Osteoadsorptive fluorogenic substrate of cathepsin k for imaging osteoclast activity and migration
In certain embodiments osteoadsorptive fluorogenic substrates of cathepsin K (or other proteases) are provided. Utilizing a bisphosphonate targeting moiety, the fluorogenic substrates provide effective bone-targeted protease sensor(s). In certain embodiments the “probes” comprise cleavable fluorophore-quencher pair linked by a cathepsin K (or other protease) peptide substrate and tethered to a bisphosphonate. Unlike existing probes that are cleared within a few days in vivo, the probes described herein (e.g., OFS-1) allow for monitoring resorption over the course of longer time periods with a single dose.
1. An osteoasorptive fluorogenic probe, said probe comprising a compound according to Formula I:
wherein:
R is CH 2 ;
n is 0 to 6;
L 1 is a linker comprising 1 to 6 carbons;
x is 0 or 1;
P is a peptide comprising a cleavage site for a protease; and
A is a quencher and B is a fluorophore, or B is a quencher and A is a fluorophore; and
A is attached directly to P or A is attached to P through a linker.
2. The probe of claim 1 , wherein said probe comprises a compound according to Formula II:
wherein:
L 2 is selected from the group consisting of a C1-C12 carbon linker, an amino acid, and a peptide; and
y is 0 or 1.
3. The probe of claim 1 , wherein said probe comprises a compound according to Formula III:
4. The probe of claim 1 , wherein A is a quencher and B is a fluorophore.
5. The probe of claim 1 , wherein P ranges in length from about 6 amino acid up to about 10 amino acids.
6. The probe of claim 1 , wherein P comprises a cleavage site for a protease selected from the group consisting of cathepsin K, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin L, tartrate-resistant acid protease (TRAP), matrix metalloproteinases (MMP)-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, MMP-14, Kallikreins (hK), hK1, PSA (hK3), hK10, hK15, seriene proteases uPA and uPAR, plasmin, ceruloplasmin (CP), thrombin, trypsin, fibroblast activation protein (FAP), and caspases.
7. The probe of claim 1 , wherein P comprises the amino acid sequence Gly-His-Pro-Gly-Gly-Pro-Gln-Gly (SEQ ID NO:1).
8. The probe of claim 1 , wherein:
said fluorophore is a fluorophore that emits in the visible spectrum or in the near infrared; and/or
said fluorophore is selected from the group consisting of FAM, BODIPY FL, Oregon Green 488, Rhodamine Green, Oregon Green 514, TET, Cal Gold, BODIPY R6G, Yakima Yellow, JOE, HEX, Cal Orange, BODIPY TMR-X, Quasar-570/Cy3, TAMRA, Rhodamine Red-X, Redmond Red, BODIPY 581/591, Cy3.5, ROX, Cal Red/Texas Red, BODIPY TR-X, BODIPY 630/665-X, Pulsar-650, Quasar-670/Cy5, Cy5.5, ALEXAFLUOR® AF647, ALEXAFLUOR® AF660, ALEXAFLUOR® AF680, ALEXAFLUOR® AF700, ALEXAFLUOR® AF750, ALEXAFLUOR® AF790.
9. The probe of claim 1 , wherein said fluorophore comprises 5-FAM or ALEXAFLUOR® AF647.
10. The probe of claim 1 , wherein:
said quencher is selected from the group consisting of BHQ-1, BHQ-3, Dabcyl, QSY 35, BHQ-0, Eclipse, QSY 7, QSY 9, BHQ-2, ElleQuencher, Iowa Black, QSY 21, TAMRA, and Blackberry Quencher BBQ-650; or
said quencher comprises BHQ-1; or
said quencher comprises BHQ-3.
11. The probe of claim 1 , wherein:
said probe comprises a fluorophore/quencher pair shown (by an X) in Table 4; or
said fluorophore comprises 5-FAM and said quencher comprises BHQ-1; or
said fluorophore comprises ALEXAFLUOR® AF647 and said quencher comprises BHQ-3; or
said fluorophore comprises 5-FAM and said quencher comprises BHQ-1.
12. The probe of claim 1 , wherein said probe comprises the structure:
13. The probe of claim 1 , wherein:
said probe accumulates on a calcium phosphate surface when administered to a mammal in vivo;
said probe said probe provides a detectable signal in vivo when said peptide is cleaved by a protease; and
said probe, when administered to a mammal in vivo, is taken up by bone.
14. A pharmaceutical formulation said formulation comprising:
a probe of claim 1 ; and
a pharmaceutically acceptable carrier.
15. A method of detecting protease activity in vivo in a mammal, said method comprising:
administering or causing to be administered to said mammal a probe of claim 1 , where the peptide in said probe comprises a cleavage site for said protease; and
detecting a signal from the fluorophore comprising said probe where said signal indicates that said protease has cleaved said peptide and provides an indication of the activity and/or location of said protease.
16. An osteoabsorptive drug delivery vehicle, said vehicle comprising a compound according to Formula IV:
wherein:
R is CH 2 ;
n, x, and y are independently 0 or 1;
L 1 is a linker comprising 1 to 6 carbons;
R 2 is H or a protecting group;
L 2 is a linker, wherein said linker is a C1-C12 carbon linker, an amino acid, or a peptide;
P is a peptide comprising a cleavage site for a protease; and
D is a therapeutic moiety wherein said therapeutic moiety is a peptide or small organic molecule.
17. The vehicle of claim 16 , wherein said vehicle comprises a compound according to Formula V:
18. The vehicle of claim 16 , wherein said vehicle comprises a compound according to Formula VI:
19. The vehicle of claim 16 , wherein:
D comprises a small organic molecule; or
D comprises an antibiotic; or
D comprises an antibiotic shown in Table 6; or
D comprises an anti-cancer agent; or
D comprises an anti-cancer agent selected from the group consisting of flourouracil (5-FU), capecitabine, 5-trifluoromethyl-2′-deoxyuridine, methotrexate sodium, raltitrexed, pemetrexed, cytosine Arabinoside, 6-mercaptopurine, azathioprine, 6-thioguanine (6-TG), pentostatin, fludarabine phosphate, cladribine, floxuridine (5-fluoro-2), ribonucleotide reductase inhibitor (RNR), cyclophosphamide, neosar, ifosfamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitosourea (BCNU), 1,-(2-chloroethyl)-3-cyclohexyl-lnitrosourea, methyl (CCNU), hexamethylmelamine, busulfan, procarbazine HCL, dacarbazine (DTIC), chlorambucil, melphalan, cisplatin, carboplatin, oxaliplatin, bendamustine, carmustine, chloromethine, dacarbazine (DTIC), fotemustine, lomustine, mannosulfan, nedaplatin, nimustine, prednimustine, ranimustine, satraplatin, semustine, streptozocin, temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA, triplatin tetranitrate, trofosfamide, uramustine, doxorubicin, daunorubicin citrate, mitoxantrone, actinomycin D, etoposide, topotecan HCL, teniposide (VM-26), irinotecan HCL (CPT-11), camptothecin, belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbine tartrate, vindesine sulphate, paclitaxel, docetaxel, nanoparticle paclitaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel, vinflunine, retinoic acid, a retinoic acid derivative, doxirubicin, vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, a camptothecin derivative, interferon, tamoxifen, and taxol.
20. A pharmaceutical formulation said formulation comprising:
a vehicle of claim 16 ; and
a pharmaceutically acceptable carrier.
21. A method of treating a pathology characterized by bone resorption in a mammal, said method comprising:
administering or causing to be administered to said mammal, an effective amount of a drug delivery vehicle of claim 16 , wherein D is selected from the group consisting of parathyroid hormone, a parathyroid hormone analog, calcitonin, a drug that induces bone growth, and an anti-cancer drug.