Crystallization method and bioavailability

Abstract

Preparation, in-vitro and in vivo characterization of novel forms of (1-hydroxy-2-imidazol-1-yl-1-phosphono-ethyl) phosphonic acid, suitable for pharmaceutical compositions in drug delivery systems for humans.

Claims

1. A crystalline molecular complex comprising zoledronic acid, DL-lysine, and water characterized by a Fourier transform infrared spectroscopy pattern corresponding to FIG. 20.

2. A pharmaceutical composition comprising the crystalline molecular complex of claim 1 and a pharmaceutically acceptable excipient.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is an oral dosage form.

4. The pharmaceutical composition of claim 3, wherein the oral dosage form is selected from a tablet, a capsule, and a liquid suspension of the crystalline molecular complex.

5. The pharmaceutical composition of claim 3, wherein the oral dosage form is an enteric coated tablet.

6. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is in the form of an injectable, a suppository, a topical, or transdermal.

7. A composition comprising a physical mixture of the crystalline molecular complex of claim 1 and an excess amount of lysine or glycine.

8. The composition of claim 7, comprising an excess amount of lysine.

9. The composition of claim 8, wherein the excess amount of lysine is DL-lysine monohydrate.

10. The crystalline molecular complex of claim 1, wherein the bioavailability of the zoledronic acid from the crystalline molecular complex is greater than the bioavailability of the zoledronic acid without DL-lysine.

11. The crystalline molecular complex of claim 1, wherein the zoledronic acid in the crystalline molecular complex has improved aqueous solubility compared to zoledronic acid without DL-lysine.

12. A method for the treatment of disease states associated with osteoporosis, hypercalcemia, cancer induced bone metastasis, Paget's disease or adjuvant or neoadjuvant cancer therapies comprising the step of administering to a patient in need thereof a therapeutically effective amount of the crystalline molecular complex of claim 1.

13. The method of claim 12, wherein said hypercalcemia is tumor induced hypercalcemia (TIH).

14. The method of claim 12, wherein said disease is cancer induced bone metastasis.

15. A method for the treatment of disease states associated with osteoporosis, hypercalcemia, cancer induced bone metastasis, Paget's disease or adjuvant or neoadjuvant cancer therapies comprising the step of administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 2.

16. A method for the treatment of disease states associated with osteoporosis, hypercalcemia, cancer induced bone metastasis, Paget's disease or adjuvant or neoadjuvant cancer therapies comprising the step of administering to a patient in need thereof a therapeutically effective amount of the composition of claim 7.

17. A method for the treatment of disease states associated with osteoporosis, hypercalcemia, cancer induced bone metastasis, Paget's disease or adjuvant or neoadjuvant cancer therapies comprising the step of administering to a patient in need thereof a therapeutically effective amount of the composition of claim 8.

18. A method for the treatment of disease states associated with osteoporosis, hypercalcemia, cancer induced bone metastasis, Paget's disease or adjuvant or neoadjuvant cancer therapies comprising the step of administering to a patient in need thereof a therapeutically effective amount of the composition of claim 9.

19. A method for enhancing the bioavailability or permeability of zoledronic acid comprising the step of administering to a patient in need thereof a therapeutically effective amount of zoledronic acid in the form of the crystalline molecular complex of claim 1.

20. A method for making the crystalline molecular complex of claim 1, comprising: slurrying zoledronic acid with DL-lysine in tetrahydrofuran and water to form a crystalline molecular complex; and separating the crystalline molecular complex from the slurry.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows PXRD diffractograms of: (A=zoledronic acid, sodium zoledronic salt and water complex), (B=NaCl), (Z1=Zoledronic acid monohydrate), (Z3=Zoledronic acid trihydrate).

(2) FIG. 2 is an FTIR spectrum of a complex comprising zoledronic acid, sodium zoledronic salt, and water.

(3) FIG. 3 shows PXRD diffractograms of: (C=ammonium zoledronic salt and water complex), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(4) FIG. 4 is an FTIR spectrum of ammonium zoledronic salt and water complex.

(5) FIG. 5 shows PXRD diffractograms of: (D=zoledronic, L-lysine, and water complex), (E=L-lysine), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(6) FIG. 6 is an FTIR spectrum of zoledronic, L-lysine, and water complex.

(7) FIG. 7 shows PXRD diffractograms of: (F=zoledronic, DL-lysine, and water complex), (G=DL-lysine), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(8) FIG. 8 is an FTIR spectrum of zoledronic, DL-lysine, and water complex.

(9) FIG. 9 shows PXRD diffractograms of: (H=zoledronic acid, zoledronic, DL-lysine, ethanol, and water complex), (G=DL-lysine), (Z1=Zoledronic acid monohydrate), (Z3=Zoledronic acid trihydrate).

(10) FIG. 10 is an FTIR spectrum of zoledronic acid, zoledronic, DL-lysine, ethanol, and water complex.

(11) FIG. 11 shows PXRD diffractograms of: (I=zoledronic, nicotinamide, and water complex), (J=nicotinamide), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(12) FIG. 12 is an FTIR spectrum of zoledronic, nicotinamide, and water complex.

(13) FIG. 13 shows PXRD diffractograms of: (K=zoledronic, adenine, and water complex), (L=adenine), (Z1=Zoledronic acid monohydrate), (Z3=Zoledronic acid trihydrate).

(14) FIG. 14 is an FTIR spectrum of zoledronic, adenine, and water complex.

(15) FIG. 15 shows PXRD diffractograms of: (M=zoledronic and glycine complex), (N=glycine), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(16) FIG. 16 is an FTIR spectrum of zoledronic and glycine complex.

(17) FIG. 17 shows PXRD diffractograms of: (0=zoledronic diammonia water complex), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(18) FIG. 18 is an FTIR spectrum of zoledronic diammonia water complex.

(19) FIG. 19 shows PXRD diffractograms of: (P=zoledronic, DL-lysine, and water complex), (G=DL-lysine), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(20) FIG. 20 is an FTIR spectrum of zoledronic, DL-lysine, and water complex.

(21) FIG. 21 shows PXRD diffractograms of: (R=zoledronic, DL-lysine, and water complex), (G=DL-lysine), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(22) FIG. 22 is an FTIR spectrum of zoledronic, DL-lysine, and water complex.

(23) FIG. 23 shows PXRD diffractograms of: (R=zoledronic, DL-lysine, and water complex), (G=DL-lysine), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(24) FIG. 24 is an FTIR spectrum of zoledronic, DL-lysine, and water complex.

(25) FIG. 25 shows PXRD diffractograms of: (Q=zoledronic, L-lysine, and water complex), (E=L-lysine), (Z1=Zoledronic acid monohydrate), and (Z3=Zoledronic acid trihydrate).

(26) FIG. 26 is an FTIR spectrum of zoledronic, L-lysine, and water complex.

(27) FIG. 27 shows the 24 hr rat plasma PK profile of parent zoledronic acid and zoledronic acid complexes delivered via IV, oral, and intraduodenal (ID) routes.

(28) FIG. 28 shows the 4 hr rat plasma PK profile of parent zoledronic acid and zoledronic acid complexes delivered orally.

(29) FIG. 29 shows the 4 hr rat plasma PK profile of parent zoledronic acid and zoledronic acid complexes delivered ID.

(30) FIG. 30 shows the 24 hr rat plasma PK profile of parent zoledronic acid and zoledronic acid complexes delivered by oral gavage.

(31) FIG. 31 shows the 4 hr rat plasma PK profile of parent zoledronic acid and zoledronic acid complexes delivered orally.

(32) FIG. 32 shows the 4 hr rat plasma PK profile of parent zoledronic acid and selected zoledronic acid complexes delivered orally.

(33) FIG. 33 shows the dog serum PK profile of parent zoledronic acid and zoledronic acid complexes delivered IV and orally.

(34) FIG. 34 shows the 4 hr dog serum PK profile of parent zoledronic acid and zoledronic acid complexes delivered IV and orally.

(35) FIG. 35 shows the dog serum PK profile of parent zoledronic acid and zoledronic acid complexes delivered IV and orally; enteric and non-enteric coated capsules.

(36) FIG. 36 shows the 6 hr dog serum PK profile of parent zoledronic acid and zoledronic acid complexes delivered IV and orally; enteric and non-enteric coated capsules.

(37) FIG. 37 shows the dog PK data for the enteric and non-enteric coated hard gelatin capsules.

(38) FIG. 38 shows the 24 hr dog serum PK profile of zoledronic acid complexes delivered IV and orally.

(39) FIG. 39 shows the 4 hr dog serum PK profile of zoledronic acid complexes delivered IV and orally.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(40) In general, active pharmaceutical ingredients (APIs) in the pharmaceutical compositions can be prepared in a variety of different forms including prodrugs, amorphous forms, solvates, hydrates, cocrystals, salts and polymorphs. The discovery of novel API forms may provide an opportunity to improve the performance characteristics of a pharmaceutical product. Additionally, discovery of drug forms expands the array of resources available for designing pharmaceutical dosage forms with targeted release profiles or other desired characteristics.

(41) A specific characteristic that can be targeted includes the crystal form of an API. The alteration of the crystal form of a given API would result in the modification of the physical properties of the target molecule. For example, various polymorphs of a given API exhibit different aqueous solubility, while the thermodynamically stable polymorph would exhibit a lower solubility than the meta-stable polymorph. In addition, pharmaceutical polymorphs can also differ in properties such as rate of dissolution, shelf life, bioavailability, morphology, vapor pressure, density, color, and compressibility. Accordingly, it is desirable to enhance the properties of an API by forming molecular complexes such as a cocrystal, a salt, a solvate or hydrate with respect to aqueous solubility, rate of dissolution, bioavailability, Cmax, Tmax, physicochemical stability, down-stream processibility (e.g. flowability compressibility, degree of brittleness, particle size manipulation), decrease in polymorphic form diversity, toxicity, taste, production costs, and manufacturing methods.

(42) In the development of orally delivered drugs, it is often advantageous to have novel crystal forms of such drugs that possess improved properties, including increased aqueous solubility and stability. In many cases, the dissolution rate increase of drugs is desired as it would potentially increase their bioavailability. This also applies to the development of novel forms of zoledronic acid which, when administered orally to a subject could achieve a greater or similar bioavailability and PK profile when compared to an IV or other formulations on a dose-for-dose basis.

(43) Cocrystals, salts, solvates and hydrates of zoledronic acid of the present invention could give rise to improved properties of zoledronic acid. For example, a new form of zoledronic acid is particularly advantageous if it can improve the bioavailability of orally delivered zoledronic acid. A number of novel zoledronic acid forms have been synthesized, characterized, and disclosed herein. Of particular interest are the zoledronic acid and the standard amino acids since they have indicated enhanced permeability compared with other molecular complexes of zoledronic acid. The mechanism of enhanced permeability of these complexes is not yet understood and, while not to be bound by this explanation, it is possible that they moderate the formation of the insoluble Ca.sup.2+ zoledronate salt resulting in more zoledronic acid to be absorbed paracellularly through the tight junctions. It must be stressed that this is a possible mechanism of enhanced permeability.

(44) Schematic diagrams for zoledronic acid:amino acid complexes (a zoledronic acid:lysine complex and a zoledronic acid:glycine complex, two embodiments of the invention) are shown below. The diagrams show a molecular structure of the complex and possible interactions between the constituents of the complex which is different from the physical mix of the constituents.

(45) 1. Zoledronic Acid: Lysine Complex

(46) ##STR00002##

(47) 2. Zoledronic Acid: Glycine Complex

(48) ##STR00003##
These represent one of the arrangements that molecules of the drug and the standard amino acids coformers could interact to form a stable complex that even when stressed thermally at elevated relative humidity (RH) environment have not displayed any signs of deterioration or disintegration to its original constituents. Such stability can be attributed to the hydrogen bonding (dashed line in the box) in these molecular complexes. When packing in a crystal structure these complexes have very different morphologies to that of its constituents or their physical mix as indicated by their powder X-ray diffraction (PXRD) patterns and therefore would possess different, unpredictable physicochemical properties.

(49) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic acid, sodium zoledronate and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 8.1, 13.3, 21.5, 24.6, and 25.6?0.2 degrees two-theta.

(50) The present invention provides a new crystal form of zoledronic acid in the form of ammonium zoledronic salt and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 11.0, 14.6, 15.4, 19.9, and 29.4?0.2 degrees two-theta.

(51) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic, L-lysine, and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 9.0, 14.4, 18.1, 26.0, and 29.6?0.2 degrees two-theta.

(52) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic, DL-lysine, and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 9.1, 14.7, 18.0, 21.2, and 26.0?0.2 degrees two-theta.

(53) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic acid, zoledronic, DL-lysine, ethanol, and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 8.8, 9.7, 17.6, 23.1, and 26.5?0.2 degrees two-theta.

(54) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic acid, nicotinamide, and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 13.1, 15.2, 21.0, 23.9, and 26.5?0.2 degrees two-theta.

(55) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic, adenine, and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 13.6, 15.9, 19.7, 27.9, and 29.5?0.2 degrees two-theta.

(56) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic and glycine complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 10.2, 17.8, 19.9, 22.9, and 28.1?0.2 degrees two-theta.

(57) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic diammonia water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 12.2, 13.0, 14.1, 17.1, and 19.3?0.2 degrees two-theta.

(58) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic, DL-lysine, and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 8.3, 11.8, 12.3, 15.8, and 20.8?0.2 degrees two-theta.

(59) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic acid, L-lysine, and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 9.6, 10.7, 14.3, 21.4, 23.5?0.2 degrees two-theta.

(60) The present invention provides a new crystal form of zoledronic acid in the form of zoledronic, DL-lysine, and water complex, characterized by an X-ray powder diffraction pattern having strong peaks at about 9.7, 10.8, 14.4, 18.9, 21.4?0.2 degrees two-theta.

(61) The present invention provides rat plasma or dog serum concentration levels and PK profiles of IV, orally and ID delivered zoledronic acid parent compound versus complexes of zoledronic acid created using the method of this invention.

(62) Accordingly, in a first aspect, the present invention includes complexes of zoledronic acid with sodium, ammonium, ammonia, L-lysine, DL-lysine, nicotinamide, adenine and glycine which are capable of complexing in the solid-state, for example, through dry or solvent-drop grinding (liquid assisted grinding), heating or solvent evaporation of their solution in single or mixed solvent systems, slurry suspension, supercritical fluids or other techniques known to a person skilled in the art.

(63) Another aspect of the invention provides zoledronic and nicotinamide complex by dissolving both compounds in water:ethylacetate (1:1 v/v) and allowing the solvent mixtures to evaporate to form crystalline material.

(64) Another aspect of the invention provides zoledronic and glycine solid complex from dissolving both compounds in water, and allowing the solvent to evaporate to form crystalline material.

(65) Another aspect of the invention provides complexes of zoledronic acid and sodium, ammonium, ammonia, L-lysine, DL-lysine, nicotinamide, adenine and glycine suitable for a pharmaceutical formulation than can be delivered orally to the human body. The pharmaceutical formulation contains a therapeutically effective amount of at least one of the novel molecular complexes of zoledronic acid according to the invention and at least one pharmaceutically acceptable carrier, (also known in the art as a pharmaceutically acceptable excipient). The novel molecular complexes of zoledronic acid are therapeutically useful for the treatment and/or prevention of disease states associated with osteoporosis, hypercalcemia (TIH), cancer induced bone metastasis, Paget's disease or adjuvant or neoadjuvant therapies, discussed above.

(66) The invention also relates to methods of treatment using novel molecular complexes of zoledronic acid of the invention or a pharmaceutical formulation containing them. A pharmaceutical formulation of the invention may be in any pharmaceutical form which contains a novel molecular complex of zoledronic acid according to the invention. The pharmaceutical formulation may be, for example, a tablet, capsule, liquid suspension, injectable, suppository, topical, or transdermal. The pharmaceutical formulations generally contain about 1% to about 99% by weight of at least one novel molecular complex of zoledronic acid of the invention and 99% to 1% by weight of a suitable pharmaceutical excipient.

(67) Complexes of zoledronic acid and sodium, ammonium, ammonia, L-lysine, DL-lysine, nicotinamide, adenine, and glycine have been observed by their PXRD patterns and FTIR spectra.

(68) Another aspect of the invention provides in-vivo data in rats concerning the oral bioavailability of zoledronic acid delivered orally and intraduodenally.

(69) Another aspect of the invention provides PK profiles of the parent compound delivered by different routes; IV, oral and ID.

(70) Another aspect of the invention provides modified oral bioavailability values of novel zoledronic acid complexes prepared by the method of invention, compared with the orally delivered parent compound.

(71) Another aspect of the invention provides the addition of excess at least one coformer to the zoledronic acid complexes, which may be the same as the coformer in the complex, a different coformer, or a mixture thereof.

(72) Another aspect of the invention provides a method where the excess cocrystal formers consist of standard amino acids.

(73) Another aspect of the invention provides modified PK profiles of zoledronic acid complexes with excess cocrystal formers, compared with that of the orally delivered parent compound.

(74) Another aspect of the invention provides improved aqueous solubility of novel zoledronic acid complexes compared with the parent compound.

(75) Another aspect of the invention provides modified oral bioavailability values of novel zoledronic acid complexes with excess cocrystal formers, compared with the orally delivered parent compound.

(76) Another aspect of the invention provides in vivo data in dogs concerning the oral bioavailability of zoledronic acid delivered IV or orally.

(77) Another aspect of the invention provides modified oral bioavailability values in dogs of novel zoledronic acid complexes prepared by the method of invention delivered in gelatin capsules compared with the orally delivered parent compound.

(78) Another aspect of the invention provides modified oral bioavailability values in dogs of novel zoledronic acid complexes prepared by the method of invention delivered in enteric coated gel capsules compared with that of the parent compound.

(79) Another aspect of the invention provides substantial improvement in oral bioavailability values in dogs of novel zoledronic acid complexes with excess cocrystal formers prepared by the method of invention delivered in hard gelatin capsules.

(80) Another aspect of the invention provides slight improvement in oral bioavailability values for zoledronic acid in dogs via zoledronic acid and novel zoledronic acid complexes orally delivered through enteric coated capsules.

(81) Another aspect of the invention provides a reduced oral bioavailability values for zoledronic acid in dogs via novel zoledronic acid complexes with excess physical mix of coformer.

(82) Another aspect of the invention provides a molecular complex comprising a bisphosphonic acid or salt thereof and at least one coformer, wherein the bioavailability of the bisphosphonic acid or salt thereof from the molecular complex is greater than the bioavailability of the bisphosphonic acid or salt thereof without the coformer. The bisphosphonic acid may be, for example, zoledronic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, residronic acid ibandronic acid or other bisphosphonic acids known in the art.

(83) Another aspect of the invention provides a method for enhancing the bioavailabilty or permeability of a bisphosphonic acid comprising the step of administering to a patient in need thereof a therapeutically effective of a bisphosphonic acid in the form of a molecular complex.

(84) The techniques and approaches set forth in the present disclosure can further be used by the person of ordinary skill in the art to prepare variants thereof, said variants are considered to be part of the inventive disclosure.

EXAMPLES

(85) The following examples illustrate the invention without intending to limit the scope of the invention.

(86) Zoledronic acid as a starting material used in all experiments in this disclosure was supplied by Farmkemi Limited (Wuhan Pharma Chemical Co.), China with purity of ca. 98% and was purified further via recrystallization from water. All other pure chemicals (Analytical Grade) were supplied by Sigma-Aldrich and used without further purification.

(87) Enteric coating of gelatin capsules was contracted out to AzoPharma, Hollywood, Fla., USA. A 10% w/w coating solution of Eudragit L100-55, and triethyl citrate, 9.09 and 0.91 w/w % respectively, in purified water and acetone was used in the Vector LDCS pan coater to achieve a uniform coating layer on the capsules. The coating uniformity and functionality for duodenal delivery was tested by 2 hr dissolution in simulated gastric fluid stirred at 75 rpm and 37? C. All capsules remained closed for the duration of this test.

(88) Solid Phase Characterization

(89) Analytical techniques used to observe the crystalline forms include powder X-ray diffraction (PXRD) and Fourier transform infrared spectroscopy (FTIR). The particular methodology used in such analytical techniques should be viewed as illustrative, and not limiting in the context of data collection. For example, the particular instrumentation used to collect data may vary; routine operator error or calibration standards may vary; sample preparation method may vary (for example, the use of the KBr disk or Nujol mull technique for FTIR analysis).

(90) Fourier Transform FTIR Spectroscopy (FTIR): FTIR analysis was performed on a Perkin Elmer Spectrum 100 FTIR spectrometer equipped with a solid-state ATR accessory.

(91) Powder X-Ray Diffraction (PXRD): All zoledronic acid molecular complex products were observed by a D-8 Bruker X-ray Powder Diffractometer using Cu K? (?=1.540562 ?), 40 kV, 40 mA. The data were collected over an angular range of 3? to 40? 2? in continuous scan mode at room temperature using a step size of 0.05? 20 and a scan speed of 6.17?/min.

Example 1: Preparation of Zoledronic Acid, Sodium Zoledronic Salt, and Water Complex

(92) 200 mg of zoledronic acid was slurried with 180 mg of sodium chloride in 1 mL of 1:1 ethanol:water overnight. The material was filtered and rinsed. The particulate material was gathered and stored in a screw cap vial for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 1 and FIG. 2, respectively.

Example 2: Preparation of Ammonium Zoledronic Salt and Water Complex

(93) 300 mg of zoledronic acid was slurried in 7N ammonia in methanol overnight. The material was filtered and rinsed. The particulate material was dissolved in water and left to evaporate at ambient conditions to obtain colorless plates after 1 week. The material was characterized by PXRD and FTIR corresponding to FIG. 3 and FIG. 4, respectively.

Example 3: Preparation of Zoledronic, L-Lysine, and Water Complex

(94) 200 mg of zoledronic acid and 54 mg of L-lysine were slurried in 2 mL of tetrahydrofuran and 200 ?l of water overnight. The solids gathered after filtration were dried and stored in a screw cap vials for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 5 and FIG. 6, respectively.

Example 4: Preparation of Zoledronic, DL-Lysine, and Water Complex

(95) 204 mg of zoledronic acid and 59 mg of DL-lysine were slurried in 2 mL of tetrahydrofuran and 200 ?l of water overnight. The solids gathered after filtration were dried and stored in a screw cap vials for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 7 and FIG. 8 respectively.

Example 5: Preparation of Zoledronic Acid, Zoledronic, DL-Lysine, Ethanol, and Water Complex

(96) 103 mg of zoledronic acid and 54 mg of DL-lysine were dissolved in 400 ?l of water, capped and stirred overnight. The next day 0.25 mL of ethanol was added drop wise. The vial was capped with a screw cap vial and after 1 day crystals appeared and were filtered off. The material was stored for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 9 and FIG. 10 respectively.

Example 6: Preparation of Zoledronic, Nicotinamide, and Water Complex by Solvent-Drop Grinding

(97) 99 mg of zoledronic acid was ground with 44 mg of nicotinamide and 40 ?l of water was added to the solid mixture. The solids gathered after grinding were stored in screw cap vials for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 11 and FIG. 12, respectively.

Example 7: Preparation of Zoledronic, Nicotinamide, and Water Complex from Solution Crystallization

(98) 25 mg of zoledronic acid and 138 mg of nicotinamide were dissolved in 2 mL of a water:ethylacetate mix (1:1 v/v). The solution was then allowed to stand for several hours to effect the slow evaporation of solvent. The solids gathered were characterized and produced very similar PXRD and FTIR patterns to that of Example 7 product.

Example 8: Preparation of Zoledronic, Adenine, and Water Complex by Solvent-Drop Grinding

(99) 96 mg of zoledronic acid was ground with 65 mg of adenine and 60 ?L of water was added to the solid mixture. The solids gathered after grinding were stored in screw cap vials for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 13 and FIG. 14, respectively.

Example 9: Preparation of Zoledronic, Adenine, and Water Complex from Solution Slurry

(100) 99 mg of zoledronic acid and 54 mg of adenine were slurried in 2 mL of a water:ethanol mix (1:1 v/v) overnight. The solids gathered after filtration were dried, characterized and produced very similar PXRD and FTIR patterns to that of Example 8 product.

Example 10: Preparation of Zoledronic and Glycine Complex

(101) 178 mg of zoledronic acid and 45 mg of glycine were slurried in 2 mL of water overnight. The solids gathered after filtration were dried and stored in a screw cap vials for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 15 and FIG. 16, respectively.

Example 11: Preparation of Zoledronic Diammonia Water Complex

(102) 1.5 g of zoledronic acid was slurried in 7N ammonia in methanol overnight. The material was filtered and rinsed. The particulate material was dissolved in water with medium heat and left to evaporate at ambient conditions to obtain colorless blocks after 1 day. The material was characterized by PXRD and FTIR corresponding to FIG. 17 and FIG. 18, respectively.

Example 12: Preparation of Zoledronic, DL-Lysine, and Water Complex

(103) 200 mg of zoledronic acid and 102 mg of DL-lysine were slurried in 2 mL of tetrahydrofuran and 400 ?l of water overnight. The solids gathered after filtration were dried and stored in a screw cap vials for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 19 and FIG. 20 respectively.

Example 13: Preparation of Zoledronic, DL-Lysine, and Water Complex

(104) 1 g of zoledronic acid and 283 mg of DL-lysine were slurried in 80 mL of tetrahydrofuran and 8 mL of water overnight. The solids gathered after filtration were dried and stored in a screw cap vials for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 21 and FIG. 22 respectively.

Example 14: Preparation of Zoledronic, DL-Lysine, and Water Complex by Antisolvent Method

(105) This complex can also be prepared by the antisolvent method by dissolving 1 g of zoledronic acid and 283 mg of DL-lysine in 5 mL of hot water and adding 40 mL of ethanol as an antisolvent stirred overnight. Similar PXRD and FTIR profiles were obtained as shown in FIGS. 23 and 24 respectively.

Example 15: Preparation of Zoledronic, L-Lysine, and Water Complex

(106) 1 g of zoledronic acid and 255 mg of L-lysine were dissolved in 60 mL of hot water. 100 mL of ethanol was then added as an anti solvent. The solids gathered after filtration were dried and stored in a screw cap vials for subsequent analysis. The material was characterized by PXRD and FTIR corresponding to FIG. 25 and FIG. 26 respectively.

Example 16: The Animal PK Studies

(107) These studies were conducted on rats and dogs as they are suitable animal models for zoledronic acid. This can be attributed to the fact that both animals have historically been used in the safety evaluation and PK screening studies and are recommended by appropriate regulatory agencies. In addition, rats and dogs have also been established as appropriate species for assessing the absorption of bisphosphonate drugs including zoledronic acid.

(108) Pure zoledronic acid and zoledronic acid complexes prepared by the methods in this invention were delivered to the rats and dogs through IV or oral routes. Additional tests included ID administration in rats and administration of enteric coated capsules in dogs. All compounds delivered were well tolerated by the animals with no adverse events or physical abnormalities noticed.

(109) Test Subjects:

(110) 8-week male Sprague-Dawley Rats (217-259 grams) were obtained from Hilltop Lab Animals, Scottdale, Pa. USA. Surgical catheters (jugular vein and intraduodenum) were implanted to the animals prior to the study. Beagle dogs from Marshall Farms, N.Y., USA, weighing from (9-12 kg) were used in this study. Surgical catheters (jugular vein) were implanted prior to the study.

(111) Housing:

(112) Rats were individually housed in stainless steel cages to prevent catheter exteriorization. Acclimation (Pre-dose Phase) was for 1 day. Dogs were already in the test facility (Absorption Systems Inc., USA) and did not need acclimation.

(113) Environment:

(114) Environmental controls for the animal room were set to maintain 18 to 26? C., a relative humidity of 30 to 70%, a minimum of 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle could be interrupted for study-related activities.

(115) Diet:

(116) For rats, water and certified Rodent Diet #8728C (Harlan Teklad) were provided. For dogs, water and the standard dog chow diet were given twice daily (every 12 hours).

(117) Fasting:

(118) All test animals were fasted overnight before IV, oral, or ID administration of zoledronic acid or zoledronic acid complexes.

(119) Routes of Rat Dosing:

(120) Zoledronic acid and its complex formulations were administered through IV, oral and ID. The doses administered to all study rats were measured as zoledronic acid, not as the complex form contained in the suspension: i. IV Administration: the dose of zoledronic acid for IV administration was 0.5 mg/kg. The dose of each rat was calculated on a per rat basis (not on an average weight of all the rats in the lot). ii. Oral gavage administration: solid suspensions were administered. The dose of each rat was calculated on a per rat basis (not on an average weight of all the rats in the lot). For solid suspensions, animals were administered 5 mg/kg of zoledronic acid or 5 mg/kg of zoledronic acid in zoledronic acid complexes contained in a suspension of PEG 400. iii. Duodenal cannula administration: solid suspensions were administered. The dose of each rat was calculated on a per rat basis (not on an average weight of all the rats in the lot). For solid suspensions, animals were administered 5 mg/kg of zoledronic acid or 5 mg/kg of zoledronic acid in zoledronic acid complexes contained in a suspension of PEG 400.

(121) Routes of Dog Dosing:

(122) Zoledronic acid and its complex formulations were administered IV and orally. The doses administered to all study dogs were measured as zoledronic acid in each complex, not as the complex form contained in the powder in the gelatin capsule or in solution for IV: i. IV Administration: The dose volume of each dog was adjusted based upon the average weight of the dog. ii. Oral administration: zoledronic acid and its equivalent of zoledronic acid complex formulations were administered through size 0 gelatin capsules based on the average weight of the dogs. iii. Oral administration with enteric coated capsules: zoledronic acid and its equivalent of zoledronic acid complex formulations were administered through size 0 enteric coated gelatin capsules based on the average weight of the dogs. iv. Oral administration of the molecular complexes with additional coformers: physical mixtures of zoledronic acid complexes with additional coformers were administered through size 0 gelatin capsules based on the average weight of the dogs.

(123) Groups:

(124) Two major groups of animals were selected for the study. Group 1, rats that contained four subgroups (I-IV) where the results of each data point on the PK profile graphs was the average drug concentration in the plasma of 3 rats. Group 2, dog PK study contained three groups with subgroups (A, B, C, D, E and F) where the results of each data point on the PK profile graphs was the average drug concentration in the serum of 5 dogs.

(125) Details of Group 1 Rat Dosing

(126) Group I (IV administration). Group members, designated IV doses are listed below

(127) TABLE-US-00001 Group # of Dose # I Designation rats Dose* volume G1 Zoledronic 3 0.5 mg/kg 1 mL Acid
IV comparator group, was conducted to calculate MAT (mean absorption time) and ka (absorption rate constant) for the oral groups.

(128) Group II (Oral Gavage): Group Designations and Oral Doses are Listed Below:

(129) TABLE-US-00002 Dose Group # of volume # II Designation Rats Dose* mL/kg Compound G2 Zoledronic 3 5 mg/kg 1 mL Zoledronic acid Acid in PEG400 G3 Solid 3 5 mg/kg 1 mL Zoledronic and suspension equivalent glycine complex in PEG400 G4 Solid 3 5 mg/kg 1 mL Zoledronic, suspension equivalent nicotinamide, and in PEG400 water complex G5 Solid 3 5 mg/kg 1 mL Zoledronic acid, suspension equivalent sodium zoledronic in PEG400 salt, and water complex G6 Solid 3 5 mg/kg 1 mL Zoledronic, suspension equivalent L-lysine, and in PEG400 water complex G7 Solid 3 5 mg/kg 1 mL Zoledronic, suspension equivalent DL-lysine,and in PEG400 water complex

(130) Group III (ID Administration): Group Designations and Oral Doses are Listed Below:

(131) TABLE-US-00003 Dose Group # of volume # III Designation rats Dose* mL/kg Compound G8 Zoledronic 3 5 mg/kg 1 mL Zoledronic acid Acid in PEG400 G9 Solid 3 5 mg/kg 1 mL Zoledronic and suspension equivalent glycine complex in PEG400 G10 Solid 3 5 mg/kg 1 mL Zoledronic, suspension equivalent nicotinamide, in PEG400 and water complex G11 Solid 3 5 mg/kg 1 mL Zoledronic acid, suspension equivalent sodium zoledronic in PEG400 salt, and water complex G12 Solid 3 5 mg/kg 1 mL Zoledronic, suspension equivalent L-lysine, and in PEG400 water complex G13 Solid 3 5 mg/kg 1 mL Zoledronic, suspension equivalent DL-lysine, and in PEG400 water complex

(132) Group IV (Oral Gavage): Group Designations and Oral Doses are Listed Below:

(133) TABLE-US-00004 Excess coformer Group # of Dose Excess amount # IV Compound rats Dose volume/kg coformer mg/kg G14 Zoledronic and 3 5 mg/kg 1 mL Glycine 45 glycine complex, equivalent solid suspension in PEG400 G15 Zoledronic and 3 5 mg/kg 1 mL Glycine 25 glycine complex, equivalent solid suspension in PEG400 G16 Zoledronic and 3 5 mg/kg 1 mL Glycine 5 glycine complex, equivalent solid suspension in PEG400 G17 Zoledronic, DL- 3 5 mg/kg 1 mL DL-lysine 39.32 lysine, and water equivalent monohydrate complex, solid suspension in PEG400 G18 Zoledronic, DL- 3 5 mg/kg 1 mL DL-lysine 28.08 lysine, and water equivalent monohydrate complex, solid suspension in PEG400 G19 Zoledronic, DL- 3 5 mg/kg 1 mL DL-lysine 5.62 lysine, and water equivalent monohydrate complex, solid suspension in PEG400 G20 Zoledronic, DL- 3 5 mg/kg 1 mL n/a n/a lysine, and water equivalent complex, solid suspension in PEG400

(134) Rat Blood Sample Collection, Handling and Analysis:

(135) Blood (approx. 300 ?L per sample) samples were withdrawn from each of 3 animals in Group I (IV administration) at eight (8) time points: 5 min, 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, and 24 hrs, after initial administration of zoledronic acid or its complexes, into EDTA plasma tubes. Plasma was collected after centrifugation at 13,000 rpm for 5 min at 4? C. and immediately frozen and stored at ?60 to ?80? C. till analysis.

(136) Samples were thawed on the day of analysis and the amount of zoledronic acid in the samples was quantified by analyzed by LC/MS/MS method.

(137) Details of Group 2 Dog Dosing:

(138) Prior to dosing, all dogs received a 20 mL dose of citric acid (24 mg/mL in water) to lower the pH of their stomach. After dosing capsules or IV, all dogs received additional 6.25 mL citric acid solution (24 mg/mL in water) as a rinse.

(139) Group A, (IV Administration). Group Members, Designated IV Doses are Listed Below:

(140) TABLE-US-00005 Group # of Dose # A Designation fasted Dogs Dose* volume Leg 1 Zoledronic 5 0.05 mg/kg 1 mL/kg Acid
IV comparator group, was conducted to calculate MAT (mean absorption time) and ka (absorption rate constant) for the oral groups.

(141) Group B (Oral Administration): Group Designations and Oral Doses are Listed Below:

(142) TABLE-US-00006 Dose of Dosing compound in # of fasted Solution Group Dosing the gelatin Dogs Conc. # B Compound Route capsules (9-12 kg) mg/mL Leg 2 Zoledronic oral 5 mg/kg 5 n/a acid equivalent Leg 3 Zoledronic oral 5 mg/kg 5 n/a and glycine equivalent complex Leg 4 Zoledronic, oral 5 mg/kg 5 n/a DL-lysine, equivalent and water complex Leg 5 Zoledronic, oral 5 mg/kg 5 n/a L-lysine, equivalent and water complex Leg 6 Zoledronic, oral 5 mg/kg 5 n/a DL-lysine, equivalent and water complex

(143) Group C (Oral Administration): Group Designations and Oral Doses are Listed Below:

(144) TABLE-US-00007 # of Dose of fasted compound in Excess Group Dogs Dosing the gelatin Excess coformer # C Compound (9-12 kg) Route capsules coformer amount Leg 7 Zoledronic acid 5 oral 56.0 mg; enteric n/a n/a monohydrate coated capsules Leg 8 Zoledronic and 5 oral 67.0 mg; enteric n/a n/a glycine complex coated capsules Leg 9 Zoledronic, 5 oral 87.7 mg DL-lysine 294.8 mg DL-lysine, and monohydrate water complex Leg 10 Zoledronic, 5 oral 87.7 mg; enteric DL-lysine 294.8 mg DL-lysine, and coated capsules monohydrate water complex Leg 11 Zoledronic, 5 oral 84.2 mg DL-lysine 294.8 mg DL-lysine, and monohydrate water complex Leg 12 Zoledronic, 5 oral 87.7 mg; enteric n/a n/a DL-lysine, and coated capsules water complex

(145) Group D, (15 Min IV Infusion): Group Members, Designated IV Doses are Listed Below:

(146) TABLE-US-00008 # of Dosing Group fasted Dogs solution # D Designation (9-12 kg) Dose* concentration Leg 13 Zoledronic 5 0.183 0.1 mg/mL Acid mg/kg IV

(147) Group E, (Oral Administration): Group Members, Designated IV Doses are Listed Below:

(148) TABLE-US-00009 # of Dose of fasted compound in Excess Group Dogs Dosing the gelatin Excess coformer # E Compound (9-12 kg) Route capsules coformer amount Leg 14 Zoledronic, 2.1 oral 35.4 mg DL-lysine 123.8 mg DL-lysine, and monohydrate water complex Leg 15 Zoledronic and 5 oral 67.0 mg DL-lysine 294.8 mg glycine complex monohydrate Leg 16 Zoledronic, 5 oral 87.7 mg DL-lysine 294.8 mg L-lysine, and monohydrate water complex Leg 17 Zoledronic, 2.1 oral 35.4 mg DL-lysine 294.8 mg DL-lysine, and monohydrate water complex

(149) Group F, (15 Min IV Infusion): Group Members, Designated IV Doses are Listed Below:

(150) TABLE-US-00010 # of Dosing Group fasted Dogs solution # F Designation (9-12 kg) Dose* concentration Leg 18 Zoledronic 5 0.12 0.1 mg/mL Acid mg/kg IV infusion

(151) After initial administration of zoledronic acid or its complexes, blood (approx. 2.5 mL per sample) was withdrawn from each of 5 animals in Group A (IV administration) at 15 time points: Pre-dose (0), 2, 5, 10, 15, 30, 45 min, 1, 1.5, 2, 4, 6, 8, 24 and 48 hrs and at 13 time points for Group B (oral administration): Pre-dose (0), 5, 10, 15, 30, 45 min, 1, 1.5, 2, 4, 6, 8, and 24 hrs. Blood samples were placed without the use of an anticoagulant and allowed to sit at room temperature for approximately 30 minutes. Samples were then centrifuged at a temperature of 4? C., at a speed of 13,000 rpm, for 5 minutes. Serum was collected and split into two aliquots and stored frozen (?80? C.) till analysis. Samples were thawed on the day of analysis and processed using analytical procedures for zoledronic acid containing an LC/MS/MS analysis method.

(152) Animal PK Studies Results

(153) Rat Study:

(154) The results of the first rat study are summarized in Table 1; the concentrations (ng/mL) of zoledronic acid in the plasma samples are the average values of the analytical results of 3 rats. In addition, the PK profiles of the IV, oral and ID groups are shown in FIG. 27. The profiles of oral and ID groups are shown in FIGS. 28 and 29. It suggests that some zoledronic acid complexes have improved oral bioavailability compared with that of the parent zoledronic acid. The complexes with improved bioavailability were further tested in a second rat PK study in which excess coformers were added to the zoledronic acid complexes and then administered to rats by oral gavage. The results of this second study are summarized in Table 2 and their PK profiles are shown in FIGS. 30, 31 and 32. These figures show improved bioavailabilities of several zoledronic acid complexes with excess coformers.

(155) Dog Study:

(156) The results of the first dog study are summarized in Table 3. The concentrations (ng/mL) of zoledronic acid are the average values of the analytical results of 5 dogs. The PK profiles of the IV and oral groups are shown in FIGS. 33 and 34 which represent the first four hours of the 48 hr PK profile. These results and FIG. 34 suggest that most if not all zoledronic acid complexes have achieved improved oral bioavailability compared to that of the parent zoledronic acid delivered orally.

(157) The results of the second dog study are summarized in Table 4; the concentrations (ng/mL) of zoledronic acid shown are the average values of the analytical results of 5 dogs. The PK profiles of the IV and oral groups are shown in FIGS. 35 and 36. FIG. 36 represents the first 6 hours of the 24 hour PK profile. These results and FIG. 35 suggest that most if not all zoledronic acid complexes have achieved improved oral bioavailability compared with that of the parent zoledronic acid delivered orally. Specifically, there was a significant improvement in zoledronic acid bioavailability for the novel zoledronic acid complexes with excess amino acid coformer (Leg 11, FIG. 37) compared to that of the parent drug. The results have also shown that there was improvement in the bioavailability of the enterically coated capsules compared with the non-enterically coated capsules (FIG. 37, Legs 7 and 2, Legs 8 and 3, Legs 12 and 4), but surprisingly the bioavailability was significantly altered when excess amino acid coformer was added to form a physical mixture to the enterically coated capsules (FIG. 37, Legs 9 and 10). The reason behind it is not fully understood.

(158) The results have shown that there is a slight increase in the oral bioavailability of zoledronic acid from the enteric coated capsules filled with neat (i.e. with no excess coformer) zoledronic acid amino acid complex. Therefore, it is expected that the excess coformer with the novel zoledronic acid complexes would also lead to increased bioavailability when delivered in enterically coated capsules. Surprisingly, when excess coformer was added to the zoledronic acid, the bioavailability of the enterically coated capsules was lower than that of the non-enterically coated capsules. This suggests that a physical powder mixture of the molecular complex and excess coformer might decrease the bioavailability when delivered to the duodenum.

(159) The analytical results of the third dog study are shown in Table 5, which contains averaged data from five dogs. The PK profiles of the IV and oral groups are shown in FIGS. 38 and 39. FIG. 39 represents the first 4 hours of the 24 hour PK profile.

(160) TABLE-US-00011 TABLE 1 Rat plasma concentrations for pure zoledronic acid and zoledronic acid complexes via different routes of delivery. Average plasma concentration Dosing Time of 3 Rats Group # Complex Route Vehicle (hour) (ng/mL) G1 Zoledronic acid IV Water 0.083333 3254.05 0.25 1950.62 0.5 1128.75 1 404.28 2 112.68 4 30.46 8 10.66 24 2.98 G2 Zoledronic acid PO PEG 0.25 330.06 400 0.5 267.45 1 138.91 2 47.72 4 11.78 8 2.00 24 0.00 G3 Zoledronic and PO PEG 0.25 648.01 glycine complex 400 0.5 435.38 1 200.88 4 12.78 8 1.46 24 0.00 G4 Zoledronic, PO PEG 0.25 434.61 nicotinamide, 400 0.5 304.94 and water 1 122.35 complex 4 7.68 8 1.82 24 0.00 G5 Zoledronic acid, PO PEG 0.25 278.47 sodium 400 0.5 280.20 zoledronic salt, 1 171.59 and water 4 13.42 complex 8 1.78 24 0.00 G6 Zoledronic, PO PEG 0.25 258.43 L-lysine, 400 0.5 249.82 and water 1 184.95 complex 4 28.70 8 3.27 24 0.00 G7 Zoledronic, PO PEG 0.25 494.31 DL-lysine, 400 0.5 379.27 and water 1 213.48 complex 4 14.57 8 3.42 24 0.00 G8 Zoledronic acid ID PEG 0.25 145.67 400 0.5 109.92 1 47.36 2 12.94 4 3.85 8 0.97 24 0.00 G9 Zoledronic ID PEG 0.25 86.51 and glycine 400 1 33.93 complex 4 1.75 8 1.55 24 0.00 G10 Zoledronic, ID PEG 0.25 69.71 nicotinamide, 400 1 21.03 and water 4 0.86 complex 8 0.00 24 0.00 G11 Zoledronic acid, ID PEG 0.25 39.99 sodium 400 1 18.50 zoledronic salt, 4 0.71 and water 8 0.00 complex 24 0.00 G12 Zoledronic, ID PEG 0.25 91.21 L-lysine, and 400 1 26.53 water complex 4 0.74 8 0.00 24 0.00 G13 Zoledronic, ID PEG 0.25 98.25 DL-lysine, 400 1 34.61 and water 4 2.65 complex 8 1.02 24 0.80

(161) TABLE-US-00012 TABLE 2 Rat plasma concentrations for zoledronic acid complexes with excess coformers, delivered by oral gavage Average plasma concentration Dosing Time of 3 Rats Group # Complex Route Vehicle (hour) (ng/mL) G14 Zoledronic PO PEG 0.0333333 14.61 and glycine 400 0.0833333 206.26 complex and 0.1666667 340.19 45 mg/kg 0.25 375.99 glycine 0.5 321.36 1 197.01 4 17.35 24 0.00 G15 Zoledronic PO PEG 0.0333333 24.48 and glycine 400 0.0833333 281.08 complex and 0.1666667 502.20 25 mg/kg 0.25 516.58 glycine 0.5 430.10 1 203.48 2 73.27 4 14.70 24 0.00 G16 Zoledronic PO PEG 0.0333333 60.03 and glycine 400 0.0833333 365.23 complex and 0.1666667 563.83 5 mg/kg 0.25 625.05 glycine 0.5 464.34 1 209.65 2 74.28 4 12.17 24 0.00 G17 Zoledronic, PO PEG 0.0333333 168.19 DL-lysine, 400 0.0833333 263.28 and water 0.1666667 440.26 complex and 0.25 456.18 39.32 mg/kg 0.5 385.57 DL-lysine 1 209.26 monohydrate 2 85.65 4 14.58 24 0.71 G18 Zoledronic, PO PEG 0.0333333 219.95 DL-lysine, 400 0.0833333 427.02 and water 0.1666667 729.65 complex and 0.25 777.54 28.08 mg/kg 0.5 632.07 DL-lysine 1 300.86 monohydrate 2 100.59 4 21.14 24 0.00 G19 Zoledronic, PO PEG 0.0333333 53.78 DL-lysine, 400 0.0833333 394.73 and water 0.1666667 649.52 complex and 0.25 669.20 5.62 mg/kg 0.5 530.00 DL-lysine 1 265.20 monohydrate 2 73.31 4 15.41 24 0.00 G20 Zoledronic, PO PEG 0.0333333 103.13 DL-lysine, 400 0.0833333 352.18 and water 0.1666667 475.33 complex 0.25 505.48 0.5 431.41 1 224.56 2 69.95 4 14.96 24 0.00

(162) TABLE-US-00013 TABLE 3 Dog serum concentrations for pure zoledronic acid and zoledronic acid complexes via different routes of delivery (IV and oral). Average serum concentration Leg Dosing Time of 5 dogs # Complex Route Vehicle (hour) (ng/mL) 1 0.05 mg/kg IV Saline 0 0.00 Zoledronic solution 0.0333 413.44 acid 0.0833 311.68 0.1667 228.97 0.25 178.63 0.5 111.11 0.75 75.91 1 56.07 1.5 30.35 2 17.61 4 4.29 8 1.13 24 0.00 48 0.00 2 56.0 mg PO n/a 0 0.00 Zoledronic 0.0833 0.00 acid 0.1667 0.00 monohydrate 0.25 0.31 capsule 0.5 110.73 0.75 97.98 1 103.60 1.5 80.57 2 75.16 4 17.86 8 2.71 24 0.56 3 67.0 mg PO n/a 0 0.00 Zoledronic 0.0833 2.45 and glycine 0.1667 12.75 complex 0.25 37.07 capsule 0.5 149.20 0.75 206.14 1 254.20 1.5 176.11 2 109.25 4 20.43 8 3.96 24 0.97 4 87.7 mg PO n/a 0 0.00 Zoledronic, 0.0833 3.11 DL-lysine, 0.1667 6.49 and water 0.25 22.55 complex 0.5 68.28 capsule 0.75 162.72 1 206.14 1.5 149.92 2 105.81 4 25.51 8 4.22 24 0.56 5 87.7 mg PO n/a 0 0.00 Zoledronic, 0.0833 0.00 L-lysine, 0.1667 3.13 and water 0.25 10.06 complex 0.5 188.52 capsule 0.75 345.28 1 318.97 1.5 180.77 2 109.23 4 23.11 8 9.73 24 1.93 6 84.2 mg PO n/a 0 0.00 Zoledronic, 0.0833 0.00 DL-lysine, 0.1667 0.20 and water 0.25 1.92 complex 0.5 106.47 capsule 0.75 120.13 1 108.13 1.5 90.45 2 54.48 4 18.14 8 4.35 24 1.06

(163) TABLE-US-00014 TABLE 4 Dog serum concentrations for pure zoledronic acid and zoledronic acid complexes via different routes of delivery IV and oral; enteric and non-enteric coated gelatin capsules. Average serum concentration Leg Dosing Time of 5 dogs # Complex Route Vehicle (hour) (ng/mL) 7 56.0 mg PO n/a 0 0.00 Zoledronic 0.1667 0.00 acid 0.25 0.00 monohydrate 0.5 0.00 enteric 0.75 0.00 coated capsule 1 9.84 1.5 86.13 2 109.37 4 107.64 6 14.15 8 4.57 24 0.50 8 67.0 mg PO n/a 0 0.00 Zoledronic 0.1667 0.00 and glycine 0.25 0.00 complex enteric 0.5 0.00 coated capsule 0.75 0.00 1 4.42 1.5 208.97 2 274.53 4 101.20 6 16.71 8 7.14 24 2.17 9 87.7 mg PO n/a 0 0.00 Zoledronic, 0.0833 13.31 DL-lysine, 0.1667 39.76 and water 0.25 120.41 complex with 0.5 364.68 294.8 mg 0.75 487.59 DL-lysine 1 499.60 monohydrate 1.5 362.16 capsule 2 254.72 4 52.22 6 16.61 8 8.93 24 2.92 10 87.7 mg PO n/a 0 0.00 Zoledronic, 0.1667 0.00 DL-lysine, 0.25 0.00 and water 0.5 0.00 complex with 0.75 3.71 294.8 mg 1 51.32 DL-lysine 1.5 403.15 monohydrate 2 309.08 enteric 4 44.83 coated capsule 6 13.15 8 7.09 24 2.66 11 84.2 mg PO n/a 0 0.22 Zoledronic, 0.1667 167.03 DL-lysine, 0.25 533.96 and water 0.5 878.63 complex with 0.75 838.82 294.8 mg 1 633.50 DL-lysine 1.5 326.63 monohydrate 2 185.44 capsule 4 46.86 6 20.26 8 11.49 24 5.95 12 87.7 mg PO n/a 0 0.57 Zoledronic, 0.1667 0.60 DL-lysine, 0.25 0.59 and water 0.5 0.61 complex enteric 0.75 0.40 coated capsule 1 132.15 1.5 566.18 2 402.12 4 65.35 6 21.02 8 12.18 24 4.33 13 0.183 mg/kg IV Saline 0 0.64 Zoledronic solution 0.0833 476.79 acid 0.1667 755.68 0.25 1057.75 0.3333 745.67 0.4167 629.22 0.5 522.78 0.75 342.58 1 245.36 1.25 182.59 1.5 139.77 2 80.87 4 23.40 8 8.78 24 3.84

(164) TABLE-US-00015 TABLE 5 Dog serum concentrations for pure zoledronic acid and zoledronic acid complexes via different routes of delivery (IV and oral). Average serum concentration Leg Dosing Time of 5 dogs # Complex Route Vehicle (hour) (ng/mL) 14 35.4 mg PO n/a 0 0.00 Zoledronic, 0.0833 0.00 DL-lysine, 0.1667 0.72 and water 0.25 11.40 complex, with 0.5 78.95 123.8 mg 0.75 126.46 DL-lysine 1 137.38 monohydrate 1.5 64.73 gelatin 2 33.38 capsule 4 6.14 8 0.89 24 0.00 15 67.0 mg PO n/a 0 0.00 Zoledronic 0.0833 2.58 and glycine 0.1667 26.13 complex, with 0.25 55.58 294.8 mg 0.5 225.41 DL-lysine 0.75 234.95 monohydrate 1 221.91 gelatin 1.5 204.90 capsule 2 117.22 4 17.79 8 3.34 24 0.77 16 87.7 mg PO n/a 0 0.00 Zoledronic, 0.0833 3.26 L-lysine, 0.1667 17.21 and water 0.25 213.77 complex, with 0.5 504.17 294.8 mg 0.75 436.00 DL-lysine 1 325.21 monohydrate 1.5 171.42 gelatin 2 100.81 capsule 4 23.38 8 4.65 24 1.48 17 35.4 mg PO n/a 0 0.00 Zoledronic, 0.0833 0.00 DL-lysine, 0.1667 13.47 and water 0.25 50.04 complex, with 0.5 146.68 294.8 mg 0.75 137.24 DL-lysine 1 116.38 monohydrate 1.5 66.70 gelatin 2 44.94 capsule 4 8.87 8 1.58 24 0.21 18 0.12 mg/kg IV Saline 0 0.00 Zoledronic solution 0.0833 309.13 acid 0.1667 524.58 0.25 717.15 0.3333 501.70 0.4167 392.35 0.5 322.84 0.75 201.78 1 132.86 1.25 93.22 1.5 69.06 2 38.38 4 9.14 8 3.24 24 1.21

(165) TABLE-US-00016 TABLE 6 Aqueous solubility of zoledronic acid (ZA) and novel zoledronic acid complexes at room temperature. Compound Conc. mg/mL mMol/L (complex) ZA monohydrate 1.57 5.41 ZA:Glycine 11.89 34.25 ZA:L-Lysine dihydrate 8.22 18.09 ZA:DL-Lysine dihydrate 6.85 15.08 ZA:DL-Lysine monohydrate 13.9 31.86