Implantable pharmaceutical composition prepared from components consisting only of calcium sulfate hemihydrate, vancomycin and gentamicin

Abstract

A pharmaceutical composition prepared from components consisting of: a) 18.4g pharmaceutical grade, phase pure calcium sulfate α-hemihydrate; b) 500mg vancomycin hydrochloride; and c) 240mg gentamicin sulfate; wherein the calcium sulfate α-hemihydrate is prepared by first converting a source of pharmaceutical grade calcium sulfate dihydrate to soluble calcium sulfate anhydrite by hydrothermal treatment in suspension, and then converting the resulting soluble anhydrous calcium sulfate to calcium sulfate α-hemihydrate by the following steps: i) adding the calcium sulfate dihydrate to a quantity of water in a water:calcium sulfate dihydrate ratio of 0.3:1-0.5:1 to form a suspension and autoclaving the suspension at 235-265oC for 1-2 hours to dehydrate to form soluble anhydrite; ii) allowing the soluble anhydrite to cool in the autoclave in suspension to rehydrate back to calcium sulfate dihydrate; iii) draining off excess water and autoclaving again at 110-150oC for 1-2 hours to convert the rehydrated calcium sulfate dihydrate to calcium sulfate α-hemihydrate. A method of manufacturing the composition and therapeutic uses thereof comprising inserting the composition into a surgical void for the treatment of infection in bone and soft tissue in a patient are also provided.

Description

Porosity and surface area of a material can be measured by the Brauner, Emmett and Teller (BET) technique or alternatively by mercury intrusion porosimetry. The BET technique involving nitrogen adsorption is widely used for detecting open pores mainly in the micro (< 2 nm) to mesopore (2-50 nm) region. The volume of nitrogen adsorbed to the surface of the material is measured at the boiling point of nitrogen (-196°C). At this temperature the nitrogen gas is below the critical temperature and so condenses on the surface in a monolayer. As the size of the nitrogen atom / molecule is known, the amount of adsorbed (condensed) gas is correlated to the total surface area of the particles including pores at the surface. Average pore size distribution can be deduced according to the Barrett-Joyner-Halenda (BJH) method. Where BET theory measures the specific surface area of materials, BJH theory is a pore size distribution determination method. This method determines pore area and specific pore volume using adsorption and desorption techniques and characterizes pore size distribution independent of external area due to particle size of the sample. Setting time of the material can be determined by a variety of methods. The surface penetration test involves applying a weighted Vicat or Gilmore needle to the surface of the material. The material is deemed to be set when it has either penetrated to a given depth (Vicat) or developed sufficient strength to support the needle and it no longer makes an indentation (Gilmore). Both the Vicat and Gilmore tests are the subject of multiple standards, including ASTM. An alternative method is acoustic monitoring, where the sound frequencies produced when dropping the material onto a hard surface from a set height are recorded. Set time is confirmed 14 1024 when the frequencies match those of a control sample that is fully set. A more accurate method for determining set time is by monitoring the temperature of the setting mass with a calibrated thermocouple inserted into the material. The calcium sulfate crystallisation reaction is exothermic, and the mass will continue to increase in temperature until the reaction has completed, at which point the material will start to reduce in temperature. The peak of the temperature curve indicates the final set time. The present invention seeks to provide an implantable composition for treatment of localised infections in the body by providing a dual antibiotic loaded material produced from materials consisting only of a pharmaceutically pure calcium sulfate a-hemihydrate, in combination with two antibiotics, vancomycin and gentamicin, in particular amounts, together providing a synergistic effect. The present invention provides a composition that not only provides a local delivery carrier for the two antibiotics but has specific properties necessary to provide a controlled reproducible dissolution rate. Pore size, surface area and compressive strength all determine the physical properties of the set beads in respect to the dissolution rate and elution kinetics of the two antibiotics. The purity of the calcium sulfate free of setting aids and other processing excipients in combination with a fixed dose of two antibiotics which together target a wider range of bacterial infections in described. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a pharmaceutical composition prepared from components consisting of 18.4g pharmaceutical grade, phase pure calcium sulfate a-hemihydrate, 500 mg of vancomycin hydrochloride; and 240 mg of gentamicin sulfate; where the calcium sulfate a-hemihydrate is prepared using the steps (i)-(iii) discussed above. The composition is typically provided in the form of beads or pellets, which are formed by setting amounts of a paste of the composition within a mould mat containing cavities of a specific size and shape to produce cylindrical beads with one hemispherical end for implantation into a surgical site. The calcium sulfate used to prepare the composition is calcium sulfate a-hemihydrate. The calcium sulfate a-hemihydrate is of a high purity exceeding the ASTM standard for calcium sulfate. The composition of the invention is prepared using only calcium sulfate a-hemihydrate, 14 1024 vancomycin hydrochloride and gentamicin sulfate, and is free of any processing and setting aids, including calcium sulfate dihydrate, potassium phosphate, sodium chloride, sodium sulfate or calcium stearate. This simplifies the process, makes it less expensive, and also reduces the variability in porosity and pore size of the composition. The vancomycin is provided in the form of its hydrochloride salt. The vancomycin hydrochloride will typically be in powder form. The gentamicin is provided in the form of its sulfate salt. The gentamicin sulfate will typically be in the form of a solution and is used for both solubilising and suspending the vancomycin hydrochloride powder and hydrating the calcium sulfate a-hemihydrate to form the composition, which is typically in the form of set beads or pellets. If the composition is in the form of beads or pellets, they are formed by measuring the gentamicin sulfate solution at the appropriate dose and adding the solution to the specified weight of vancomycin powder. The vancomycin partly dissolves in the gentamicin solution to produce a suspension containing the two antibiotics. The suspension is then applied to the specified weight of calcium sulfate a-hemihydrate powder and mixed, typically for about 20 seconds. The resultant paste is then applied to a bead mould and allowed to set. Once set, the resultant beads are then removed from the mould and are ready for implantation into the surgical site. The beads possess a specific surface area and pore size distribution allowing for a predictable and controlled rate of dissolution and drug elution when measured in vitro. In one specific embodiment, the beads are cylindrical, having a diameter between 2.8 mm and 3.2 mm at their widest point. One end of the bead is hemispherical. The bead mould will typically contain a total of 837 cavities to incorporate all the paste. The bead mould is typically a two-sided mould with bead cavities on both sides. This facilitates easier removal of the beads by adding flexibility to the mould. The cavities are typically identical on both sides. In one specific embodiment, the bead mould is square in shape having sides of 152.5 mm and a depth of 7.5 mm. The physical properties of the beads are a product of the antibiotic doses and the weight of the calcium sulfate hemihydrate powder used to make them. The ratio of drug concentration to hemihydrate powder (i.e. the liquid :powder ratio referenced above) is critical to the 14 1024 performance of the beads once implanted. The liquid:powder ratio is linked to bead set time, compressive strength and elution profile of both pharmaceutical ingredients. As discussed above, the porosity of the composition is determined in part by the volume of aqueous solution containing the active pharmaceutical ingredients. Adding too little of the aqueous solution to the powder results in only a partial conversion to calcium sulfate dihydrate, producing a weaker, more soluble biphasic material. Conversely, too much liquid results in surplus water for crystallisation which will not take part in the reaction and remains as free water in the material, contributing to the subsequent porosity and leaving a weaker material. The liquid to powder ratio is an important parameter to produce a finished material with the ideal surface area and pore size, critical factors in the dissolution of the beads and consequently the elution of the added pharmaceutical ingredients. The ideal liquid to powder ratio to fully convert the calcium sulfate a-hemihydrate is in the range of 0.26-0.35:1, more typically 0.28-0.32:1. The liquid to powder ratio of other commercially available calcium sulfate products use ratios which are either lower or higher than this, as shown in Table 1 below, due to them being manufactured in different ways to the calcium sulfate a-hemihydrate used in the present invention. In one specific embodiment, the dose of gentamicin sulfate in solution is 40 mg / ml. 6 ml of the gentamicin sulfate in solution is used to partially dissolve the vancomycin hydrochloride at a dose of 500 mg and the resultant aqueous suspension contains 83.33 mg / ml of vancomycin hydrochloride. This will fully hydrate 18.4g of calcium sulfate a-hemihydrate powder. In the specific embodiment of 18.4g calcium sulfate alpha hemihydrate with 500g vancomycin and 240mg gentamicin prepared as described, the set beads typically have specific properties in terms of BET surface area of between 0.2 m2 / g and 0.8 m2 / g; more typically between 0.4 m2 / g and 0.6 m2 / g. According to one embodiment of the invention, the beads or pellets have a BJH adsorption average pore diameter (4v / A) area deduced according to the Barrett-Joyner-Halenda (BJH) pore size distribution method between 10 nm and 25 nm. The beads when produced according to the aforementioned embodiments, have a setting time of under 20 minutes as defined as the time required from the combination of the aqueous suspension to the calcium sulfate alpha-hemihydrate until hardening is achieved. The disclosed embodiments produce antibiotic loaded calcium sulfate dihydrate beads that maintain sufficient compressive strength that is not compromised by the addition of antibiotics. According to one embodiment, the compressive strength is between 10 and 20 MPa at 1 hour after mixing. The beads produced using the aforementioned method may be used in surgical procedures as part of an infection management strategy where they absorb and elution the full amount of vancomycin and gentamicin at the surgical site reducing the risk of systemic toxicity compared to systemic administration of the two antibiotics. BRIEF DESCRIPTION OF THE FIGURES 14 1024 Figure 1 illustrates the variation in dissolution between two commercially available calcium sulfates and the calcium sulfate a-hemihydrate of the invention, with different liquid / powder ratios as shown in Table 1: Table 1. Three calcium sulfates manufactured with different liquid / powder ratios Liquid / Powder Ratio Sample 1 (Synthecure®) 0.240:1 Sample 2 (the calcium sulfate a-hemihydrate of the invention) 0.320:1 Sample 3 (InterSep®) 0.395:1 Figure 2 shows a plan view, sectional side view and perspective view of a 3mm bead 2 made of the composition of the invention. The diameter of the bead at its widest point is 3mm. Figure 3 shows a plan view of a mould 4 used to make the beads. The mould has a plurality of cavities 6 thereon, which here each have a diameter of 3mm. 14 1024 The bead mould 4 is typically a two-sided mould with bead cavities 6 on both sides, as shown in Figure 4, which shows two sectional side views of the bead mould 4, one with a wider perspective, and the other showing two corresponding cavities 6 on each side of the mould. Having the cavities 6 on both sides of the mould 4 facilitates easier removal of the beads by adding flexibility to the mould 4. The cavities 6 are identical on both sides of the mould 4. Figure 5 shows another perspective view of the bead mould 4, showing the mould with its arrangement of cavities 6. EXAMPLES EXAMPLE 1 Demonstrate synergistic effect of VG over G against Gram positive staph strains 18.4 g of calcium sulfate a-hemihydrate was combined with either 240 mg (6 ml) of gentamicin sulfate liquid or 240 mg (6ml) of gentamicin sulfate liquid with 500 mg of vancomycin hydrochloride powder and formed into 6mm beads and allowed to set. Agar plates were seeded with 0.2 ml of an actively growing suspension of the relevant organism. The plates were transferred to an incubator for 30 minutes. The plates were then removed from the incubator and the beads samples were placed on the surface. The plates were then re-incubated. On removal from the incubator, the plates were examined for the absence of growth as seen by a clear zone around the bead. Any zones of inhibition (ZOI) were measured and photographed. Zone sizes are shown in Tables 2-4. Table 2. ZOI: Staphyloccocus aureus ATCC 6538 Zone (mm) Test Sample (6mm bead) Sample 1 Sample 2 Sample 3 CS + Gentamicin 15-16 15-16 17-18 CS + Vancomycin + Gentamicin 20-21 20-21 20-21 Table 3. ZOI: Staphyloccocus epidermidis ATCC 12228 Zone (mm) Test Sample (6mm bead) Sample 1 Sample 2 Sample 3 CS + Gentamicin 25-26 23-24 23-24 CS + Vancomycin + Gentamicin 25-26 26-27 26-27 Table 4. ZOI: MRSA NCTC 12493 Zone (mm) Test Sample (6mm bead) Sample 1 Sample 2 Sample 3 CS + Gentamicin 16-17 14-15 16-17 CS + Vancomycin + Gentamicin 21-22 21-22 20-21 14 1024 EXAMPLE 2 Demonstrate surface area 18.4 g of calcium sulfate a-hemihydrate was combined with 240 mg (6 ml) of gentamicin sulfate liquid with 500 mg of vancomycin hydrochloride powder and formed into 3mm beads and allowed to set. A sample set of 3 beads were selected at random for analysis. The beads were vacuum degassed at room temperature and then isotherms were obtained using a nitrogen adsorption instrument (Tristar II Plus. Micromeritics Ltd.) Results are shown in Table 5. Table 5. BET surface area of three lots of calcium sulfate combined with vancomycin and gentamicin Sample BET Surface Area (m2 / g) Lot 1 Sample 1 0.346 Lot 1 Sample 2 0.352 Lot 1 Sample 3 0.372 Lot 2 Sample 1 0.353 Lot 2 Sample 2 0.355 Lot 2 Sample 3 0.361 Lot 3 Sample 1 0.292 Lot 3 Sample 2 0.289 Lot 3 Sample 3 0.294 EXAMPLE 3 Demonstrate BJH pore size distribution 18.4 g of calcium sulfate hemihydrate was combined with 240 mg (6 ml) of gentamicin liquid with 500 mg of vancomycin powder and formed into 3mm beads and allowed to set. A sample set of 3 beads were selected at random for analysis. The beads were vacuum degassed at room temperature and then isotherms were obtained using a nitrogen adsorption instrument (Tristar II Plus. Micromeritics Ltd.) Results are shown in Table 6. 14 1024 Table 6. BJH Adsorption Average Pore Diameter of three lots of calcium sulfate combined with vancomycin and gentamicin Sample BJH Adsorption Average Pore Diameter (4V / A) (nm) Lot 1 Sample 1 13.6327 Lot 1 Sample 2 13.8784 Lot 1 Sample 3 13.4841 Lot 2 Sample 1 19.4566 Lot 2 Sample 2 19.1605 Lot 2 Sample 3 17.5870 Lot 3 Sample 1 18.0439 Lot 3 Sample 2 19.1441 Lot 3 Sample 3 17.1265 EXAMPLE 4 Demonstrate Compressive Strength 18.4 g of calcium sulfate hemihydrate was combined with 240 mg (6 ml) of gentamicin liquid with 500 mg of vancomycin powder and applied to a mould mat to form cylinders of approximately 7 mm in diameter and 10 mm in height. The samples were then individually placed between compression plates on a single-column materials testing machine (Zwickiline, Zwick Roell) with a maximum deformation of 2 mm and upper force limit of 2000N and compressed at 0.5 mm / minute. Fmax was recorded (N) and converted to MPa. The compressive strength was recorded as 14.56 MPa (n=5) compared to unloaded calcium sulfate cylinders with a compressive strength of 18.3 MPa (n=5). It is of course to be understood that the present invention is not intended to be restricted to the foregoing examples which are described by way of example only.

Claims

1. A pharmaceutical composition prepared from components consisting only of:a) 18.4 g pharmaceutical grade, phase pure calcium sulfate a-hemihydrate;b) 500 mg of vancomycin hydrochloride; andc) 240 mg of gentamicin sulfate;wherein the calcium sulfate a-hemihydrate is prepared by first converting a source of pharmaceutical grade calcium sulfate dihydrate to soluble calcium sulfate anhydrite by hydrothermal treatment in suspension, and then converting the resulting soluble anhydrous calcium sulfate to calcium sulfate a-hemiliydrate by the following steps:t) adding the calcium sulfate dihydra te to a quantity of water in a water:calcium sulfate dihydrate ratio of 0.3:1 0.5:1 to form a suspension and autoclaving the suspension at 235-265°C for 1-2 hours to dehydrate to form soluble anhydrite;it) allowing the soluble anhydrite to cool in the autoclave in suspension to rehydrate back to calcium sulfate dihydrate;iii) draining off excess water and autoclaving again at H0-150°C for 1-2 hours to convert the rehydrated calcium sulfate dihydrate to calcium sulfate a-hemihydrate;and wherein the gentamicin sulfate in the form of a solution and the vancomycin hydrochloride are combined together to form a suspension containing vancomycin and gentamicin, and the suspension is combined with the calcium sulfate a-hemihydrate in powder form in a liquid:powder ratio of 0.26-0.35:1 parts by weight, wherein the liquid is the suspension containing the vancomycin and gentamicin.

2. The composition according to claim 1, wherein the liquid:powder ratio is 0.28-0.32:1 parts by weight.

3. The composition according to any preceding claim, wherein the vancomycin hydrochloride is provided in a powder form.14 10244. The composition according to any preceding claim, wherein the composition is in the form of beads or pellets.

5. The composition according to claim 4, wherein the beads or pellets are between 2.8 and 3.2 mm in diameter at their widest point.

6. The composition according to claim 4 or claim 5, wherein the beads or pellets have a compressive strength of between 10 MPa and 20 MPa at 1 hour after setting.

7. The composition according to any of claims 4-6, wherein the beads or pellets have a BET specific surface area obtained using the Brunauer Emmett-Teller (BET) Method of between 0.2 m2 / g and 0.8 m2 / g.

8. The composition according to any of claims 4-7, wherein the beads or pellets have a BJH adsorption average pore diameter (4v / A) area, deduced according to the Barrett-Joyner-Halenda (BJH) pore size distribution method, of between 10 nm and 25 nm.

9. A method of manufacturing a composition according to any preceding claim, the method comprising the steps of:a) mixing 500 mg of vancomycin hydrochloride with 240 mg of gentamicin sulfate, the gentamicin sulfate being in the form of a liquid, to produce a suspension containing vancomycin and gentamicin;b) combining said suspension with a powder consisting of 18.4 g of pharmaceutical grade, phase pure calcium sulfate a-hemihydrate to form a mixture, wherein the suspension is combined with the calcium sulfate a-hemihydrate in powder form in a liquid:powder ratio of 0.26-0.35:1 parts by weight, said liquid being the suspension containing the vancomycin and gentamicin;c) forming said mixture into beads or pellets which are allowed to set and solidify using a plurality of moulds; andd) producing beads or pellets of the composition.

10. The method according to claim 9, wherein:a) the vancomycin hydrochloride is provided in the form of a powder;14 1024andb) the beads or pellets of the composition are individually shaped as a cylinder with a single hemispherical end.

11. The method according to claim 9 or 10, wherein the liquid:powder ratio is 0.28-0.32:1 parts by weight.

12. The method according to any of claims 9-11, wherein the gentamicin is in the form of an aqueous solution.

13. The method according to claim 12 wherein the concentration of gentamicin in the aqueous solution is from 25-50 mg / ml.

14. The method according to claim 12 or claim 13, wherein the volume of the aqueous solution to receive the vancomycin for a single dose of the composition is between 5.5 ml and 7.5 ml.

15. The method according to any of claims 9-14, wherein the beads or pellets are prepared using a mould mat containing cavities wherein the size, spacing and geometric arrangement of cavities is such that any straight line drawn along the full length of the mold mat parallel to an edge of the mold mat in at least one direction and within the arrangement of cavities on the mat will always intersect at least one cavity.

16. The method according to claim 15, wherein the mould mat contains cavities on both sides of the mat.

17. The method according to any of claims 9-16, wherein the beads or pellets set in a time of between 10 and 20 minutes as measured by the temperature rise method.

18. The method according to claim 17, wherein the setting time is determined by the insertion of a thermometer probe inside the setting composition to track changes in temperature over time.

19. A composition according to any of claims 1-9 for use in the treatment of infection in bone and soft tissue in a patient.14 1024

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