A RADIOPAQUE POLYMERIC LIQUID EMBOLIC SYSTEM

Abstract

The invention relates to radiopaque liquid embolic composition comprising tetra iodo compound, 4,4-bis (4-hydroxy-3,5 diiodo phenyl) valeric acid (IBHV) of Formula I, covalently linked to ethylene vinyl alcohol copolymer (EVOH) and dissolved in a water miscible organic liquid.

Claims

1. A radiopaque liquid embolic composition comprising tetra iodo compound, 4,4-bis (4-hydroxy-3,5 diiodo phenyl) valeric acid (IBHV) of Formula I, covalently linked to ethylene vinyl alcohol copolymer (EVOH) and dissolved in a water miscible organic liquid.

2. The radiopaque liquid embolic composition as claimed in claim 1, wherein said organic liquid is selected from Dimethyl sulfoxide (DMSO) or N-methyl pyrrolidone (NMP).

3. The radiopaque liquid embolic composition as claimed in claim 1, wherein the polymer content is in the range of 10-45% (w/v) which helps to set different viscosity ranges for embolizing composition.

4. The radiopaque liquid embolic composition as claimed in claim 1, wherein the iodine content in the polymer is in the range of 30-70% (w/w) which helps to attain different opacity ranges for the embolizing composition.

5. The radiopaque liquid embolic composition as claimed in claim 1, wherein the embolic compositions can be modified to get viscosities in the range 5 centistokes to 500 centistokes.

6. The radiopaque liquid embolic composition as claimed in claim 1, wherein the embolic compositions can be used for embolizing blood vessels, tumor, aneurism and arteriovenous malformations.

7. A process for the preparation of the liquid embolic composition as claimed in claim 1 comprising the steps of iodinating 4,4-bis (4-hydroxyphenyl) valeric acid (BHV) with sodium iodide followed by treatment with Sodium hypochlorite and an acid to obtain the tetraiodocompound, 4,4-bis(4-hydroxy-3,5 diiodophenyl) valeric acid (IBHV), dissolving ethylene vinyl alcohol copolymer (EVOH) and IBHV in an organic solvent to form a mixture, adding 1,3-Dicyclohexylcarbodiimide and 4-Dimethylaminopyridine to this mixture and stirring followed by pouring the reaction mixture into water to precipitate the polymer and dissolving the polymer in an organic solvent to obtain the liquid embolic composition

8. The process as claimed in claim 7, wherein said organic solvent is selected from Dimethyl sulfoxide (DMSO) and N-methyl pyrrolidone (NMP).

9. The process as claimed in claim 7, wherein the ethylene vinyl alcohol copolymer has ethylene content in the range 20 mol % to 50 mol % which is utilized for increasing the grafting efficiency of 4,4-bis (4-hydroxy-3,5 diiodo phenyl) valeric acid onto EVOH copolymer.

Description

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0020] FIG. 1: Scheme of the grafting reaction

[0021] FIG. 2: .sup.1H NMR of the grafted copolymer.

[0022] FIG. 3: Thermogravimetric analysis of the parent polymer, i.e., ethylene vinyl alcohol (EVOH) copolymer (FIG. 3 (a)) and grafted copolymer (FIG. 3(b)).

[0023] FIG. 4: Differential scanning calorimetry of the grafted copolymer.

[0024] FIG. 5: Differential thermal analysis of the grafted copolymer.

[0025] FIG. 6: Solution of Iodo compound grafted EVOH being injected into saline.

[0026] FIG. 7: Computerized X-ray images of IBHV-g-EVOH.

[0027] FIG. 8: Representative images showing the survival of cells after contacting with the 50% extract of IBHV-g-EVOH.

[0028] FIG. 9: MTT assay of L929 cells after contact with 50%, 25%, 12.5% and 6.25% extracts of test material.

BRIEF DESCRIPTION OF THE INVENTION

[0029] Thus according to this invention is provided a radiopaque liquid embolic composition and a process for the preparation thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0030] According to this invention is provided a radiopaque liquid embolic composition and a process for the preparation thereof.

[0031] In accordance with this invention is provided a radiopaque, homogenous, non-adhesive, non-toxic polymeric material composition suitable for the embolization of blood vessels. As the first step an aromatic acid is iodinated to make it radiopaque and this iodo compound is reacted with ethylene vinyl alcohol copolymer (EVOH) to make the polymer radiopaque. The radiopaque polymer is dissolved in a water miscible organic liquid to convert it into a liquid embolic composition and this composition precipitates into a solid mass when it comes in contact with physiological fluids. The precipitated mass is non-cytotoxic, non-hemolytic and biologically stable. The embolic composition is suitable for blocking the blood vessels, aneurisms, supply vessels to tumors and arteriovenous malformations.

[0032] The injectable embolizing composition has a polymer content in the range of 10-45% (w/v) which helps to set different viscosity ranges for embolizing composition. The iodine content in the polymer is in the range of 30-70% (w/w) which helps to attain different opacity ranges for the embolizing composition. The embolic compositions can be modified to get viscosities in the range 5 centistokes to 500 centistokes.

[0033] The injectable embolizing composition can be used for embolizing blood vessels, tumor, aneurism and arteriovenous malformations.

[0034] Several variables control the efficacy of the solution as a liquid embolic agent

[0035] Iodine content in the liquid embolic composition

[0036] Vinyl alcohol content in the copolymer

[0037] The ratio of polymer to iodocompound taken for synthesis

[0038] Concentration of grafted polymer in the water miscible organic solvent

[0039] Viscosity of the embolic system

[0040] Sterilization technique used

[0041] The grade of EVOH is defined by the mole % of ethylene content present in the copolymer. Several grades of EVOH are available (for example, 27 mol %, 32 mol & 38 mol %). The viscosity, solubility in the organic solvents, grafting reaction with the iodo compound, etc. depend on the ethylene content. Suitable solvents for the EVOH and the iodinated polymer are Dimethyl sulfoxide (DMSO) or N-methyl pyrrolidone (NMP).

[0042] First object of the present invention is the synthesis of tetra iodo compound, namely, 4,4-bis (4-hydroxy-3,5 diiodo phenyl)valeric acid of the chemical formula 1

##STR00001##

Synthesis of 4,4-bis (4-hydroxy-3,5 diiodophenyl) valeric acid (IBHV)

[0043] Iodination of 4,4-bis (4-hydroxyphenyl) valeric acid results the formation of 4,4-bis (4-hydroxy-3,5 diiodophenyl) valeric acid. The tetraiodocompound, 4,4-bis(4-hydroxy-3,5 diiodophenyl) valeric acid (IBHV) is synthesized by iodinating 4,4-bis (4-hydroxyphenyl) valeric acid (BHV). For this, BHV, an alkali and sodium iodide are dissolved in an alcohol. Sodium hypochlorite is added dropwise and stirred for 1 hr. An acid is added to precipitate the IBHV formed. This is washed and dried. The yield of the product obtained was 80%.

[0044] The reaction temperature has a strong influence on the iodination. The purity of the compound can be assessed by High Performance Liquid Chromatography. The compound can be characterized by FTIR spectroscopy, .sup.1HNMR spectroscopy, Mass spectroscopy, Thermogravimetric analysis. Analysis: Molecular weight 789.69 g. In mass spectrum it is obtained as 812.69 (M+Na.sup.+ ion) & 834.67 (M+K.sup.+ ion). Thin layer chromatography (TLC) ethyl acetate/water (AcOEt/H.sub.2O: 3/8) R.sub.f=0.46. For BHV, R.sub.f=0.22

[0045] .sup.1H NMR (DMSO) 9.48 (s, —COOH) 7.45 (s, —C.sub.6H.sub.5) 3.38 (s, —OH) 2.5 (triplet, CH—COOH) 1.4, 1.9, 2.1 ppm (multiplet, —CH.sub.3).

[0046] The absence of aromatic proton peaks at 6.65 & 6.9 ppm in the .sup.1HNMR spectrum indicates the successful iodination of 4,4-bis (4-hydroxyphenyl) valeric acid.

Grafting of 4,4-bis (4-hydroxy-3,5 diiodophenyl) Valeric Acid onto EVOH

[0047] The grafting reaction can be carried out in the solution state. This is achieved by dissolving the EVOH and 4,4-bis (4-hydroxy-3,5 diiodophenyl) valeric acid in DMSO or NMP as illustrated in the scheme (FIG. 1). A suitable catalyst such as dimethyl amino pyridine is suitable for controlling the reaction. When the reaction conditions are favorable the iodo compound reacts with the EVOH polymer resulting the formation radiopaque polymer. When the reaction is complete the product may be purified by thorough washing with distilled water. The product may be obtained in the powder form by drying in an air oven. The grafted polymer can be characterized by FTIR spectroscopy, .sup.1HNMR spectroscopy, UV-Visible spectroscopy, Thermogravimetric analysis, Differential Scanning calorimetry & Differential thermal analysis. FTIR spectrum gives the ester C═O & C—O stretch at 1767 cm.sup.−1 and 1116 cm.sup.−1 respectively. Intensity of the —OH stretching vibration was found to decrease as the grafting progresses. In vitro cell culture cytotoxicity test using in L929 cells indicated that the compound was non-cytotoxic.

[0048] Radiopaque Liquid Embolic Composition

[0049] Radiopaque liquid embolic composition can be formulated by dissolving suitable concentrations of iodocompound grafted EVOH in DMSO or NMP. This composition is suitable for the occlusion of blood vessels, aneurysm and arteriovenous malformation by precipitation in the presence of blood or other physiological fluids. It is achieved by injecting the embolic composition at the required site with the aid of a catheter. The radiopacity of the system is measured by Computed Tomography (CT) scan. In a typical example, a solution of about 35% radiopaque polymer exhibits 2965 Hounsfield Units radiopacity which is adequate for imaging. Higher or lower Radiopacity can be achieved by manipulating the solution concentrations and/or iodine content in the composition. The viscosity of the composition can be adjusted by varying the concentration of the polymer in the solution or by changing the ethylene content of EVOH. The concentration of the polymer and the grade of EVOH affect the precipitation behavior of the system.

[0050] The tetraiodocompound, 4,4-bis(4-hydroxy-3,5 diiodophenyl) valeric acid (IBHV) was synthesized by iodinating 4,4-bis (4-hydroxyphenyl) valeric acid (BHV). For this, BHV, an alkali and sodium iodide were dissolved in an alcohol. Sodium hypochlorite was added dropwise and stirred for 1 hr. An acid is added to precipitate the IBHV formed. This is washed and dried. The yield of the product obtained was 80%.

[0051] Analysis: Molecular weight 789.69 g. In mass spectrum it is obtained as 812.69 (M+Na.sup.+ ion) & 834.67 (M+K.sup.+ ion). Thin layer chromatography (TLC) ethyl acetate/water (AcOEt/H.sub.2O: 3/8) R.sub.f=0.46. For BHV, R.sub.f=0.22

[0052] .sup.1H NMR (DMSO) 9.48 (s, —COOH) 7.45 (s, —C.sub.6H.sub.5) 3.38 (s, —OH) 2.5 (triplet, CH—COOH) 1.4, 1.9, 2.1 ppm (multiplet, —CH.sub.3).

[0053] EVOH and IBHV are dissolved in dimethyl sulfoxide and 1,3-Dicyclohexylcarbodiimide and 4-Dimethylaminopyridine is added to this mixture and stirred for about 72 hrs. The reaction mixture is poured into water to precipitate the polymer. The precipitate is washed initially with water and later with methanol and finally dried in an air oven.

[0054] Analysis: .sup.1H NMR (DMSO) (FIG. 2) 2.5 ppm (proton adjacent to —COO group) 7.45 ppm (s, —C.sub.6H.sub.5) 3.35 ppm (phenolic proton) 1.4 ppm, 1.9 ppm, 2.1 ppm (methyl protons).

[0055] Thermal stability is analyzed by thermogravimetric analysis. The parent polymer, i.e., ethylene vinyl alcohol (EVOH) copolymer, was thermally stable upto 297° C. (FIG. 3 (a)) but grafting decreased the thermal stability to 169° C. (FIG. 3(b)).

[0056] The glass transition temperature of the grafted polymer is analyzed by differential scanning calorimetry. Its glass transition temperature (Tg) was found to be 51.1° C. (FIG. 4). EVOH had a Tg of 62° C.

[0057] From the differential thermal analysis traces showed that the melting point of IBHV-g-EVOH is 162.79° C. (FIG. 5). EVOH has a melting point of 172.23° C. Grafting decreases the melting point.

[0058] The iodo compound grafted EVOH is subjected to various tests for its physical and biological properties.

[0059] A solution of iodo compound grafted EVOH is dissolved in dimethyl sulfoxide. This solution is taken in a syringe and injected into saline. As the solution comes in contact with saline it forms into a solid mass.

[0060] The invention will now be explained in greater details with the help of the following non limiting examples. The examples are indicative of the invention but do not limit the scope of the claimed invention.

EXAMPLE

Example 1

[0061] The tetraiodocompound, 4,4-bis(4-hydroxy-3,5 diiodophenyl) valeric acid (IBHV) was synthesized by iodinating 4,4-bis (4-hydroxyphenyl) valeric acid (BHV). For this, 2 g BHV, 0.13 g sodium hydroxide and 6.3 g sodium iodide were dissolved in 50 mL methanol. Sodium hypochlorite (70 mL) was added dropwise and stirred for 1 hr. HCl (10% solution) was added to precipitate the IBHV formed. This was washed with distilled water and dried at 65° C. The yield of the product obtained was 80%.

[0062] Analysis: Molecular weight 789.69 g. In mass spectrum it was obtained as 812.69 (M+Na.sup.+ ion) & 834.67 (M+K.sup.+ ion). Thin layer chromatography (TLC) ethyl acetate/water (AcOEt/H.sub.2O: 3/8) R.sub.f=0.46. For BHV, R.sub.f=0.22

[0063] .sup.1H NMR (DMSO) 9.48 (s, —COOH) 7.45 (s, —C.sub.6H.sub.5) 3.38 (s, —OH) 2.5 (triplet, CH—COOH) 1.4, 1.9, 2.1 ppm (multiplet, —CH.sub.3).

Example 2

[0064] EVOH (1 g) and IBHV (5 g) were dissolved in dimethyl sulfoxide (120 ml). Added 1,3-Dicyclohexylcarbodiimide (1.43 g) and 4-Dimethylaminopyridine (0.847 g) to this mixture and stirred for about 72 hrs. The reaction mixture was poured into water to precipitate the polymer. The precipitate was washed initially with water and later with methanol and finally dried in an air oven.

[0065] Analysis: .sup.1H NMR (DMSO) (FIG. 2) 2.5 ppm (proton adjacent to —COO group) 7.45 ppm (s, —C.sub.6H.sub.5) 3.35 ppm (phenolic proton) 1.4 ppm, 1.9 ppm, 2.1 ppm (methyl protons).

[0066] Thermal stability was analyzed by thermogravimetric analysis. The parent polymer, i.e., ethylene vinyl alcohol (EVOH) copolymer, was thermally stable upto 297° C. (FIG. 3 (a)) but grafting decreased the thermal stability to 169° C. (FIG. 3(b)).

[0067] The glass transition temperature of the grafted polymer was analyzed by differential scanning calorimetry. Its glass transition temperature (Tg) was found to be 51.1° C. (FIG. 4). EVOH had a Tg of 62° C.

[0068] From the differential thermal analysis traces showed that the melting point of IBHV-g-EVOH was 162.79° C. (FIG. 5). EVOH has a melting point of 172.23° C. Grafting decreases the melting point.

Example 3

[0069] A solution (35%) of iodocompound grafted EVOH was dissolved in dimethyl sulfoxide. This was taken in a syringe and injected into saline. As the solution comes in contact with saline it formed into a solid mass (FIG. 6).

Example 4

[0070] Different solutions of grafted polymer (about 5 mL each) in DMSO were prepared and subjected to computerized X-ray imaging procedure in a computed tomography machine. In one composition, a 10% solution of IBHV-g-EVOH showed radiopacity of 1331 Hounsfield Units (HU). In another composition a 30% solution of IBHV-g-EVOH showed radiopacity of 2976 HU. In a third composition, a 35% solution of IBHV-g-EVOH showed 2976HU radiopacity. In yet another composition, a 15% solution of IBHV-g-EVOH showed radiopacity of 1922 HU (FIG. 7).

Example 5

[0071] Biocompatibility evaluation of the grafted copolymers were carried out by performing in vitro cell culture cytotoxicity tests using L929 cell lines in accordance with ISO 10993-5 standard guidelines. For this purpose at first the grafted copolymer was sterilized by ethylene oxide. Extracts of the grafted copolymer was prepared by incubating 0.2 g of IBHV-g-EVOH in 1 mL physiological saline at 37±1° C. for 24±2 h at an extraction ratio of 1.25 cm.sup.2/ml. Ultra high molecular weight polyethylene and dilute phenol were used as negative and positive controls, respectively. Results showed that extract of IBHV-g-EVOH did not cause any cytotoxic response. Representative images showing the survival of cells after contacting with the 50% extract of IBHV-g-EVOH is shown in the FIG. 8. The MTT assay of L929 cells after contact with 50%, 25%, 12.5% and 6.25% extracts of test material showed 99.66%, 101.34%, 101.51%, 99.20% metabolic activity, respectively (FIG. 9). It also revealed the non cytotoxic nature of the grafted copolymer. So from test on extract method and MU assay evaluation it is clear that the iodocompound grafted copolymer is non-cytotoxic.

Example 6

[0072] The sterile samples as described in example 5 were subjected to hemolysis test according to the procedure described in ISO 10993-4:2017 (Selection of tests for interaction of materials with blood). The percentage hemolysis in plasma samples after exposure to IBHV-g-EVOH was 0.02%. The result is the average of the experiment repeated at 3 times. According to ISO 10993-4: 2017, hemolysis rate less than 0.1% is considered non hemolytic.

Example 7

[0073] The solid mass obtained in example 3 was washed well with deionised water and lyophilized until it is free of water. The dried samples were weighed and placed in phosphate buffered saline in a shaking incubator at 37° C. and with an rpm of 80 for 3 months. The samples were washed with distilled water, lyophilized again to dry and weighed. None of the samples show any weight loss indicating that the samples are stable at these conditions.