POLYMERS AND MICROSPHERES

Abstract

New cationic polymers are provided that are suitable for the preparation of microspheres. The microspheres are capable of loading and eluting anionic species such as drugs and find use in i.a. embolotherapy

Claims

1. A polymer comprising a macromer, which macromer comprises 1,2 or 1,3 diol groups and pendent, cross linkable groups, the pendant cross linkable groups being cross linked by a cationically charged vinylic co-monomer of the formula I ##STR00009## wherein X is a linear or branched C.sub.1-6 alkylene, C.sub.2-6 alkenylene or C.sub.2-6 alkynylene group; R.sup.1, R.sup.2 and R.sup.3 are the same or different and selected from C.sub.1-4 alkyl groups; R.sup.4 is H or C.sub.1-4 alkyl.

2. A polymer according to claim 1 wherein the macromer comprises 1,3 diol groups.

3. A polymer according to claim 1 wherein the macromer comprises polyvinyl alcohol.

4. A polymer according to claim 1 wherein the macromer comprises pendant cross linkable groups of the formula: ##STR00010## wherein Q is a linear or branched C.sub.1-C.sub.8 alkylene group; R.sup.5 is H, a C.sub.1-6 alkyl, or a C.sub.3-6 cycloalkyl; R.sup.6 is an olefinically unsaturated electron attracting copolymerizable radical having up to 25 carbon atoms; and R.sup.7 is H or a C.sub.1-6 alkyl.

5. A polymer according to claim 4 wherein R.sup.6 is a group of the formula III ##STR00011## wherein p is 0 or 1; and R.sup.9 is H or C.sub.1-4 alkyl; and wherein, when p is 0, then R.sup.8 is ##STR00012## and when p is 1, R.sup.8 is a C.sub.1-4 alkylene group.

6. A polymer according to claim 1, further comprising one or more iodines covalently bound to the polymer.

7. A polymer according to claim 6 comprising groups of the formula IV ##STR00013## wherein Z is a group comprising one or more covalently bound iodines

8. A polymer according to claim 1, which is in the form of a hydrogel.

9. A polymer according to claim 1 comprising one or more pharmaceutical actives.

10. A polymer according to claim 9 wherein the pharmaceutical active is bound within the polymer by ionic interactions.

11. A microsphere comprising a polymer according to claim 1.

12. A microsphere according to claim 11 wherein the polymer comprises between 5 and 75 weight % cationic co-monomer

13. A microsphere according to claim 11 further comprising a pharmaceutical active and/or an imaging agent.

14. A microsphere according to claim 13 wherein the pharmaceutical active and/or imaging agent is reversibly bound within the polymer by ionic interactions.

15. A composition comprising one or more microspheres according to claim 11.

16. A composition according to claim 15 comprising no ruptured microspheres.

17. A composition according to claim 15 comprising a pharmaceutically acceptable diluent.

18. A method of treatment comprising providing a microsphere according to claim 11 and delivering said microsphere to a blood vessel of a patient in need thereof, thereby embolising the blood vessel.

19. A microsphere according to claim 11 for use in a method of embolotherapy.

20. A microsphere according to claim 11 for use in a method comprising the direct injection of the microsphere to a site within the body of a patient.

21. A sealed vessel comprising one or more sterile microspheres according to claim 11 lyophilised and under a pressure of less than 0.9 bar.

Description

FIGURES

[0068] FIG. 1 shows a microsphere prepared according to example 1 and having a disrupted outer layer.

[0069] FIG. 2 shows optical photomicrographs of a) APTA.sub.16, b) APTA.sub.27, c) APTA.sub.43 and d) APTA.sub.60.

[0070] FIG. 3 shows Confocal Laser Scanning Microscopy images of microspheres prepared according to Example 2 after exposure to FITC-Dextrans of a variety of molecular weights.

[0071] FIG. 4 gives the structures of 4 sulphonic acid dyes used as model compounds in loading and elution studies (a). 1-pyrenesulfonic acid sodium salt (P1): (b) 6,8-dihydroxypyrene-1,3-disulfonic acid disodium salt (P2): (c) 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (P3) and (d) 1,3,6,8-pyrenetetrasulfonic acid hydrate tetrasodium salt (P4).

[0072] FIG. 5 illustrates the fraction of P1, P2, P3 and P4 dyes eluted from APTA.sub.43 microspheres in 200 mL of PBS (meanrange, n=3). Initial loading was 8.6 umols per mL of microspheres.

[0073] FIG. 6 illustrates the fraction of P1 eluted from APTA.sub.16, APTA.sub.43 and APTA.sub.60 microspheres in 200 mL of PBS (averagerange, n=3). Initial loading was 8.6 umols per mL of microspheres.

EXAMPLES

Example 1

[0074] (i) Synthesis of PVA Macromer

[0075] Macromer may be prepared essentially according to Example 1 of WO04071495. Mowiol 8-88 PVA powder (88% hydrolised, 12% acetate content, average molecular weight about 67,000D) (150 g) (Clamant, Charlotte, N.C. USA) is added to a 2 litre glass reaction vessel. With gentle stirring, 1000 ml water is added and the stirring increased to 400 rpm. To ensure complete dissolution of the PVA, the temperature is raised to 999 C. for 2-3 hours. On cooling to room temperature N-acryloylaminoacetaldehyde (NAAADA) (Ciba Vision, 10 Germany) (2.49 g or 0.104 mmol/g of PVA) is mixed in to the PVA solution followed by the addition of concentrated hydrochloric acid (100 ml). The reaction proceeds at room temperature for 6-7 hours and is then stopped by neutralisation to pH 7.4 using 2.5M NaOH.

[0076] Diafiltration is performed using a stainless steel Pellicon 2 Mini holder stacked with 0.1 m.sup.2 cellulose membranes having a molecular weight cut off of 3000 (Millipore Corporation, Bedford, Mass. USA). The macromer solution is circulated over the membranes at approximately 50 psi. When the solution has been concentrated to about 1000 ml the volume is kept constant by the addition of water at the same rate that the filtrate is being collected to waste until 6000 ml extra has been added. Once achieved, the solution is concentrated to 20-23% solids with a viscosity of 1700-3400 cP at 25 C.

[0077] (ii) Preparation of Polymer Microspheres

[0078] Microspheres were synthesised in a redox catalysed reaction in a water in oil type system.

[0079] Organic Phase:

[0080] 600 g n-butyl acetate and 11.5 g of a 10% (w/w) cellulose acetate butyrate (CAB) in ethyl acetate were added to a glass 1 L jacketed vessel connected to a heater-chiller unit and stirred at approximately 300 rpm at 25 C. and purged with N.sub.2.

[0081] Aqueous Phase:

[0082] A known amount of PVA macromer (21 g non-volatile weight), 1.3 g ammonium persulphate (APS), the appropriate amount of 3-acrylamidopropyl)trimethylammonium chloride (APTA) solution and an additional amount of purified water were mixed together and added to the reaction vessel. Water was added so that the total amount of water in the formulation was approximately 130 g.

[0083] Polymerisation was activated through the addition of 1.6 mL TMEDA. An excess amount of N,N,N,N-tetramethylethlenediamine (TMEDA) to APS was used to ensure complete reaction of APS. The reaction was allowed to continue for three hours at 55 C. under an inert N.sub.2 atmosphere. The microspheres were then purified by washing in ethyl acetate and acetone to remove residual CAB, before hydration and washing in water. The microspheres were heat extracted by boiling in an 80 mM disodium hydrogen phosphate in 0.29% (w/w) NaCl solution before rehydration in water, followed by equilibration in saline.

[0084] Microspheres were produced in a range of sizes, typically between 100 to 1200 m when hydrated in saline, and were separated into size ranges using sieves. In all formulations the total water content, weight of macromer and APS remained the same. Notation for the formulations represents the ratio of weight percentage (wt %) for APTA to macromer used in synthesis e.g. APTA.sub.45 denotes 45 wt % APTA to 55 wt % macromer. Table 1 gives the weight percentage (wt %) of APTA versus macromer in example microsphere formulations.

TABLE-US-00001 TABLE 1 Formulation APTA (wt %) Macromer (wt %) APTA.sub.0 0 100 APTA.sub.16 16 84 APTA.sub.27 27 73 APTA.sub.43 43 57 APTA.sub.60 60 40 APTA.sub.86 86 14 APTA.sub.100 100 0

[0085] Gravimetric analysis was used to determine the exact mass of polymer per volume of hydrated microspheres. A volume of microspheres fully hydrated in water, was measured out using a measuring cylinder, transferred to a vial and the water removed. The microspheres were dried under vacuum at 80 to 120 C. until a constant weight was reached. The weight of the remaining polymer was recorded and the mass per volume of microspheres determined.

[0086] Equilibrium water content measured for each of APTA.sub.16, APTA.sub.43 and APTA.sub.60 spheres was between 98 and 99% (n=7)

Example 2. Molecular Weight Cut-Off of Matrices

[0087] Molecular weight cut-off data was determined for each matrix formulation by exposure of microspheres, fully swollen in water, to FITC-Dextran conjugates (FITC-D) with molecular weights between 4 kDa and 250 kDa. The diffusion of FITC-Ds into the interior of the microspheres was monitored using Confocal Laser Scanning Microscopy (CLSM). Representative images of centralised regions of interest are shown in FIG. 3 for APTA.sub.16, APTA.sub.43 and APTA.sub.60. A summary of the maximum molecular weight cut-off range, above which FITC-Ds were not observed in the centre of microspheres, is presented in Table 2.

TABLE-US-00002 TABLE 2 Formulation Molecular Weight Cut-Off Range (kDa) APTA.sub.16 40-70 APTA.sub.43 70-250 APTA.sub.60 70-250

Example 3: Loading of Small Molecules into the Polymer Matrix

[0088] The loading and elution properties of the microspheres of the invention were characterised using a series of commercially available pyrene sulfonic acid sodium salts as model anionic drugs. The chemical structures of each dye; 1-pyrenesulfonic acid sodium salt (P1), 6,8-dihydroxypyrene-1,3-disulfonic acid disodium salt (P2), 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (P3) and 1,3,6,8-pyrenetetrasulfonic acid hydrate tetrasodium salt (P4) are shown in FIG. 4.

[0089] (i) Loading

[0090] A measuring cylinder was used to aliquot a volume of microspheres fully hydrated in saline (e.g. 1 mL). The microspheres were then transferred to a vial and the saline solution removed. A solution of the model compound was prepared by dissolving the compound in deionised water. The solution was then added to the vial containing the slurry of microspheres. The vial was then rolled to mix at room temperature, whilst loading was monitored by removing aliquots of the loading solution.

[0091] The maximum binding capacity of each formulation was determined by mixing the microsphere slurry for 72 hours with excess test compound. The remaining solution was removed from the slurry and the microspheres were washed with water to remove residual unbound compound. The binding capacity was determined by complete elution in 500 mL of a saturated KCl solution in water mixed in 50:50 ratio with ethanol.

[0092] Samples were analysed by UV/Vis. spectrophotometry against a standard curve prepared for each compound. Maximum absorbance at 375 nm for P1, 411 nm for P2, 404 nm for P3 and 376 nm for P4 were used. Table 3 gives the loading capacity of the 4 dyes in 3 different microsphere formulations.

TABLE-US-00003 TABLE 3 Measured bound loading capacity (mg .Math. mL.sup.1) of Formulation Dye microspheres APTA.sub.16 P1 9.1-9.6 P2 10.8-11.2 P3 6.3-7.3 P4 6.3-6.8 APTA.sub.43 P1 22.9-24.1 P2 22.8-24.5 P3 16.7-18.3 P4 17.1-18.4 APTA.sub.60 P1 31.2-32.2 P2 30.9-33.7 P3 21.2-21.5 P4 22.1-22.9

[0093] (ii) Elution

[0094] Microspheres of each polymer formulation were loaded with equal quantities of each dye. 1 ml samples of dye-loaded microspheres were added to 200 mL of PBS in an amber jar. The microsphere suspensions were rolled to provide continuous mixing. At each time point the eluent was sampled and assayed by UV/Vis spectrophotmetery as above. The volume of sampled eluent was replaced with fresh PBS to maintain the elution volume. FIG. 5 illustrates the elution profiles of each dye from APTA.sub.43 microspheres.

[0095] There is a difference in elution rate of the individual dyes. The monovalent dye P1 has the fastest rate of elution as 80% of the initial loaded amount was released within 60 minutes in comparison to 9% of the divalent dye P2 and approximately 3% of P3 and P4. As an illustration the elution profiles of dye P1 from APTA.sub.16, APTA.sub.43 and APTA.sub.60, are compared in FIG. 6.