Injectable calcium-phosphate cement releasing a bone resorption inhibitor

Abstract

The present invention relates to a macroporous, resorbable and injectable apatitic calcium-phosphate cement with a high compressive strength useful as bone cement and releasing a bone resorption inhibitor, preparation method and uses thereof.

Claims

1. A composition comprising a calcium-phosphate bone cement releasing a gem-bisphosphonic compound comprising a pulverulent solid phase, wherein the pulverulent solid phase comprises a gem-bisphosphonic compound chemically associated to calcium-deficient apatite (CDA), and wherein the pulverulent solid phase comprises up to 0.15% by weight of gem-phosphonic compound.

2. The composition according to claim 1, wherein the bisphosphonic compound thereof is selected in the group consisting of etidronate, clodronate, pamidronate, alendronate, risedronate, tiludronate, ibandronate, zoledronate, incadronate, olpadronate, and neridronate.

3. The composition according to claim 2, wherein the bisphosphonic compound is alendronate.

4. The composition according to claim 1, wherein the pulverulent solid phase further comprises one or more calcium and/or phosphate compounds selected from the group consisting of hydroxyapatite (HA), -tricalcium phosphate (-TCP), -tricalcium phosphate (-TCP), amorphous calcium phosphate (ACP), monocalcium phosphate monohydrate (MCPH), dicalcium phosphate anhydrous (DCPA), calcium deficient apatite (CDA), CaCO.sub.3 and mixtures thereof.

5. The composition according to claim 1, wherein the pulverulent solid phase comprises between 30 and 80 wt.-% of -TCP.

6. The composition according to claim 1, wherein the pulverulent solid phase further comprises at least one biopolymer.

7. The composition according to claim 1, wherein the pulverulent solid phase comprises -tricalcium phosphate (-TCP), dicalcium phosphate dehydrate (DCPD), calcium deficient apatite (CDA), monocalcium phosphate monohydrate (MCPH), and a biopolymer.

8. The composition according to claim 7, wherein the biopolymer is hydroxypropylmethylcellulose.

9. A composition comprising a pulverulent solid phase according to claim 1 and a liquid phase comprising a Na.sub.2HPO.sub.4 aqueous solution, a NaH.sub.2PO.sub.4 aqueous solution or a citric acid aqueous solution.

10. The composition according to claim 9, wherein the liquid phase consists in a Na.sub.2HPO.sub.4 aqueous solution.

11. The composition according to claim 9, wherein the pH of the liquid phase is between 5 to 10.

12. The composition according to claim 9, wherein the liquid phase/solid phase (L/S) ratio is between 0.2 to 0.9 ml/g.

13. The composition according to claim 9, which is injectable.

14. The composition according to claim 9, which has an initial setting time of less than one hour.

15. The composition according to claim 9, which has after setting a compressive strength of above about 10 MPa.

16. A kit for preparing a calcium-phosphate bone cement paste releasing a gem-bisphosphonic compound comprising a pulverulent solid phase and a liquid phase, wherein the pulverulent solid phase comprises a gem-bisphosphonic compound chemically associated to calcium-deficient apatite (CDA) and wherein the liquid phase comprises a Na.sub.2HPO.sub.4 aqueous solution, a NaH.sub.2PO.sub.4 aqueous solution or a citric acid aqueous solution, and wherein the pulverulent solid phase comprises up to 0.15% by weight of gem-phosphonic compound.

17. The kit according to claim 16, wherein the pulverulent solid phase comprises between 30 and 80 wt.-% of -TCP.

18. The kit according to claim 16, wherein the pulverulent solid phase comprises -tricalcium phosphate (-TCP), dicalcium phosphate dehydrate (DCPD), calcium deficient apatite (CDA), monocalcium phosphate monohydrate (MCPH), and a biopolymer.

Description

FIGURES

(1) FIG. 1: .sup.31P VACP MAS NMR spectrum of modified CDA [10.4 wt % alendronate], showing the alendronate component associated to CDA (see example 1). The spectra were recorded at a spinning frequency of 12 kHz and a magnetic field of 7.0 T.

(2) FIG. 2: .sup.31P MAS spectra of cements (see example 5) after a one week setting time. reference=no alendronate added, solid=alendronate powder mixed with the cement solid component, solution=alendronate dissolved in the cement liquid component, CDA=alendronate chemically associated to the CDA component

(3) FIG. 3: (upper view) .sup.31P single pulse MAS-NMR spectrum of modified -TCP [4.7 wt % alendronate], (bottom view) .sup.31P VACP MAS NMR spectrum of modified -TCP [4.7 wt % alendronate] (see example 2).

(4) FIG. 4: Scanning Electron Microscopy (observation in the backscattered electron mode) view of a ewe vertebral body implanted with a 3 g-dose of unloaded CPC, 12 weeks after implantation.

(5) FIG. 5: Scanning Electron Microscopy (observation in the backscattered electron mode) view of a ewe vertebral body implanted with a 3 g-dose of alendronate-loaded CPC (0.13 wt %), 12 weeks after implantation (see example 8).

EXAMPLES

Example 1: Preparation of CDA Modified with Alendronate

(6) A suspension of calcium phosphate was prepared by introducing 100 mg of CDA into 8.75 ml of ultrapure water mixed with 1.25 ml of a 0.02 mol.Math.l.sup.1 sodium alendronate aqueous solution. The suspension was placed in a tube maintained at room temperature, and was stirred with a rotary stirrer at 16 rpm for 5 days. The suspension was then centrifuged and the most part of the supernatant was removed. The solid residue was filtered off, washed several times with small portions of ultrapure water, and then dried at room temperature. The resulting solid contained 7.4 wt % alendronate.

Example 2: Preparation of -TCP Modified with Alendronate

(7) In addition to example 1, the bisphosphonate can also be chemically associated to one of the other components of the solid phase (CaCO.sub.3, DCPA, -TCP . . . ). For example, in the case of -TCP, a suspension of the calcium phosphate support was prepared by introducing 100 mg of -TCP into 8.75 ml of ultrapure water mixed with 1.25 ml of a 0.02 mol.Math.l.sup.1 sodium alendronate aqueous solution. The suspension was placed in a tube maintained at room temperature, and was stirred with a rotary stirrer at 16 rpm for 2 days. The suspension was then centrifuged and the most part of the supernatant was removed. The solid residue was filtered off, washed several times with small portions of ultrapure water, and then dried at room temperature. The resulting solid contained 4.7 wt % alendronate.

Example 3: Preparation of DCPD Modified with Alendronate

(8) In addition to example 1, the bisphosphonate can also be chemically associated to one of the other components of the solid phase. For example, in the case of DCPD, a suspension of the calcium phosphate support was prepared by introducing 100 mg of DCPD into 9 ml of ultrapure water mixed with 1 ml of a 0.02 mol.Math.l.sup.1 sodium alendronate aqueous solution. The suspension was placed in a tube maintained at room temperature, and was stirred with a rotary stirrer at 16 rpm for 2 days. The suspension was then centrifuged and the most part of the supernatant was removed. The solid residue was filtered off, washed several times with small portions of ultrapure water, and then dried at room temperature. The resulting solid contained 5.3 wt % alendronate.

Example 4: Preparation of CaCO3 Modified with Alendronate

(9) In addition to example 1, the bisphosphonate can also be chemically associated to one of the other components of the solid phase. For example, in the case of CaCO.sub.3, a suspension of the calcium phosphate support was prepared by introducing 100 mg of CaCO.sub.3 into 8.5 ml of ultrapure water mixed with 1.5 ml of a 0.02 mol.Math.l.sup.1 sodium alendronate aqueous solution. The suspension was placed in a tube maintained at room temperature, and was stirred with a rotary stirrer at 16 rpm for 2 days. The suspension was then centrifuged and the most part of the supernatant was removed. The solid residue was filtered off, washed several times with small portions of ultrapure water, and then dried at room temperature. The resulting solid contained 5.0 wt % alendronate.

Example 5: Preparation of an Injectable CPC Releasing Alendronate

(10) The solid phase of the cement consists of alpha-tertiary calcium phosphate -TCP, CaHPO.sub.4, CaCO.sub.3 and some precipitated hydroxyapatite CDA.

(11) The solid phase composition is the same for all samples: 62.4 wt % (249.6 mg) -TCP 26.8 wt % (107.2 mg) DCPA (CaHPO.sub.4) 8.8 wt % (35.2 mg) CaCO.sub.3 2 wt %(8 mg) CDA.

(12) -TCP was prepared by using an appropriate mixture of CaHPO.sub.4 and CaCO.sub.3, heating it at 1300 C. for at least 6 h and quenching it in air down to room temperature.

(13) Three ways are used to combine alendronate with the cement samples. alendronate is dissolved in the cement liquid phase (up to 1.2 mg in 120 L see Table IV); or alendronate is added to the solid phase (0.1-10 mg for 400 mg see Table IV); or alendronate is chemically associated to (i) CDA as prepared in Example 1 replacing partially the CDA of the solid phase (see Table II) (ii) -TCP as prepared in Example 2 replacing partially the -TCP of the solid phase (see Table III).

(14) Seven concentrations of alendronate have been used: 0,100 wt % (0.40 mg) 0.060 wt % (0.25 mg) 0.025 wt % (0.10 mg) 0.25 wt % (1 mg) 0.3 wt % (1.2 mg) 2.5 wt % (10 mg) 3.9 wt % (15.7 mg)

(15) Three liquid phases were chosen to prepare different cement formulations: 2.5 Na.sub.2HPO.sub.4 by weight in water, 2.5% NaH.sub.2PO.sub.4 by weight in water or 85 mM citric acid in water.

(16) The liquid/powder ratio L/P of cements was taken to be either 0.30 ml/g for samples prepared with Na.sub.2HPO.sub.4 and NaH.sub.2PO.sub.4 and 0.25 ml/g for samples prepared with citric acid.

(17) The powders are finely ground during 10 minutes.

(18) Then, the liquid phase is added dropwise and the two phases are mixed with a spatula or a pestle.

(19) The mixing sets in moulds.

Example 6: Setting Time Assays of the Samples of Example 5

(20) Setting times were determined with Gillmore needles following the standard ASTM C266-89.

(21) Tables II and III and IV summarize the results.

(22) TABLE-US-00002 TABLE II Initial and final setting times with alendronate chemically associated to CDA m(Alend) tf tf Liquid phase [mg] pH ti [min] [min] ti [min] [min] T [ C.] Na.sub.2HPO.sub.4 control 8.5 30 75 20 (2.5% by 0.1 8.5 30 80 30 70 20 weight) 0.25 8.5 40 90 30-35 80 20 L/S = 0.3 0.5 8.5 40 85 35 75 20 NaH.sub.2PO.sub.4 control 5 20 60 21 (2.5% by 0.1 4.5 20 60 20 60 21 weight) 0.25 4.5 20 60 20 60 21 L/S = 0.3 0.5 4.5 25 80 20 60 21

(23) TABLE-US-00003 TABLE III Initial and final setting times with alendronate chemically associated to -TCP m(Alend) ti tf T Liquid phase [mg] pH [min] [min] [ C.] NaH.sub.2PO.sub.4 control 5 20 60 21 (2.5% by 0.5 5 30 70 21 weight) obtained by mixing L/S = 0.3 4.24 wt % of modified -TCP [4.7 wt % alendronate] in pure -TCP.sup.a 0.5 5 35 70 21 obtained by using only modified -TCP [0.2 wt % alendronate].sup.b 1 5 35 70 21 obtained by mixing 8.48 wt % of modified -TCP [4.7 wt % alendronate] in pure -TCP.sup.c 10 5 35 85 21 obtained by using only modified -TCP [4 wt % alendronate].sup.d Note a: After one week incubation, a full transformation of -TCP was observed, and the resulting cement showed good mechanical properties. Note b: After one week incubation, the self-setting of the cement was very poor, while X-Ray diffraction gave evidence that the transformation of -TCP was very low. Note c: After one week incubation, a full transformation of -TCP was observed, and the resulting cement showed quite good mechanical properties, although the cement was a little more crumby than in the case of note a. Note d: After one week incubation, the self-setting of the cement was very poor, while X-Ray diffraction gave evidence that the transformation of -TCP was very low. From notes b and d, it can be deduced that if the entire -TCP component is modified with a bisphosphonate, the self-setting properties of the cement are strongly inhibited.

(24) TABLE-US-00004 TABLE IV Initial and final setting times with alendronate dissolved in the liquid phase or added to the solid phase Alendronate dissolved Alendronate added T = 22 C. in the liquid phase to the solid phase Liquid m(Alend) tf tf phase [mg] pH ti [min] [min] pH ti [min] [min] Na.sub.2HPO.sub.4 control 8.5 25-30 75 8.5 25-30 75 (2.5% by 0.4 6.5-7.0 45 80 8.5 35 95 weight) 0.25 6.5-7.0 45 80 8.5 40 90 L/S = 0.3 0.1 6.5-7.0 40 90 8.5 30 95 **NaH.sub.2PO.sub.4 control 5 25 65-70 5 25 65-70 (2.5% by 10.sup.a 5 45 95 weight) 1.2.sup.b 4.5 65 >100 L/S = 0.3 0.4 4.5 35 75 5 30 80 0.25 4.5 45 90 5 30 80-85 0.1 4.5 35 60 5 35 80 citric acid.sup.c control 2 12 53 2 12 53 (85 mM) 0.4 2 35 65 2 13 60 L/S = 0.25 0.25 2 20 55 2 15 60 0.1 2 25 55 2 15 60 Note .sup.aIn that case, the amount of -TCP transformed is low, and after one week incubation, the material is obtained as a chewy paste with poor mechanical properties. Note .sup.bIn that case, the amount of -TCP transformed is low, and after one week incubation, the material is obtained as a soft paste with very poor mechanical properties. Note .sup.cUnder incubation conditions, a swelling of the preparation is observed, and after one week poor mechanical properties were observed for the material that is brittle and crumby, although a full transformation of -TCP was evidenced by X-ray diffraction.

Example 7: RMN Assays (Concerning the Samples of Example 5)

(25) The cement samples obtained after 7 days incubation were studied using solid-state magic angle spinning (MAS) NMR spectrometry. The experiments were carried out on a Bruker Advance 300 spectrometer, operating at 7.0 T (.sup.1H and .sup.31P Larmor frequencies of 300 and 121.5 MHz), using 4 mm double-resonance and triple-resonance MAS probes.

(26) The .sup.31P-{.sup.1H} cross-polarisation (CP) MAS experiments were performed using a ramped cross polarization with a contact time of 1 ms. .sup.1H decoupling was achieved using the SPINAL64 sequence with a .sup.1H nutation frequency of 70 kHz. The recycle delay was set to 2 s. Longitudinal relaxation times T.sub.1 for .sup.31P sites in the modified -TCP samples were measured and found to vary between 10 and 300 s (v.sub.0(.sup.31P)=121.5 MHz). The .sup.31P single pulse spectra were thus obtained by recording a single scan after a delay of 600 s.

Example 8: Preparation of a Second Type of Injectable CPC Releasing Alendronate

(27) The solid phase of the cement consists of alpha-tertiary calcium phosphate -TCP, DCPD, MCPH, HPMC and some precipitated hydroxyapatite CDA.

(28) The solid phase composition is the same for all samples: 78 wt % (7.8 g) -TCP 5 wt % (0.5 g) DCPD (CaHPO.sub.4.2H.sub.2O) 5 wt % (0.5 g) MCPH (Ca(H.sub.2PO.sub.4).sub.2.H.sub.2O) 10 Wt % (1 g) CDA. 2 wt % (0.2 g) HPMC (hydroxypropylmethylcellulose).

(29) -TCP was prepared by using an appropriate mixture of CaHPO.sub.4 and CaCO.sub.3, heating it at 1300 C. for at least 6 h and quenching it in air down to room temperature.

(30) Three ways are used to combine alendronate with the cement samples. alendronate is dissolved in the cement liquid phase (up to 40 mg in 5 mL see Table V) alendronate is added to the solid phase (13.3-40 mg for 10 g see Table V) alendronate is chemically associated to (i) CDA as prepared in Example 1 replacing partially the CDA of the solid phase (see Table VI) (ii) -TCP as prepared in Example 2 replacing partially the -TCP of the solid phase (see Table VII) (iii) DCPD as prepared in Example 3 replacing partially the DCPD of the solid phase (see Table VIII)

(31) Three concentrations of alendronate have been used: 0.133 wt % (13.3 mg) 0.266 wt % (26.6 mg) 0.4 wt % (40.0 mg)

(32) The liquid phase chosen to prepare different cement formulations was 5% Na.sub.2HPO.sub.4 by weight in water. The liquid/powder ratio L/P of cements was taken to be 0.50 ml/g. The powders are finely ground during 30 minutes.

(33) Then, the liquid phase is added dropwise and the two phases are mixed with a spatula or a pestle.

(34) The mixing sets in moulds.

Example 9: Setting Time Assays Related to Example 8

(35) The properties of the cements were studied using Vickers microindentation (maximal compressive strength), powder X-ray diffraction and .sup.31P solid state NMR (transformation ratio of -TCP to CDA), and texture analyses (evaluation of the initial setting time). The latter method consists in measuring the compression force necessary to extrude the cement dough (initial setting time=the time to reach a force value >25 Newton) versus time.

(36) Tables V, VI, VII and VIII summarize the results.

(37) TABLE-US-00005 TABLE V Setting and mechanical properties of cements with alendronate dissolved in the liquid phase or added to the solid phase Maximal Transformation m(Alend) compressive Initial setting of -TCP to [mg] strength [MPa] time [min] CDA Alendronate dissolved in the liquid phase 0 (control) 11 1 15 high 13.3 18 3 65 high 26.6 19 1 >100 high 40.sup.b 20 3 >>250 high Alendronate added to the solid phase 0 (control) 11 1 15 high 13.3 11 1 45 high 26.6 Not measurable.sup.a >100 high 40.sup.b Not measurable.sup.a >>100 Very high Note a: in that case, after two days incubation, the material is obtained as a brittle and crumby material, leading to non-reproducible data. Note b: the presence of alendronate is detected on .sup.31P.sup.1H VACP NMR spectra, as a broad signal at ca. 18 ppm, very similar to that present in FIG. 1, thus suggesting that the bisphosphonate is chemisorbed on the surface of the CDA resulting from the transformation of the -TCP component.

(38) TABLE-US-00006 TABLE VI Setting and mechanical properties of cements with alendronate chemically associated to CDA Alendronate associated to CDA Maximal Initial Transformation m(Alend) compressive setting of -TCP to [mg] strength [MPa] time [min] CDA 0 (control) 11 1 15 high 13.3 16 2 40 high 26.6 19 2 90-100 high 40.sup.a 18 2 >>90 high Note a: The presence of alendronate is detected on .sup.31P.sup.1H VACP NMR spectra, as a broad signal at ca. 18 ppm, very similar to that present in FIG. 1, thus suggesting that the bisphosphonate is chemisorbed on the surface of the CDA resulting from the transformation of the -TCP component.

(39) TABLE-US-00007 TABLE VII Setting and mechanical properties of cements with alendronate chemically associated to -TCP. Alendronate associated to -TCP Maximal Initial setting Transformation m(Alend) compressive time of -TCP to [mg] strength [MPa] [min] CDA 0 (control) 11 1 15 high 13.3 13 1 40 high 26.6 12 1 >120 high 40 12 1 >>120 high 13.3, obtained by mixing 14 1 17 high 6 wt % of modified -TCP [2.85 wt % alendronate] in pure -TCP 26.6, obtained by mixing 14 1 30 high 12 wt % of modified - TCP [2.85 wt % alendronate] in pure - TCP 40, obtained by mixing 18 15 1 75 high wt % of modified -TCP [2.85 wt % alendronate] in pure -TCP

(40) TABLE-US-00008 TABLE VIII Setting and mechanical properties of cements with alendronate chemically associated to DCPD. Alendronate associated to DCPD Maximal compressive Initial Transformation m(Alend) strength setting of -TCP [mg] [MPa] time [min] to CDA 0 (control) 11 1 15 high 13.3 22 3 60 high 26.6 20 1 >>90 high 40 20 2 >>90 high

(41) For the different cases corresponding to Tables V-VIII (0.133 wt % alendronate relative to the solid phase), cement blocks obtained after 2 hours incubation were immerged in a 0.9 wt % NaCl aqueous solution at 37 C. for 5 days. The blocks were then dried and cut, before SEM (Scanning Electron microscopy) observations. In all cases, homogeneously dispersed macropores (20 to 100 m) were observed, resulting from the degradation of the HPMC component.

Example 10: In Vivo Assays in Ewes Related to Example 8

(42) Six 10-years-old ewes are used for this experiment. The animals had free access to normal diet. The animals were randomly implanted with alendronate-loaded or unloaded CPC. 3 g-doses of alendronate-loaded CPC were prepared according to example 8 with 4 mg of alendronate chemically associated to CDA (see example 1). Each ewe received 33 g-doses (shared inside 3 vertebral bodies) of either alendronate-loaded or unloaded CPC. Animals were sacrificed 3 months after implantation. Each implanted vertebral body was analysed using: 1. Scanning Electron Microscopy (observation in the backscattered electron mode) 2. Micro-CT scan (histomorphometric measurements).

(43) Auto-induced macroporosity, direct interface with bone and significant surface osteoconduction were observed on SEM images in both CPC and alendronate-loaded CPC (FIGS. 4 and 5). Significant implant resorption and volumic osteconduction were observed for alendronate-loaded CPC implants (FIG. 5). Comparative histomophometric analysis of the trabecular bone structures surrounding CPC and alendronate-loaded CPC implants demonstrated (N=18, p<0.05) clearly that the bone architecture is reinforced by alendronate-loaded CPC implants (see table below).

(44) TABLE-US-00009 Bone Trabecular Trabecular Volume (%) space (m) number (m.sup.1) CPC 17.7 1.5 494.9 5.8 0.95 0.07 CPC + 28.4 2.7 420.7 8.0 1.53 0.22 alendronate