Non-isotopic detection of osteoblastic activity in vivo using modified bisphosphonates
The present invention is directed to a non-isotopic methods for the in vitro and in vivo detection of hydroxyapatite-positive cells and structures.
1. A contrast agent represented in the general formula [I] or pharmaceutically acceptable salts thereof:
wherein
D represents a near infrared fluorescent moiety having excitation and emission spectra between about 650 nm and about 1000 nm;
R represents a linking group that covalently links the dye (D) and bisphosphonate moiety;
R 1 represents H, —OH, or a halogen; and
R 2 represents, independently for each occurrence, a free electron pair, hydrogen, or a pharmaceutically acceptable counterion.
2. The agent of claim 1 , wherein R is an amine substituted lower-alkyl which forms an amide bond with a pendant group of the fluorescent moiety.
3. The agent of claim 2 , wherein R is —(CH 2 ) L —NH—, where L is an integer from 1 to 6.
4. The agent of claim 1 , wherein R 1 is —OH or —Cl.
5. The agent of claim 1 , wherein D is a near-infrared fluorescent dye.
6. The agent of claim 5 , wherein D is a polysulfonated indocyanine dye.
7. The agent of claim 1 , wherein the fluorescent moiety is represented by the formula [II]:
wherein
L 1 -L 7 are each, independently, a substituted or unsubstituted methine, provided that one of L 1 -L 7 is substituted with the linker group R which is attached to the bisphosphonate;
R 3 , independently for each occurrence, is a substituted or unsubstituted alkyl;
A and B are each, independently, 5-7 membered substituted or unsubstituted aromatic rings;
X and Y are the same or different and each is a group of the formula —O—, —S—, —CH═CH— or —C(R 4 ) 2 —;
R 4 , independently for each occurrence, is a hydrogen or substituted or unsubstituted lower alkyl; and
r is 0, 1 or 2.
8. The agent of claim 7 , wherein the fluorescent moiety has one or more of the following features: (a) is free ofacarboxylic acid group in a molecule; (b) r is 1; (c) includes 4 or more sulfonic acid groups; (d) includes 10 or less sulfonic acid groups; (e) A and B are, independently, benzo or naphtho rings; and (f) X and Y arc —C(CH 3 ) 2 —.
9. The agent of claim 1 , wherein the fluorescent moiety is represented by the formula [III]
wherein
X and Y are the same or different and each is a group of the formula —O—, —S—, —CH═CH— or C(R 4 ) 2 —;
R 4 , independently for each occurrence, is a hydrogen or substituted or unsubstituted lower alkyl; and
R 6 , independently for each occurrence, is hydrogen or —SO 3 R 7 ;
R 7 , independently for each occurrence, is hydrogen or a pharmaceutically acceptable counter ion;
m is 0, 1, 2, 3, 4 or 5.
10. The agent of claim 1 , wherein the near-infrared fluorescent moiety has an extinction coefficient of at least 100,000 M −1 cm −1 in aqueous medium.
11. The agent of claim 1 , wherein the near-infrared fluorescent moiety has a quantum efficiency, Φ F , of at least 25%.
12. The agent of claim 1 , having a half-life in the human 20 body of at least 10 minutes.
13. A contrast agent comprising a bisphosphonate covalently linked to a near-infrared fluorescent moiety, wherein the contrast agent (a) has an extinction coefficient of at least 100,000 M −1 cm −1 in aqueous medium, and (b) has a half-life in the human body of at least 10 minutes.
14. The contrast agent of claim 13 , wherein the bisphosphonate is selected from the group consisting of alendronate, clodronate, EB-1053, etidronate, ibandronate, incadronate, neridronate, olpadronate, phosphonate, pamidronate, risedronate, tiludronate, YH 529 and zoledronate.
15. A method for manufacturing a composition for in vivo imaging comprising formulating a contrast agent of claim 1 in a pharmaceutically acceptable excipient.
16. A kit for in vivo imaging comprising a contrast agent of claim 1 in association with instructions for administering the contrast agent to a patient.
17. A method for in vivo imaging of tissue with exposed hydroxyapatite, comprising,
(i) administering to an animal a contrast agent of claim 1 in an amount sufficient to render hydroxyapatite-containing tissue detectable by a fluorescence detector;
(ii) obtaining a fluorescent image of the animal, or at least a portion thereof, at a wave length(s) which detects the contrast agent;
(iii) constructing an image of the animal including the pattern of distribution of the contrast agent.
18. The method of claim 17 , for evaluating a bone for its condition and/or biomechanical property.
19. The method of claim 18 , for determining bone matrix density.
20. The method of claim 18 , for detecting changes in bone matrix volume.
21. The method of claim 18 , as part of a protocol for diagnosing osteoporosis.
22. The method of claim 18 , for determining at least one of anisotropic elastic constants, bone strength, or fracture risk.
23. The method of claim 17 , for diagnostic detection of bone diseases accompanied with abnormality of calcium hydroxyapatite.
24. The method of claim 23 , for detecting the presence of osteoblastic metastase.
25. The method of claim 17 , for detecting of vessel micro-calcification.
26. The method of claim 17 , wherein the animal is a human.
27. The agent of claim 7 , wherein A and B are independently selected from the group consisting of benzo- and naptho-rings.