CONJUGATED BISPHOSPHONATES FOR THE DIAGNOSIS AND THERAPY OF BONE DISEASES

Abstract

The invention relates to a compound V for complexing metallic isotopes, comprising a chelator X and one or more targeting vectors conjugated with the chelator X, said targeting vectors having the structure -L.sub.1-R.sub.1-L.sub.2-R.sub.2-L.sub.3-R.sub.3, wherein R.sub.3 contains a bisphosphonate. A pharmaceutical consists of the compound V and a metallic isotope which is complexed with compound V.

##STR00001##

Claims

1. Compound V for complexing metallic isotopes, comprising a chelator X and one or more targeting vectors conjugated with the chelator X, said targeting vectors having the structure -L.sub.1-R.sub.1-L.sub.2-R.sub.2-L.sub.3-R.sub.3, wherein L.sub.1 is selected from the group consisting of: amide, phosphinate, alkyl, triazole, thiourea, ethylene, maleimide, —(CH.sub.2).sub.k— and —(CH.sub.2CH.sub.2O).sub.k—, where k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L.sub.2 is selected from —(CH.sub.2).sub.m— and —(CH.sub.2CH.sub.2O).sub.m—, where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and L.sub.3 is selected from —(CH.sub.2).sub.n— and —(CH.sub.2CH.sub.2O).sub.n—, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, characterized in that ##STR00057## R.sub.2 is selected from the group consisting of: a substituent group of a: furan, azole, oxazole, thiophen, thiazole, azine, oxazine, thiazine, naphthalene, quinoline, chromene, or thiochromene; and ##STR00058##

2. Compound V according to claim 1, characterized in that the chelator X is selected from the group consisting of: EDTA (ethylenediamine-tetraacetate), EDTMP (diethylenetriamine penta(methylene phosphonic acid)), DTPA (diethylenetriamine pentaacetic acid) and its derivatives, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTAGA (dodeca-1-glutaric acid-1,4,7,10-tetraamine-triacetic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) and other DOTA derivatives, TRITA (trideca-1,4,7,10-tetraamine tetraacetic acid), TETA (tetradeca-1,4,8,11-tetraamine-tetraacetic acid) and its derivatives, NOTA (nona-1,4,7-triamine-triacetic acid) and its derivatives, NOTAGA (1,4,7-triazacyclononane, 1-glutaric acid,4,7-acetic acid), NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)hypophosphorous acid]-7-[methylene(2-carboxyethyl)hypophosphorous acid]) and its derivatives, PEPA (pentadeca-1,4,7,10,13-pentaamine pentaacetic acid) and its derivatives, HEHA (hexadeca-1,4,7,10,13,16-hexaamine hexaacetic acid) and its derivatives, HBED (hydroxybenzyl-ethylenediamine) and its derivatives, DEDPA and its derivatives, H.sub.2DEDPA (1,2-[{6-(carboxylate-)pyridine-2-yl}methylamine]ethane), DFO (deferoxamine) and its derivatives, Deferiprone, CP256 (4-acetylamino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-ylmethyl)-carbamoyl]-ethyl}-heptane diacid bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridine-2-ylmethyl)-amide]) and its derivatives, YM103; TRAP (triazacyclononane-hypophosphorous acid) and its derivatives, TEAP (tetraazycyclodecane-hypophosphorous acid) and its derivatives, AAZTA (6-amino-6-methylperhydro-1,4-diazepine-N,N,N′,N′-tetraacetic acid) and derivatives such as DATA; SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) and salts thereof.

3. Compound V according to claim 1 characterized in that the compound V has a structure according to Formula I ##STR00059##

4. Compound V according to claim 1, characterized in that the compound has a structure according to Formula II ##STR00060##

5. A pharmaceutical composition comprising the compound V according to claim 1, and a metallic isotope M complexed with the compound V.

6. The pharmaceutical composition according to claim 5, characterized in that the metallic isotope M is selected from the group consisting of: .sup.44Sc, .sup.47Sc, .sup.55Co, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.89Zr, .sup.86Y, .sup.90Y, .sup.90Nb, .sup.99mTc, .sup.111In, 35Sm, .sup.159Gd, .sup.149Tb, .sup.160Tb, .sup.161Tb, .sup.165Er, .sup.166Dy, .sup.166Ho, .sup.175Yb, .sup.177Lu, .sup.186Re, .sup.168Re, .sup.213Bi and .sup.225Ac.

7. A method for producing a pharmaceutical composition according to claim 5, comprising the following steps: (a) providing a solution S containing the compound V; (b) providing a metallic isotope M, such as .sup.68Ga(III); and (c) ligating the metallic isotope M with the compound V to form a complex MV of the metallic isotope M with the compound V in a solution F.

8. The method according to claim 7, characterized in that, in step (b), the metallic isotope M is provided in a solution.

9. The method according to claim 7, characterized in that, in step (b), a radionuclide generator with a mother nuclide and a metallic isotope M formed via decay of the mother nuclide is provided, and, in step (c), the metallic isotope M is separated from the mother nuclide with the solution S.

10.-11. (canceled)

12. A method of medical treatment or medical imaging comprising administration of a pharmaceutical composition according to claim 5 to a subject.

13. (canceled)

14. The method according to claim 12 wherein the treatment is treatment of a bone disease.

15. The method according to claim 5 wherein the treatment is treatment of a bone tumors.

16. The method according to claim 15, wherein the treatment is treatment of a diseases of unmanifested bone metastases.

17. The method according to claim 16, wherein the treatment comprises accumulation of the pharmaceutical composition in a tumor cell in order to inhibit farnesyl pyrophosphate synthesis (FPPS).

18. The method according to claim 12, wherein the medical imaging comprises positron emission tomography, single photon emission computer tomography, magnetic resonance tomography (nuclear magnetic resonance tomography) or optical imaging.

19. The pharmaceutical composition according to claim 5 further comprising an artificial bone substance, a bone cement, or a bone implants.

20.-23. (canceled)

Description

EXAMPLES

[0094] The following examples illustrate embodiments and elements of the present invention, but do not limit the subject matter of the invention to the embodiments and elements illustrated in the examples. If DOTA or its conversion or use is described in the examples, the chelator DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) can alternatively be used instead of the chelator DOTA.

Example 1: Synthesis of the Compound DOTA.SUP.ZOL

[0095] Instruments and Chemicals Used

[0096] ESI-MS: Agilent Technologies 6130 Quadrupole LC/MS Spectrometer or Finnigan MAT-95 Spectrometer. NMR spectrometer: Bruker 600 (Bruker BioSpin AG, Fallanden, Switzerland). DC or Radio DC: Merck Silica on aluminum foil, eluent: 0.1 M citrate pH=4 or acetylacetone:acetone:concentrated HCl (10:10:1). Detector: Canberra Packard Instant Imager. Radio HPLC: Waters-system 1525, column: MultoKrom (CS chromatography) RP18, 5μ, 250×4 mm. Eluent: A (10 mM tetrabutylammonium citrate pH=4.5), B (acetonitrile). Gradient 1 cm.sup.3/min (1 mL/min) 70(A):30(B) at 20(A):80(B). Detector: Berthold Technologies (Dresden). .sup.68Ga/.sup.68Ge generator: Eckert & Ziegler AG (Berlin). .sup.177Lu(III) in 0.05 M HCl: itm AG (Munich). μPET: Siemens Focus 120. PET data were processed with Pmod software and OSEM 2D reconstruction. The radioactivity in tissue samples was decay-corrected with an auto gamma counter (WIZARD2, Perkin Elmer, Germany).

##STR00051##

[0097] The primary N-w-acetylhistamine as a starting compound is prepared from histamine with the aid of acetic anhydride. The compound is commercially available (Sigma-Aldrich), but may also be synthesized according to known literature specification from van der Merwe et al., Hoppe-Seyler's Zeitschrift fir Physiologische Chemie [Journal of Physiological Chemistry], 177, 1928, 305. DOTA-NHS ester is likewise commercially available, but may also be synthesized according to the following literature specification from Rasaneh et al., Nucl. Med. Biol., 36, 2009, 363-369.

[0098] Instead of the chelator DOTA, the chelator DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) can alternatively be used that likewise is commercially available or may be synthesized accordingly.

1-(benzyl acetate) 4-(ethylacetamide)-imidazole (1)

[0099] Nω-acetylhistamine 1 g (6.53 mmol) is dissolved in 50 mL dry DMF and 4.4 g (13 mmol) cesium carbonate is added. The solution is stirred in an argon atmosphere and ice cooling. 2.2 g (13 mmol) benzyl bromoacetate dissolved in 50 mL dry DMF is slowly added by drops to the suspension. The mixture is stirred for 12 hours before activated carbon is added. The solids are subsequently filtered out, and the solvent is removed in a vacuum. The raw product is recrystallized out of acetyl acetate, and 1.14 g (58%) of 1-(benzyl acetate) 4-(ethylacetamide)-imidazole (1) is obtained as a faintly yellow solid.

[0100] .sup.1H-NMR (CDCl.sub.3, 300 MHz): δ 1.97 (s, 3H, CH.sub.3—CO), 2.76 (t, JH=6.3 Hz, 2H, CH.sub.2—CH.sub.2), 3.53 (q, JH=6.0 Hz, 2H, CH.sub.2—CH.sub.2), 4.70 (s, 2H, N—CH.sub.2—CO), 5.22 (s, 2H, Bn-CH.sub.2—CO), 6.53 (bs, 1H, NH), 6.75 (d, JH=1.3 Hz, Imidazole-H), 7.39 (m, 5H, Benzyl), 7.44 (d, JH=1.3 Hz, .sup.1H, Imidazole-H).). FD-MS(+): cald 301.14 obsd 302.3 (M+H.sup.+), 603.2 (2M+H.sup.+).

1-(1-hydroxy-ethane-1,1-bis(phosphonic acid))4-(ethylamine)-imidazole (3)

[0101] 300 mg (1 mmol) of (1) is dissolved in 20 mL dry methanol and is added Pd/C (10% w). The suspension is stirred for 12 hours under hydrogen atmosphere (0.5 MPa (5 bar)). After the removal of the solvent and the solids, the unprotected acid (2) (208 mg, 98%) was directly converted further. 1 mL methanesulfonic acid and 164 mg (2 eq.) phosphorous acid were added while stirring. The mixture was heated to 75° C., and 300 mg (2.2 eq.) phosphorus trichloride was slowly added by drops in an inert gas atmosphere. After 12 hours, the reaction mixture was cooled to room temperature and added to 2 mL ice water. The solution was subsequently heated for 24 hours with recycling. After the addition of activated carbon, all solids were filtered out and concentrated sodium hydroxide was added by drops to the solution until a white solid began to precipitate. The suspension was stored for 24 hours at 4° C. to complete the precipitation. In the final step, the obtained solid was recrystallized from boiling water, and 88.6 mg (28%) of the aforementioned hydroxybisphosphonate (3) was obtained.

[0102] .sup.1H-NMR (D.sub.2O/NaOD, 300 MHz): δ 2.46 (m, 2H, CH.sub.2—CH.sub.2), 2.66 (m, 2H, CH.sub.2—CH.sub.2), 4.28 (m, 2H, N—CH.sub.2 phosphonate), 6.89 (s, 1H, Imidazole-H), 7.54 (s, 1H, Imidazole-H). .sup.31P-NMR (D.sub.2O/NaOD, 162.05 MHz): δ 14.4. ESI-MS(+): cald 315.04 obsd 316.05 (M+H.sup.+), 338.04 (M+Na.sup.+).

[0103] DOTA.sup.ZOL

[0104] 15.75 mg (0.05 mmol) (3) is suspended in 1 mL water, and triethylamine (TEA) is added until all solids have dissolved. 38 mg (0.05 mmol) DOTA-NHS ester dissolved in 0.5 mL water is slowly added by drops to the bisphosphonate solution. The reaction mixture is stirred at 50° C. for 24 hours. The pH value is regularly monitored and kept between 8 and 9 via addition of TEA. The raw product is separated from the educts via preparative HPLC (Phenomenex Synergy Hydro-RP 80, 10μ, 250×30 mm, eluent: H.sub.2O+0.1% TFA). In a second step, the raw product is additionally purified via solid phase extraction (NH.sub.2 phase, Merck LiChroprep NH.sub.2). After washing the solid phase with water/methanol/water, the product is eluted from the solid phase via a solution of H.sub.2O+2% TFA. After lyophilizing, 5.6 mg (15.7%) of a white solid is obtained.

[0105] .sup.1H-NMR (D.sub.2O/NaOD, 300 MHz): δ 2.42 (m, 2H, CH.sub.2—CH.sub.2), 2.61 (m, 2H, CH.sub.2—CH.sub.2), 2.9-3.5 (b, 16H, cyclen-CH.sub.2), 3.75 (bs, 8H, —CH.sub.2—CO), 4.55 (m, 2H, N—CH.sub.2 phosphonate), 7.28 (s, 1H, imidazole-H), 8.54 (s, 1H, imidazole-H). .sup.31P-NMR (D.sub.2O/NaOD, 162.05 MHz): δ 14.3. ESI-MS(+): cald 701.2 obsd 702.5 (M+H.sup.+), 351.1 (M+2H.sup.+).

[0106] Alternatively, the chelator DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) can be used instead of the chelator DOTA.

Example 2: Synthesis of [.SUP.68.Ga]DOTA.SUP.ZOL

[0107] 25 nmol DOTA.sup.ZOL are dissolved in 500 μL sodium acetate buffer (0.5 M, pH=4) and added to 400 μL 68Ga(III) solution. The mixture is heated for 15 min at 98° C. The reaction solution is subsequently filtered in a sterile manner. The radiochemical purity is determined—via thin-layer chromatography and HPLC—to be greater than/equal to 95%.

Example 3: Synthesis of [.SUP.177.Lu]DOTA.SUP.Z.OL

[0108] 10 nmol DOTA.sup.ZOL per 1 GBq .sup.177Lu(III) is dissolved in 1 mL sodium acetate buffer (0.1 M, pH=5.0) and added to .sup.177Lu(III) solution. The mixture is heated for 30 min at 98° C. The reaction solution is subsequently filtered in a sterile manner. The radiochemical purity is determined—via thin-layer chromatography and HPLC—to be greater than/equal to 98%.

Example 4: NHS Coupling of DOTAGA in Slightly Basic Aqueous Solution

[0109] ##STR00052##

Example 5: NCS Coupling of an HEHA Derivative in Slightly Basic Aqueous Solution

[0110] ##STR00053##

Example 6: Squaric Acid Coupling of DO3A Derivative in Slightly Basic Aqueous Solution

[0111] ##STR00054##

Example 7: Mannich Reaction of DO3A in Acid Solution

[0112] ##STR00055##

Example 8: In Vivo Experiments in Rats

[0113] Under isoflurane anesthesia, 15-18 MBq of the .sup.68Ga-marked compound or of the .sup.177Lu-marked compounds, diluted in isotonic saline solution, was applied into the tail vein of healthy Wistar rats (N=5) having a weight between 140 and 220 g. The rats were killed 60 min after injection, organ samples were removed and weighed, and the accumulation of the marked bisphosphonate in the tissue was determined in SUV (standardized uptake value) according to the formula: SUV=(activity per g of tissue)/(injected activity) x body weight.

[0114] The known α-Hydroxy-BP BPAPD and a pamidronate-DOTA conjugate (DOTA.sup.PAM) were chosen as comparison substances. These compounds represent the compounds known in the prior art that are mentioned in the preceding, which have obviously lost their affine amino function due to the derivation with the bifunctional chelator.

##STR00056##

[0115] Measurement results regarding organ distribution of the .sup.68Ga-marked bisphosphonates described in the preceding are summarized in the following Tables 1 and 2.

TABLE-US-00002 TABLE 1 Ex vivo biodistribution of [.sup.68Ga]BPAPD, [.sup.68Ga]DOTA.sup.PAM and [.sup.68Ga]DOTA.sup.ZOL in Wistar rats after 60 min. SUV Organ [.sup.68Ga]BPAPD [.sup.68Ga]DOTA.sup.PAM [.sup.68Ga]DOTA.sup.ZOL Lung 0.43 (0.08) 0.53 (0.16) 0.45 (0.11).sup.  Liver 0.37 (0.11) 0.43 (0.04) 0.28 (0.03).sup.‡ Spleen 0.23 (0.08) 0.31 (0.04) 0.17 (0.02).sup.‡ Kidneys 0.56 (0.08) 0.48 (0.06) 0.53 (0.04).sup.  Muscle 0.17 (0.02) 0.09 (0.02) 0.08 (0.02).sup.† Heart 0.32 (0.09) 0.23 (0.02) .sup. 0.14 (0.04).sup.†‡ Blood 0.86 (0.21) 0.60 (0.03) 0.47 (0.19).sup.† Intestine 0.26 (0.05) 0.28 (0.12) 0.14 (0.08).sup.  Femur 3.21 (0.29) 4.53 (0.17) .sup. 5.40 (0.62).sup.†‡ Data presented in SUV (standard deviation) from five animals. .sup.†P < 0.05 vs. [.sup.68Ga]BPAPD; .sup.‡P < 0.05 vs. [.sup.68Ga]DOTA.sup.PAM.

TABLE-US-00003 TABLE 2 Bone/organ ratios of [.sup.68Ga]BPAPD, [.sup.68Ga]DOTA.sup.PAM and [.sup.68Ga]DOTA.sup.ZOL in Wistar rats after 60 min. Bone-to-organ ratios [.sup.68Ga]BPAPD [.sup.68Ga]DOTA.sup.PAM [.sup.68Ga]DOTA.sup.ZOL Lung 7.47 8.61 12.06 Liver 8.68 10.02 19.21 Spleen 13.96 14.51 31.71 Kidneys 5.73 9.44 10.22 Muscle 18.88 47.96 65.96 Heart 10.03 19.49 37.97 Blood 3.73 7.61 11.47 Intestine 12.35 16.11 38.67

TABLE-US-00004 TABLE 3 Pharmacological parameters (2-Compartment Model) from the in vivo μPET experiments with [.sup.68Ga]BPAPD, [.sup.68Ga]DOTA.sup.PAM and [.sup.68Ga]DOTA.sup.ZOL in Wistar rats. Elimination half-life [.sup.68Ga]BPAPD [.sup.68Ga]DOTA.sup.PAM [.sup.68Ga]DOTA.sup.ZOL t.sub.1/2(α) 5 min 4.5 min 5 min t.sub.1/2(β) 4 h 4.5 h 2.3 h

[0116] Alternatively, the chelator DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) can be used instead of the chelator DOTA.

Example 9: Accumulation at Bone Metastases

[0117] In comparison to current, particularly successful PSMA tracers (PSMA=Prostate Specific Membrane Antigen), radiopharmaceuticals of the [M]DOTA.sup.ZOL type, as well as derivatives derived from these (for example, DOTAM-based derivatives), show a markedly more intensive accumulation at bone metastases in the same patient (factors 2 through 8) simultaneously with significantly reduced accumulation in healthy organs (see example 9). The table shows the measured uptake (as SUV max values) of [.sup.68Ga]DOTA.sup.ZOL and [.sup.68Ga]HBED-PSMA.sup.CC in direct comparison in a patient with prostate carcinoma and bone metastases.

TABLE-US-00005 TABLE 4 SUV max. values of [.sup.68Ga]DOTA.sup.ZOL and [.sup.68Ga]HBED-PSMA.sup.CC in direct comparison in a patient with prostate carcinoma and bone metastases. [.sup.68Ga]HBED- PSMA.sup.CC PET/CT [.sup.68Ga]DOTA.sup.ZOL Day n + 2 PET/CT (meaning 2 Day n days later) Organ SUV max Bone C4 vertebral body 12.38 7.97 lesions and hip pedicle 8th right rear rib 7.99 7.12 T9 vertebral body 17.08 7.60 T11 vertebral body 32.29 12.98 L2 vertebral body 68.92 8.85 L3 vertebral body 62.96 9.46 L4 vertebral body 34.51 10.98 L5 vertebral body 21.94 19.99 Sacrum 17.98 14.96 Right ischium 32.55 14.96 Left bones, 62.32 8.99 upper ischium, and pubic bone Right acetabulum 25.39 11.71 Soft tissue Right parotid gland 1.22 5.80 Left parotid gland 1.46 6.48 Liver 2.85 6.48 Spleen 4.50 3.27 Right kidney 9.34 22.72 Left kidney 4.12 5.75 Bladder 15.53 22.23

BRIEF DESCRIPTION OF FIGURES

[0118] Accompanying Figures illustrate embodiments and elements of the present invention, but do not limit the subject matter of the invention to the embodiments and elements illustrated in the Figures. If DOTA or its conversion or use is described in the Figures, the chelator DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) can alternatively be used instead of the chelator DOTA.

[0119] FIG. 1: Comparison of [.sup.99mTc]MDP and .sup.223RaCl.sub.2 on day 1, 2, and day 6 after the injection, from O. Sartor, P. Hoskin, Ø. S. Bruland, Targeted radio-nuclide therapy of skeletal metastases, Cancer Treatment Reviews, 2013; 39: 18-26.

[0120] FIG. 2: μPET exposures of various .sup.68Ga(III)-marked macrocyclic bisphosphonates in healthy Wistar rats after 60 min, in Maximum Intensity Projection operating mode.

[0121] FIG. 3 schematically shows a first device 1 for producing a radiopharmaceutical made up of the compound V, as described in detail above.

[0122] FIG. 4 schematically shows a second device 2 for producing a radiopharmaceutical made up of the compound V according to the invention and a metallic radioisotope M, as described in detail above.

[0123] FIG. 5: Distribution of [.sup.177Lu]DOTA.sup.ZOL in a patient with disseminated bone metastases: [.sup.177Lu]DOTA.sup.ZOL scintigraphy on a patient with prostate carcinoma 6 hours after injection. In a first therapeutic applications [sic], within two months, the PSA value (which represents an important marker in monitoring the progress of the prostate carcinoma) could be lowered from 478 ng/mL initially to 88 ng/mL after only one treatment with 5.5 GBq [.sup.177Lu]DOTA.sup.ZOL. .sup.177Lu-DOTAM.sup.ZOL may be used equivalently.

[0124] FIG. 6: PET/CT scan of a prostate carcinoma patient with bone metastases, examined with .sup.68Ga-DOTA.sup.ZOL. .sup.68Ga-DOTAM.sup.ZOL may be used equivalently.