adjacent to, a predetermined location, such as a target tissue, optionally in conjunction with a biopsy of the target tissue.

The amount of formulation to ster to the mammal or individual in step (c) can be ined by one of skill in the art, taking into consideration the nature ofthe investigation and the size ofthe area to be imaged. Typically, at least 100 uL formulation is administered. In various specific embodiments, the method comprises stration of between 100 uL and 20 mL, such as between 200 pL and 10 mL, such as between 200 pL and 2 mL.

In step (c), an X-ray image is typically recorded of at least a part of the body ofthe mammal including the predetermined location. In particular embodiments, steps (c) and (d) may be performed simultaneously, so that image-recording and execution of radiotherapeutic treatment is integrated and performed sequentially or simultaneously.

W0 2014!187962 Use of the formulation as a tissue sealant The present invention also provides the formulation as defined herein above for use as a tissue sealant, e.g. for needle canals formed by biopsy in conjunction with an imaging ure according to the invention.

The tissue sealant may e an effective amount of a hemostatic agent, e.g. an agent selected from coagulation factors, coagulation tors, platelet activators, vasoconstrictors and fibrinolysis inhibitors, e.g. hrine, adrenochrome, collagens, thrombin, fibrin, fibrinogen, oxidized cellulose and chitosan.

Specific embodiments of the invention As said above, the present invention is in one embodiment an X—ray contrast composition for local administration, wherein the X-ray contrast composition ts contrast properties and wherein at least 60% of an administrated amount of said X-ray contrast composition remains more than 24 hours within 10 cm from an injection point when the X-ray contrast ition is administrated to a human or animal body. There are various forms of injection forms and routes possible, such as, but not limited to, utane injection, using a scope (bronchoscope, gastroscope, or any other flexible wired systems used to navigate inside a body), spraying orjust adding on a open wound, attached to another such system, intracranial injection, inside air and fluent filled organs or cavities (e.g. bladder, stomach), or inside non lly or medically created cavities.

Furthermore, there are various forms of dosing such as, but not limited to, fast injections ('bolus’), pulling back to needle while injecting, slowly injection on the site (e.g. less than 5 seconds, 60 seconds, 120 seconds, 5 minutes, 10 minutes or less than 20 minutes), pulsating the injection, pushing the needle forward, and pump giving a constant pressure for a defined period. Furthermore, there are various devices that may be used such as, but not limited to, needle with 1 or more holes on the side ofthe needle forming multiple smaller s, flexible, multiple chamber s. In one embodiment, the present invention has gelating ties and is a liquid before stration and has the ability to transform into a gel after administration. In one specific ment, the present invention has gelating W0 2014!187962 properties and is a homogeneous liquid before administration and has the ability to transform into a gel after administration. Furthermore, in one ment the present invention is a non-colloidal x-ray contrast agent as part of a homogeneous liquid x-ray contrast composition that gels upon injection into a human or animal subject. In yet another specific embodiment the X-ray contrast composition is a liquid before administration into a human or animal body that increases in viscosity by more than 100 centipoise (cP), such as e.g. more than 1,000, more than 2,000 or more than 5,000 centipoise (cP), after administration into a human or animal body.

According to another specific embodiment of the present invention the X—ray contrast composition is a liquid before administration into a human or animal body that increases in viscosity by more than 10,000 centipoise (cP) after administration into a human or animal body. In another ic embodiment the present invention has a viscosity of less than 10,000 centipoise (cP) at 20°C.

Furthermore, from one perspective of the present invention, the X—ray contrast composition comprises an X-ray contrast agent that is part of the X-ray contrast ition and said X—ray contrast agent is an organic substance.

According to one specific embodiment, the organic substance is the contrast ”agent” and the X-ray contrast composition ses alginate and chitosan. In another specific embodiment the X—ray contrast agent comprises one or more natural polymers, synthetic polymers, oligomers, lipids, saccharides, disaccharides, polysaccharides, peptides or any combination thereof and as mentioned before these may be the contrast ”agent”. In yet another specific ment of the present invention the X-ray contrast agent comprises one or more iodinated polymers, ers, lipids, saccharides, disaccharides, polysaccharides, peptides, or a derivative or a combination f. Further, in one embodiment the X-ray contrast agent is an inorganic acid or salt, such as auric acid.

The present ion may in one ment comprise particles for various purposes. One purpose may be an additive contrast effect; another purpose may be to potentiating the effect and a third e may be as a carrier of e.g. medication or other substances. ing to one specific embodiment of the present ion, the X-ray contrast composition comprises nanoparticles comprising gold (Au). In yet W0 2014!187962 another embodiment the X-ray st composition also ses particles in the size range from 1 — 1000 nm, such as nanoparticles in the size range from 2 to 500 nm and in one specific ment the nanoparticles comprises gold (Au) as the prefered X—ray attenuating element. In yet another embodiment, the X—ray contrast composition comprising nanoparticle that may be an MRI, PET, ultrasound, fluorescence, radiofrequency, visible light contrast agent. Furthermore, in one ic embodiment the nanoparticle is an MRI or PET contrast agent or a ation of the above mentioned imaging modalities.

The present invention may in one embodiment comprise solid particles coated with PAM (MW 3500). By choosing PNIPAM as the coating material various interesting properties can be introduced to the particles. PNIPAM is more hydrophobic compared to e.g. PEG but still water soluble, which enables efficient and straightforward le coating in aqueous solution without prior tion to organic solvents. Additionally, by having PNIPAM as the coating material results in a nano ite which can be lyophilized into a powder without inducing particle aggregation etc. which is not possible with other polymers e.g. PEG. Having the solid particles in a powder form is advantageous from multiply perspectives in terms of increased stability, easy storage and straight forward formulation procedures.

Furthermore, by having PNIPAM as the only polymer on the solid particles enables the particles to be suspended in c solvents such as e.g. EtOH for a prolonged period of time without aggregation due to the increased hydrophobicity of the particle introduced by the PNIPAM polymer. By having PNIPAM attached to the solid particles, as the only r in the formulation, the hydrophobic interactions with the gel forming solution in terms of e.g. sucrose acetate isobutyrate (SAIB) is sed resulting in a able system with very high particle retention. Choosing a more hydrophilic coating material for the particles would induce the release of the solid particles from the gel matrix which can be an advantage or a disadvantage depending on the desired properties of the formulation.

As mentioned previously the present invention may have gelating properties and the gelling may be initiated by various factors such as, but not limited to, temperature, hydration, enzymatic activation, ion concentration and/or pH. In one W0 2014!187962 embodiment the X—ray contrast ition ts gel—formation in se to a temperature in the range of 35 to 40°C. In another embodiment the X-ray contrast composition exhibits gel-formation in response to hydration. In yet another ment the X—ray contrast composition exhibits gel—formation in response to an ion-concentration in the range of 1 uM to 500 mM, such as in the range of 1 mM to 200 mM. In one embodiment the ions are divalent ions, such as calcium ions. In one embodiment the X—ray contrast composition exhibits gel—formation in response to a pH in the range of 6 to 8. In yet another embodiment, the X-ray contrast composition exhibits gel—formation in response to contacting with an initiator and here an initiator can be many different things such as, but not limited to, ions, or a chemical reactive compound that cross link other molecules.

In one embodiment, the X—ray st composition ing to the present invention may comprise radioactive compounds, paramagnetic compounds, fluorescent compounds or ferromagnetic compounds, or any mixture thereof.

As mentioned previously, the X-ray contrast composition may also act as a carrier of substances such as, but not limited to, pharmaceutical nces. The nce may be in the composition or in or coated/linked to the nanoparticles.

The substance may also be other types of additives. Examples of substance could be, but is not limited to, substances suitable for chemotherapy, gemcitabine, cisplatin, doxorubicin, doranidazole, hormones or odies. In one embodiment the X—ray composition comprise at least one pharmaceutical substance. In one specific ment the X—ray contrast composition comprises particles in the size range from 1 — 1000 nm, such as nanoparticles in the size range from 2 to 500 nm and wherein the particle contains at least one pharmaceutical substance.

In one embodiment a polymer may be used to work as a stabilizer between gel and biological surrounding and ore, the X-ray contrast composition may also comprises a molecule that increase gel stability in the human or animal body, such as an interfacially active molecule, such as an amphiphilic molecule, such as an emulsifier. Therefore in one embodiment the X—ray contrast ition comprises thylene glycol-b-caprolactone) (PEG-PCL), sucrose acetate isobutyrate (SAIB), poly(D,L-lactic acid) (PLA), or actic—co-glycolic acid) (PGLA), or a combination W0 2014!187962 2014/060673 f. In one embodiment of the present invention poly(D,L-lactic acid) (PLA) is added to sucrose acetate isobutyrate (SAIB) gel causing a reduction of burst release of said encapsulated contents e.g. particles drugs etc. Further, in one embodiment, the X—ray contrast ition comprises sucrose e isobutyrate (SAIB) or a derivative thereof and in one specific embodiment of the present invention, the X- ray contrast composition comprises an iodinated derivate of sucrose e isobutyrate (SAIB). Furthermore in another specific embodiment of the t invention the X-ray contrast ition comprises an iodinated derivate of sucrose acetate isobutyrate (SAIB) doped into sucrose acetate yrate . This has been evaluated for stability and the amount of this iodo—SAIB/SAIB that can be doped into SAIB, is at least 50 w/w%.

The iodo-SAIB provides high X-ray contrast. The iodo-SAIB compound is poorly soluble in ethanol and is a white solid whereas SAIB is highly soluble in ethanol and is a thick oil. However, a mixture of ethanol and SAIB can solubilize the iodo-SAIB very nicely. This means that the SAIB helps solubility of iodo-SAIB, which is an interesting e and which provides an injectable solution which forms a biodegradable, amorphous carbohydrate glass matrix after administration (through a thin needle, thinner than 20 gauge) that can function as a high st X-ray marker. When injected into mice, the iodo—SAIB/SAIB provides high contrast and has the desirable stability properties. rmore, the gel is homogeneous. In one embodiment of the present invention the X-ray contrast composition comprises an iodinated derivate of sucrose acetate isobutyrate (SAIB) solubilized in a mixture of ethanol and sucrose acetate isobutyrate (SAIB).

One way of ning and also storing the composition may be, held in the interior ofa syringe. This indicates a possible life of at least 6 months. One embodiment of the present invention is a kit comprising a syringe, a needle used for injection into a body or surgical related procedures such as but not limited to biopsy adapted to the open end of said syringe, and a composition ing to the present invention.

In one embodiment of the present invention, the X-ray contrast composition comprises an iodinated derivate of sucrose acetate isobutyrate (SAIB) and contains a W0 2014!187962 pharmaceutical substance. In another embodiment the X—ray contrast composition ses an iodinated derivate of sucrose acetate isobutyrate (SAIB) and ns particle that ns a pharmaceutical substance. In yet another embodiment, the X—ray contrast composition ses an ted derivate of sucrose acetate isobutyrate (SAIB) solubilised in a mixture of ethanol and sucrose acetate isobutyrate (SAIB) and contains a pharmaceutical substance. Furthermore, in one specific embodiment of the present invention, the X-ray contrast composition comprises an iodinated derivate of sucrose acetate isobutyrate (SAIB) solubilised in a mixture of ethanol and sucrose acetate isobutyrate (SAIB) and ns a particle that contains a pharmaceutical substance.

The ed use of the present invention is for radio therapy or image— guided radiation therapy, but not exclusively, other uses are thinkable such as, but not limited to, 2D X-ray scans, for use in g, diagnostics, treatment and/or quality rating of radiation therapy. The present invention may be used as a tissue marker and/or for use as a controlled drug release ition.

In one embodiment the X—ray contrast composition according to the present invention is for use in administration of an amount of 0.01 — 5.0 mL and in one specific embodiment the X-ray contrast composition is for use in administration wherein the amount is 0.1 — 1.0 mL. In one embodiment the present invention may be used as a tissue sealant.

In one embodiment the X—ray contrast composition according to the present invention, the X-ray st composition is erally administered to a predetermined location of the body of a mammal, and wherein an X-ray image of at least a part of the body of the mammal ing the predetermined location is recorded. Further, an embodiment ofthe invention may comprise a method of recording an X-ray image of the body of a mammal, comprising the steps of a. providing an X-ray contrast composition comprising an organic X—ray agent in a gel-forming system; b. administering the X—ray contrast composition to a predetermined location ofthe mammal, and W0 2014l187962 C. recording X—ray-based images of at least a part of the body which comprises the predetermined location.

In another ment, the invention comprise a method of joint herapy and X—ray imaging of a target tissue in a mammal, comprising the steps of a. providing an X-ray contrast ition comprising an organic X—ray agent in a gel-forming system; administering the X—ray contrast composition to a predetermined target tissue of the mammal, recording X—ray-based images, of at least a part of the body which comprises the target tissue, thereby providing a definition of the ta rget , a nd d. using the definition of the target tissue obtained in c) to direct external beam radiotherapy to the target tissue.

Steps (c) and (d) may potentially be performed simultaneously.

In another embodiment, the ion comprise a method for directing local administration ofa pharmaceutical agent to a target tissue in a mammal, comprising the steps of a. providing an X-ray contrast composition comprising an organic X-ray agent in a gel—forming system; administering the X-ray contrast composition to a predetermined target tissue of the mammal, recording X—ray-based images, of at least a part of the body which comprises the target , thereby ing a definition of the ta rget tissue, a nd d. using the X—ray contrast composition in b) to further comprise an pharmaceutical agent for delivery of a pharmaceutical agent to a predetermined target tissue of the mammal.

Steps (c) and (d) may potentially be med simultaneously.

In one specific embodiment of the present invention the target tissue comprises undesirably growing cells and in another specific ment the target tissue comprises tumor cells.

W0 2014!187962 Detailed description ofthe drawings Figure 1. Illustrates various mechanisms of gel-formation including thermo-, ion-, pH-, enzymatically-, tor— and hydration responsive gel-forming systems.

Figure 2. Illustrates various thermo responsive gel—forming systems which can exhibit an inverse l transition.

Figure 3. Illustrates various ion ive gel-forming systems which form gels in high salt concentration.

Figure 4. Illustrates various pH sensitive gel-forming systems which form hydrogels at specific pH intervals.

Figure 5. Illustrates s enzymatically sensitive rming systems which form hydrogels in presence of specific enzymes.

Figure 6. Illustrates the use of e acetate isobutyrate (SAIB) as a hydration sensitive gel-forming system. SAIB dissolved in organic solvent such as ethanol have a low viscosity suitable for injection trough thin needles. Upon hydration the ethanol diffuses out of the matrix resulting in a highly viscous hydrophobic gel le for ulation of contrast agents.

Figure 7. Illustrates various iodo-SAIB derivates which may be used for x—ray attenuation.

Figure 8. rates a tic scheme for the synthesis of 2-(2,4,6- triiodophenoxy)acetic acid (3) Figure 9. Illustrates a synthetic scheme for the synthesis of 6,6'—(2,4,6- triiodophenoxy)acetoxy-isobutyric—Sucrose (8) Figure 10. Illustrates CT-contrast of iodinated gels with 10-, 25-, or 50w% (8) ((w% is the weight of the atom/molecule (in this case iodine) divided by the total weight of the material times 100)) and a negative control containing MQ-HZO were ized in a clinical CT—scanner at different energies; 80-, 100—, 120- and 140kV, all ZOOmAs, 2 mm (col 40 x 0.6mm).

Figure 11. Illustrates AuNP synthesis and characterization. A) Synthetic scheme for the synthesis of PNIPAM-coated AuNPs using a seeding approach,- B) AuNP characterization by UV-Vis; C) AuNP terization by DLS; D) AuNP characterization by Z—potential.

W0 2014!187962 Figure 12. Illustrates the enhanced stability of PNIPAM coated AuNPs. A) UV- Vis of PNIPAM coated AuNPs before(stock)/after lization and re-suspension in ous EtOH (concentration of AuNP in the range of 1.0-5.0 mg Au/mL); B) DLS of PNIPAM coated AuNPs before(stock)/after lyophilization and re—suspension in anhydrous EtOH (concentration of AuNP in the range of 1.0-5.0 mg .

Figure 13. rates the accumulative release of PNIPAM3500- and PE65000 coated AuNPs from gels composed of SAIB/EtOH/PLA (75:20:5) + 3.0w% PNIPAM3500 or PE65000 coated AuNPs.

Figure 14. Illustrates a ultrasonography imagee of Formulation B (SAIB/8/EtOH (55:25:20)) (250pL) in vitro. Gel present at the bottom ofa glass beaker under water.

Figure 15. Illustrates MicroCT images of Formulation B (SAIB/8/EtOH (55:25:20)) (200pL) administered by aneous injection to healthy NMRI mice.

A) CT—image recorded 24h p.i.; B) CT—image recorded 48 p.i.

Figure 16. A) MicroCT image of SAIB/8/EtOH (65:15:20) injected sq. in immunocompetent mice; B) MicroCT image of SAIB/8/EtOH (50:30:20) injected sq. in immunocompetent mice; C) Ex vivo visualization of SAIB/8/EtOH (50:30:20) present in the s.q. compartment 14w p.i. and D) Gel implants composed of SAIB/8/EtOH (50:30:20) removed after 14w implantation in immunocompetent mice.

Figure 17. A) Series of MicroCT images of SAIB/8/EtOH (50:30:20) injected sq. in mice. MicroCT scans recorded with short time intervals to monitor the gelation kinetics of the iododinated gel; B) Gelation kinetics of SAIB/8/EtOH (50:30:20) (50uL) implanted sq. in immunocompetent mice and C) 14w ation profiles of iododinated gels composed of SAIB/8/EtOH (65:15:20) or SAIB/8/EtOH (50:30:20) after s.q. tation (50uL).

Figure 18. Illustrates a CT—image of Formulation B (SAIB/8/EtOH (55:25:20)) administrated intratumoral to a companion dog can Staffordshire terrier, 9 years, 34kg) with a mast cell tumor t between the front legs.

W0 87962 Examples Example 1 — lodo—SAIB gel formation and CT-contrast in vitro Materials Chemicals were purchased from Sigma—Aldrich Inc. (Br¢ndby, k) unless otherwise stated. 2-(2,4,6—triiodophenoxy)acetic acid (3) and 6,6'-(2,4,6— triiodophenoxy)acetoxy-isobutyric-Sucrose (8) was synthesized in two and four steps, respectively, as outlined in Figure 7 and Figure 8.

Synthesis ,6—triiodophenoxy)acetic acid (3). 2,4,6—triiodophenol (1) (10.00g, 21.2mmol) was dissolved in dry DMF (75mL) under Nz-atmosphere. To this solution, tert—butyl bromoacetate (4.20mL, 28.46mmol) and K2C03 (8.79g, 63.6mmol) were added and the stirred ght at rt. The solvent was removed in vacou and the remaining yellow oil re-dissolved in EtOAc (150mL) and washed with MQ-HZO (3x150mL). The organic phase was dried with MgSO4, filtrated and concentrated in vacou to give tert—butyl 2—(2,4,6-triiodophenoxy) acetate (2) as a light yellow oil which was used in the next step without further purification. 2 was dissolved in CHzClz (60mL) and trifluoroacetic acid (30mL) was added. The mixture stirred for 1h at rt after which the t was removed in vacou to give a white solid. The crude product was re-crystallized from EtOH to give 2—(2,4,6-triiodophenoxy)acetic acid (3) as fine white needles (9.58g, 85% (2 ). 1H-NMR (300MHz, MeOD): 5 6.58 (s, 2H), 2.95 (s, 2H). TOF MS (DHB+Na): Chemical Formula: C8H5|3Na03, calculated mass; 552.83; found: 553.08 (M+Na+). 6,6'-TBDP$-Sucrose (5). Sucrose (4) (3.00g, 8.76mmol) was dissolved in dry pyridine (54.0mL) under Nz—atmosphere. To this solution tert-butyldiphenylchloro— silane (TBDPS-Cl) (2.51mL, ol) and a catalytic amount of DMAP (107.5mg, 0.88mmol) were added and the mixture heated at 70°C for 3h. After cooling to rt, TBDPS-Cl (2.51mL, ol) was added and the mixture stirred overnight at rt. The solvent was removed in vacou and the crude product purified by flash chromato- graphy using a stepwise gradient starting from,- i) EtOAc, ii) EtOAc/Acetone/HZO (100:100:1) and iii) EtOAc/Acetone/HZO (10:10:1) as eluent to give BDPS- Sucrose (5) as a white solid (4.66g, 65%). Rf = 0.40 (EtOAc/Acetone/HZO (100:100:1)).

W0 2014!187962 MALDI-TOF MS a): Chemical Formula: Na011Si2, calculated mass; 841.08; found: 841.81 (M+Na+). 6,6'-TBDPS-isobutyric-Sucrose (6). 6,6'-TBDPS-Sucrose (5) (3.00g, 3.66mmol) was dissolved in dry pyridine (45.0mL) under Nz—atmosphere. To this solution isobutyric anhydride (15.00mL, 90.4mmol) was added and the e stirred at rt overnight. Additional isobutyric anhydride (5.0mL, 15.06mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP) (50mg, 0.41mmol) were added and the mixture heated to 70°C for 6h. The solvent was removed in vacou and the crude product purified by flash chromatography using hexanezEtOAc (5:1) as eluent to give 6,6'-TBDPS-isobutyric-Sucrose (6) as clear viscous oil (4.54g, quantitative). Rf = 0.48 (hexane:EtOAc (5:1). MALDI-TOF MS (DHB+Na): Chemical Formula: C68H94Na017Si2, calculated mass; 1262.62; found: 2 ). 6,6'-OH-isobutyric-Sucrose (7) 6,6'-TBDPS-isobutyric-Sucrose (6) (217.2g, 0.175mmol) was dissolved in THF (940mL) and stirred at RT. Glacial acetic acid (42.1g, 0.701mol) was added to the flask followed by addition of tetrabutyl- ammonium fluoride trihydrate 3H20) g, 0.701mol) in THF (692mL). The solution was d at RT for 15h after which heptanes (2085mL) and phosphate buffer (0.5M, 2111mL) (HZKPO4 (177.2g) and HK2P04 (343.3g) in MQ-HZO L)), pH 7.0) was added. The organic phase was collected and washed with additionally two portions of phosphate buffer (0.5M, 2111mL). The crude product purified by flash chromatography using a gradient starting from hexanes:EtOAc (7:3) then hexanes:EtOAc (6:4) as eluent to give 6,6'—OH-isobutyric—Sucrose (7) as clear viscous oil (106.1g, 79%). Rf = 0.21 (hexane:EtOAc (3:1). 1H-NMR (300MHz, DMSO-de): 6 5.75 (d,J = 6.1 Hz, 1H), 5.50 (d,J = 3.6 Hz, 1H), 5.40 (d, J = 7.7 Hz, 1H), 5.31 (t,J = 7.4 Hz, 1H), 5.18 (t, J = 9.8 Hz, 1H), 4.87 (t, J = 5.5 Hz, 1H), 4.70 (dd, J = 10.4, 3.7 Hz, 1H), 4.29 (d,J = 11.9 Hz, 1H), 4.11 (dd, J = 12.0, 5.5 Hz, 1H), 3.69—3.44 (m, 4H), 2.64—2.49 (m, 6H), 1.13—0.96 (m, 36H). MALDI-TOF MS (DHB+Na): Chemical Formula: C36H53Na017, calculated mass; 785.83; found: 785.82 (M+Na+). 6,6’-(2,4,6—triiodophenoxy)acetoxy—isobutyric-Sucrose (8) H—isobutyric- Sucrose (7) (800mg, 1.05mmol) was dissolved in dry DMF L) under N2- atmosphere. To this solution a pre-mixed mixture of 2-(2,4,6-triiodophenoxy)acetic W0 2014!187962 acid (3) (1.67g, 3.15mmol), EDC-HCI (622mg, 3.15mmol) and DMAP (769mg, 6.29mmol) in dry DMF (10.0mL) were added and the reaction stirred at rt overnight.

The solvent was removed in vacou and the remaining yellow oil re-dissolved in CH2C|2 (40mL) and washed with MQ—HZO (3x40mL). Organic phase was dried with MgSO4, filtrated and reduced in vacou to give light yellow oil. Final purification was achieved by flash tography using hexanezEtOAc (5:1) as eluent to give 6,6'- (2,4,6-triiodophenoxy)acetoxy-isobutyric—Sucrose (8) as white foamy solid (1.56g, 83%). Rf = 0.31 (hexane:EtOAc (5:1). 1H-NMR (300MHz, MeOD): 6 8.05 (s, 2H), 8.04 (s, 2H), 5.68 (d,J= 3.7 Hz, 1H), 5.56 (d, J = 7.3 Hz, 1H), 5.54— 5.48 (m, 1H), 5.43 (t,J = 7.2 Hz, 1H), 5.37 (t,J = 9.8 Hz, 1H), 5.03 (dd,J = 10.2, 3.7 Hz, 1H), 4.70—4.06 (m, 12H), 2.73—2.45 (m, 6H), 1.36—1.04 (m, 36H). MALDI-TOF MS (DHB+Na): Chemical Formula: C52H64|6Na021, calculated mass; 1809.47; found: 1809.59 (M+Na+).

Gel preparation Three sucrose e isobutyrate (SAIB)—based formulations (600mg each) with increasing amounts of 6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-sucrose were prepared as ed in the table below. 6,6'—(2,4,6-triiodophenoxy)— ation SAIB EtOH acetoxy-isobutyric-sucrose (8) SAIB/8/EtOH 420mg 60mg 120mg (70:10:20) SAIB/8/EtOH 330mg 150mg 120mg :20) SAIB/8/EtOH 180mg 300mg 120mg (30:50:20) W0 2014!187962 SAlB-solution (90w/w in EtOH) was weighted off and mixed with 8 and anhydrous EtOH (see table above). The mixtures were nized on a ball-mill homogenizer for 60min (30s'1) and centrifuged for 20s at 5000RPM to remove air bubbles from the formulations. All formulations were homogenous clear solutions with sing viscosity as a on of the concentration of 8 — all injectable trough 256 hypodermic s.

Iodinated gels (500pL) from formulation A—C were prepared by injection into MQ-HZO (5.0mL) containing plastic vials at 37°C. The aqueous solutions were replaced three times and the gels stored at 37°C for 12 days prior to ualization and HU-contrast measurements in a clinical CT-scanner.

CT—contrast of iodinated gels in vitro The three formed iodinated gels with 10—, 25-, or 50w% 8 and a negative control containing MQ-HZO were visualized in a clinical CT-scanner at different energies; 80—, 100—, 120— and 140kV, all 200mAs, 2 mm (col 40 x 0.6mm). The obtained contrast in Hounsfield unit (HU) plotted as a function of energy is illustrated in Figure 10 and listed in the table below. Excellent contrast g from 1.300-10.500HU was observed dependent on the w% of8 and the applied energy. w% iodine Formulation (before 80kV 100kV 120kV l40kV injection) A 4.26w% 2500HU 1800HU 1500HU 1300HU B 10.65w% 5000HU 4500HU 3500HU 3000HU C 21.30w% 10500HU 8800HU 6200HU 5900HU As may be understood from above, according to one specific embodiment of the present invention, the X-ray st composition is a liquid before administra- tion into a human or animal body and having an iodine concentration of more than 1.5 w% before injection, such as 2-30 w%, such as 3-25 w%, such as 4-25 w%.

W0 2014!187962 Example 2 — Synthesis and improved properties of PNIPAM-coated AuNP Materials Chemicals were sed from Sigma-Aldrich Inc. (Br¢ndby, Denmark) unless otherwise stated. x3H20 was purchased from Wako Chemicals GmbH (Neuss, Germany) and SH—PNIPAM (MW 3500, PDI = 1.24) was purchased from Polymer Source (Dorval, Canada).

AuNP synthesis, PNIPAM coating and particle characterization All glassware was cleaned with aqua regia prior to use. Trisodium citrate (10mL, 38.8mM) was rapidly injected into a refluxing solution of HAuCl4*3H20 (100mL, 1.0mM) under vigorous stirring. An immediately color change from light yellow to wine red was observed and the reflux was continued for 15min after which the solution was cooled to rt. The ed AuNP—seeds (20mL) were added to a boiling on of *3HZO (2500mL, 0.296mM) under vigorous stirring.

Subsequently, trisodium citrate L, 38.8mM) was added and the mixture refluxed for 30min resulting in a clear color change from wine red to purple.

Additional trisodium citrate (100mL, 38.8mM) was added as stabilizer and the mixture heated for additional 1h. The AuNP solution was cooled to rt and SH- PNIPAM3500 (40mg, 11.4pmol) (6 molecules pr/nm2 AuNP e area) dissolved in EtOH (5.0mL) was added. The reaction mixtures d overnight at rt (Figure 11a).

The PNIPAM-coated AuNPs was extensively washed with MQ-HZO and up- concentrated to approx. 2.3mL etically 65mg AuNP/mL) by centrifugation (4.500RPM, 45min/cycle). The AuNP-seeds, the citrate stabilized AuNPs and the purified up-concentrated PNIPAM-coated AuNP were all terized by UV-Vis (Figure 11b), DLS (Figure 11c) and the ntial was measured (Figure 11d). The [Au]-concentration of the up-concentration PNIPAM-coated AuNPs were ined by ICP—MS using a Au3+'standard (1000mg/mL) in 5% HCl spiked with 0.5ppt Ir as internal standard. Up—concentrated PNIPAM—coated AuNPs were dissolved in aqua regia and diluted with 5% HCl to theoretically 666ppt Au3+. The concentration of the PNIPAM-coated AuNPs was determined to 64mg Au/mL. The PNIPAM coated AuNPs were stored at 5°C until further use.

W0 87962 Lyophilization of PNIPAM coated AuNP and stability in organic solvent PNIPAM coated AuNPs (see synthesis above) were d to 1.0—, 2.5- or 5.0 mg Au/mL (500uL each) with MQ—HZO and snap-frozen in liquid nitrogen for 2 minutes. The samples were lyophilized overnight (p < 6.0x10'2 mbar) to form dark colored shiny s. The lyophilized PNIPAM coated AuNPs were re—dissolved in EtOH (0.50 mL) and ed for a few seconds. The particles tely re- dispersed within s to give dark colored solutions. The particle morphology was evaluated by UV-Vis (figure 12a) and DLS (figure 12b). No sign of aggregation or instability was observed for the PNIPAM—coated AuNPs neither during lyophilization or EtOH solubilization. The lyophilized powder could easily be stored and weighted off at a later time—point.

Example 3 — Controlling particle retention in SAIB gels based on particle hydrophobicity Chemicals were purchased from Sigma—Aldrich Inc. (Brondby, Denmark) unless otherwise stated. HAuCl4x3HZO was purchased from Wako Chemicals GmbH (Neuss, Germany), SH-PNIPAM (MW 3500, PDI = 1.24) was purchased from Polymer Source (Dorval, Canada) and MeO—PEGgooo—SH was purchased from Rapp Polymere GmbH (Tuebingen, Germany).

AuNP synthesisI PEGM g and particle characterization PEGylated AuNPs (PEG5000) were prepared as outlined for the PNIPAM coated AuNP in Example 2 using SH—PEGSOOO as particle coating r. PEGylated particles were characterized by UV—Vis (A = 539nm) and DLS (59.7i0.9nm) and the tration determined by ICP—MS (82.6mg Au/mL).

In vitro release of AuNP from SAIB EtOH PLA els ations (1000mg each) consisting of SAIB/EtOH/PLA (7522025) + 3.0w% PNIPAM3500 or PE65000 coated AuNP was prepared as outlined in the table below.

PNIPAM3500' PEGSOOO' Formulation SAIB EtOH PLA AuNP AuNP D 750mg 200mg 50mg 30mg - E 750mg 200mg 50mg — 30mg W0 2014!187962 The gel components were mixed and nized by a ball homogenizer (45min, 30s'1) to give a clear homogenous solution. AuNPs (PNIPAM3500 or PEGsooo) were erred into ous EtOH, mixed with the gel solution and vortexed.

In vitro release study was carried out by injection of the formulations (3x200uL each) into MQ-HZO (10.0mL for PNIPAM—AuNP) or PBS-containing (for PEG-AuNP) glass vial at 37°C. Small aliquots ) were removed as a function of time and replaced with fresh aqueous solutions. The amount of released AuNPs was measu- red by correlating the UV-Vis absorbance with a standard curve based on the corre- sponding particles (figure 13). A burst release (20%) of the encapsulated hydrophilic PEGylated particles was observed within the first few hours whereas the more hydrophobic PNIPAM coated AuNP remained encapsulated in the SAIB— amorphous glass matrix due to the enhanced hydrophobic interactions with the gel matrix.

Example 4 — AIB gel formation with PNIPAM-coated AuNP in vitro Materials Chemicals were purchased from Sigma-Aldrich Inc. (Brondby, k) unless otherwise stated. HAuCl4x3HZO was purchased from Wako Chemicals GmbH (Neuss, Germany) and PAM (MW 3500, PDI = 1.24) was purchased from Polymer Source l, Canada). 6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric- sucrose (8) was synthesized as described in e 1.

AuNP synthesisI PNIPAM coating and particle characterization PNIPAM coated AuNPs were prepared as described in Example 2.

Gel preparation A formulation consisting of SAIB/8/EtOH (55:25:20) + 3.0w% PNIPAM-AuNP was prepared as outlined in the table below. 6,6'-(2,4,6-triiodophenoxy)- PNIPAM- Formulation SAIB EtOH acetoxy—isobutyric-sucrose (8) AuNPs /EtOH 165 mg 75mg 60mg 9mg (55:25:20) + 3.0w% PNIPAM-AuNP W0 2014!187962 olution (90w/w% in EtOH) was weighted off and mixed with 8 (see table above). The e were homogenized on a ball-mill homogenizer for 60min (30s‘1) and centrifuged for 205 at 5000RPM to remove air bubbles from the formulations. PNIPAM coated AuNPs (141uL, 64mg L) was diluted with MQ- H20 (1659uL) and lyophilized to give a shinny powder. The lyophilized PNIPAM— coated AuNPs was re-dispersed anhydrous EtOH (52.8uL) and mixed with the other gel components.

In vitro release of AuNP in MQ-HZQ An iodinated gel (200uL) with 3.0w% PNIPAM-coated AuNPs (Formulation F) were prepared by injection into a MQ-HZO (10.0mL) containing glass vial at 37°C.

Small aliquots (1.0mL) were removed as a function of time and replaced with fresh MQ-HZO. The amount of released AuNPs was measured by correlating the UV-Vis absorbance with a standard curve based on the PNIPAM—coated AuNPs. No release of PNIPAM-coated AuNPs was observed throughout the experiment. Formulation F was a homogenous dark colored solution injectable trough 256 hypodermic needles.

Example 5 — Visualization of iodo—SAIB gels using onography in vitro Materials Chemicals were purchased from Sigma—Aldrich Inc. by, Denmark) unless otherwise stated. 6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric—Sucrose (8) was synthesized as described in Example 1.

Gel preparation A formulation consisting of /EtOH (55:25:20) (350mg) was prepared as bed in Example 1 (Formulation B). The iodo—SAIB gel (250uL) was injected into MQ—HZO (500mL) in a glass beaker and the gel was allowed to set for 5 days prior to visualization by ultrasonography. ound imaging ofthe iodo-SAIB gel was ted by an Ultrasound Scanner (BK Medical, Herlev, k) with the following settings: Res/Hz 2/21Hz, B Gain 83%, Dynamic range 80dB, Noise reject 10, Noise cutoff 32. The iodo—SAIB gel was clearly visible using ultrasonography as illustrated in Figure 14.

W0 2014!187962 Example 6 — Iodo—SAIB gels as iniectable CT-contrast agent in vivo — Visibility study in immunocompetent mice Materials als were sed from Sigma—Aldrich Inc. (Br¢ndby, Denmark) unless otherwise . 6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-sucrose (8) was synthesized as described in Example 1. Healthy female NMRI (Naval Medical Research Institute) mice were purchased from Taconic (Borup, Denmark).

Gel preparation A formulation consisting of SAIB/8/EtOH (55:25:20) (900mg) was ed as described in e 1 (Formulation B).

Animal setup Formulation B 8/EtOH (55:25:20)) was strated to healthy female NMRI mice (n = 3) by subcutaneous injection (200uL each) under anaesthesia.

MicroCT imaging of iniectable iodo—SAIB gels The iodinated gels were visualized over time by computed tomography (CT).

Images were acquired with a MicroCAT® II system (Siemens Medical solutions, Malvern, USA). Excellent CT-contrast was achieved using Formulation B (SAIB/8/EtOH :20)) as illustrated in Figure 15A-B (CT-images recorded 24h pi and 48 pi.) Example 7 — Iodo—SAIB gels as able CT-contrast agent in vivo — long term ity and visibility study in immunocompetent mice Materials Chemicals were purchased from Sigma-Aldrich Inc. (Br¢ndby, Denmark) unless otherwise stated. 6,6'-(2,4,6—triiodophenoxy)acetoxy-isobutyric—Sucrose (8) was synthesized as described in Example 1. Healthy female NMRI (Naval Medical Research Institute) mice were purchased from Taconic (Borup, Denmark).

Gel preparation Formulation consisting of a) SAIB/8/EtOH (65:15:20) (750mg) and b) SAIB/8/EtOH (50:30:20) (750mg) were prepared as described in Example 1.

W0 2014!187962 Animal setup Both formulations; a) SAIB/8/EtOH (65:15:20) and b) SAIB/8/EtOH (50:30:20) were administrated to healthy female NMRI mice (n = 2><8mice) by subcutaneous injection (50uL each) under anesthesia.

MicroCT imaging of iniectable iodo-SAIB gels and mplantation visualization The iodinated gels were visualized over time by computed tomography (CT).

Images were acquired with a MicroCAT® II system (Siemens Medical ons, Malvern, USA). Excellent CT-contrast was achieved using both formulations: a) SAIB/8/EtOH (65:15:20) and b) SAIB/8/EtOH (50:30:20) as rated in Figure 16A—B.

The obtained CT- contrast was found the scale with the formulated amount of iodo- SAIB (8) in the formulation. After 14w of implantation the animals were sacrificed and the gels removed from the s.q. compartment (Figure 16C-D). The iodinated gels were well-defined gels that could easily be removed and transferred without disruption of the gels. They were furthermore soft enough to be deformed using a scalpel.

Gelation kinetics of iniectable iodo-SAIB gels The gelation kinetics of the ted gels composed of SAIB/8/EtOH (50:30:20) was monitored by running multiply micro-CT scans within the first few hours of ion (Figure 17A). Based on these images the total volume ofthe iodinated gel as a function of time was calculated as illustrated in Figure 178.

Gelation of the iodinated gel is caused by efflux of EtOH from the gel matrix which takes place within the first two hours p.i. causing a rapid increase in hte viscosity of the iodinated gel and an increase of CT-contrast by approximately 35% due to contraction of the gel.

Degradation profile of iniectable iodo-SAIB gels over 14w The degradation profile of iodinated gels composed a) /EtOH (65:15:20) and b) SAIB/8/EtOH (50:30:20) were monitored by microCT scanning over a period of 14w. Based on these images the total volume of the iodinated gels as a on of time were calculated as illustrated in Figure 17C. No difference in degradation e between the two ations was ed and a steady—state degradation e was observed for both formulations. A volume loss, with a 95% W0 2014!187962 confidence interval, of -0.09176uL/day was observed for both formulations after the initial EtOH efflux phase.

Example 8 — |odo-SA|B gels as iniectable CT-contrast agent in vivo — visibility study in canine with spontaneous tumor Materials Chemicals were purchased from Sigma-Aldrich Inc. (Br¢ndby, k) unless otherwise stated. 6,6'-(2,4,6-triiodophenoxy)acetoxy-isobutyric-Sucrose (8) was synthesized as described in e 1.

Gel preparation A formulation consisting of SAIB/8/EtOH :20) (350mg) was prepared as described in Example 1 (Formulation B).

Animal setup Formulation B (SAIB/8/EtOH (55:25:20)) was administrated to a ion dog (American Staffordshire r, 9 years, 34kg) with a mast cell tumor present between the front legs. The iodo—SAIB gel was administrated by intratumoral injection (SOOuL) using a 256 needle.

CT imaging of iniectable iodo-SAIB gels in canine The iodo-SAIB gel was visualized computed tomography (CT). Images were ed with a Single slice Siemens CT—scanner (Siemens Medical solutions, Malvern, USA). Excellent CT-contrast was achieved using Formulation B (SAIB/8/EtOH (55:25:20)) as illustrated in Figure 18 (CT—image recorded 24h p.i.).

Claims (18)

Claims 1.

1. An X-ray contrast composition for local administration, n the X-ray contrast composition exhibits st properties and wherein at least 60% of an 5 administrated amount of said imaging contrast composition remains more than 24 hours within 10 cm from an injection point when the X-ray contrast composition is strated to a human or animal body, wherein the X-ray st composition is a liquid before administration and has the ability to transform into a gel after administration, and wherein the X-ray st composition comprises an iodinated 10 derivate of sucrose acetate isobutyrate (SAIB) or an ted derivate of sucrose acetate isobutyrate (SAIB) doped into sucrose acetate isobutyrate (SAIB), and wherein the X-ray contrast composition also comprises a rming component comprising poly(ethylene -b-caprolactone) (PEG-PCl), e acetate isobutyrate (SAIB), poly(D,L-lactic acid), poly(lactic-co-glycolic acid) (PLGA), chitosan, 15 PEG-PPG-PEG, PLGA-g-PEG, PEG-PLGA-PEG, PNIPAM, PEG/PLLA mulitiblock copolymer, PLGA-PEG-PLGA, multi-arm PLGA-PEG, poly(1,2-propylene phosphate, P(NIPAM-co-AA), poly[(Val-Pro-Gly-Val-Gly)-co-(Pro-Hyp-Gly)10], cyclotriphosphazenes, cellulose (MC), hydroxyl propyl methylcellulose (HPMC), PCL-PEG, PAA-g-pluronic, PCL-PEG-PCL, PEG-PCL-PEG, te, FEK16, PVA, 20 gelrite, OSM-PCLA-PEG-PCLA-OSM, PAA-PEG-PAA, or PAE-PCL-PEG-PCL-PAE, or a combination thereof.

2. The X-ray contrast composition according to claim 1, wherein the X-ray contrast composition increases in viscosity by more than 1,000 centipoise (cP) after 25 administration into a human or animal body.

3. The X-ray contrast composition according to claim 1, wherein the X-ray contrast composition has a viscosity of less than 10,000 centipoise (cP) at 20°C.

4. The X-ray st composition according to claim 1, wherein the X-ray contrast composition ses an X-ray contrast agent that is part of the X-ray contrast composition and said X-ray contrast agent is an organic substance.

5 5. The X-ray contrast agent according to claim 4, wherein the X-ray contrast agent comprises one or more natural polymers, synthetic polymers, oligomers, lipids, saccharides, disaccharides, polysaccharides, peptides or any combination thereof, or wherein the X-ray contrast agent comprises one or more iodinated polymers, oligomers, , saccharides, disaccharides, polysaccharides, peptides, or 10 a tive or a combination thereof.

6. The X-ray contrast composition according to claim 1, wherein the X-ray st composition exhibits gel-formation in response to a temperature in the range of 35 to 40°C, in response to hydration, in response to an ncentration in 15 the range of 1 uM to 500 mM, in response to a pH in the range of 6 to 8 and/or in response to contacting with an initiator.

7. The X-ray contrast composition according to claim 1, wherein the X-ray st composition also comprises, radioactive compounds, paramagnetic 20 compounds, fluorescent compounds or ferromagnetic compounds, or any mixture thereof, and/or wherein the X-ray contrast composition also comprises at least one pharmaceutical nce.

8. The X-ray contrast composition according to claim 1, wherein the gel-forming 25 component ses poly(ethylene glycol-b-caprolactone) (PEG-PCl), sucrose acetate isobutyrate (SAIB), ,L-lactic acid), or actic-co-glycolic acid) (PGLA), or a combination thereof.

9. The X-ray contrast composition according to claim 1, wherein the X-ray 30 contrast composition comprises an iodinated derivate of sucrose acetate isobutyrate (SAIB) solubilized in a mixture of ethanol and sucrose acetate isobutyrate (SAIB).

10. The X-ray contrast composition according to claim 1, wherein the X-ray contrast composition comprises an ted derivate of sucrose acetate isobutyrate (SAIB) and contains a pharmaceutical nce or particle that contains a 5 pharmaceutical substance.

11. The X-ray contrast composition ing to claim 1, wherein the X-ray contrast composition comprises an iodinated derivate of e acetate isobutyrate (SAIB) solubilised in a mixture of ethanol and sucrose acetate isobutyrate (SAIB) and 10 contains a pharmaceutical substance or a particle that ns a pharmaceutical substance.

12. The X-ray contrast composition according to claim 1, for use in radio therapy, for use in imaging, diagnostics, treatment and/or quality rating of radio therapy, for 15 use as a tissue marker and/or for use as a controlled drug e composition.

13. The X-ray contrast ition according to claim 1, wherein the composition is formulated for parenteral stration to a human or animal body. 20

14. A kit comprising a syringe, a needle used for injection into a body or surgical related procedures, such as but not limited to biopsy adapted to the open end of said syringe, and an X-ray contrast composition ing to any one of the previous claims. 25

15. A method of recording an X-ray image of the body of a non-human mammal, comprising the steps of a. providing an X-ray contrast composition as claimed in any one of claims 1 – 13, b. administering the X-ray contrast composition to a predetermined 30 location of the mammal, and c. recording X-ray-based images of at least a part of the body which comprises the predetermined location.

16. A method of joint radiotherapy and X-ray imaging of a target tissue in a non- 5 human , comprising the steps of a. providing an X-ray contrast composition as claimed in any one of claims 1 – 13, b. administering the X-ray st composition to a predetermined target tissue of the mammal, 10 c. recording X-ray-based images, of at least a part of the body which ses the target tissue, thereby providing a definition of the target tissue, and d. using the definition of the target tissue obtained in c) to direct external beam radiotherapy to the target .

17. A method for directing local administration of a pharmaceutically active agent to a target tissue in a non-human mammal, comprising the steps of a. providing an X-ray contrast composition as claimed in any one of claims 1 – 13, 20 b. administering the X-ray contrast ition to a predetermined target tissue of the , c. recording X-ray-based images, of at least a part of the body which comprises the target tissue, thereby providing a definition of the target tissue, and 25 d. using the X-ray contrast composition in b) to further comprise an active pharmaceutical agent for delivery of an active pharmaceutical agent to a predetermined target tissue of the mammal.

18. The method according to any one of claims 16 or 17, wherein the target 30 tissue comprises undesirably growing cells.