The present invention relates generally to optical dye bioconjugates useful in medical diagnosis and/or therapy.
Several dyes that absorb and emit light in the visible and near-infrared region of electromagnetic spectrum are currently being used for various biomedical applications due to their biocompatibility, high molar absorptivity, and/or high fluorescence quantum yields. The high sensitivity of the optical modality in conjunction with dyes as contrast agents parallels that of nuclear medicine, and permits visualization of organs and tissues without the undesirable effect of ionizing radiation.
Cyanine dyes with intense absorption and emission in the near-infrared (NIR) region are particularly useful because biological tissues are optically transparent in this region (B. C. Wilson, Optical properties of tissues. Encyclopedia of Human Biology, 1991, 5, 587-597). For example, indocyanine green, which absorbs and emits in the NIR region, has been used for monitoring cardiac output, hepatic functions, and liver blood flow (Y-L. He, et al., Measurement of blood volume using indocyanine green measured with pulse-spectrometry: Its reproducibility and reliability. Critical Care Medicine, 1998, 26(8), 1446-1451; J. Caesar, et al., The use of Indocyanine green in the measurement of hepatic blood flow and as a test of hepatic function. Clin. Sci. 1961, 21, 43-57), and its functionalized derivatives have been used to conjugate biomolecules for diagnostic purposes (R. B. Mujumdar, et al., Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters. Bioconjugate Chemistry, 1993, 4(2), 105-111; U.S. Pat. No. 5,453,505; WO 98/48846; WO 98/22146; WO 96/17628; WO 98/48838).
A major drawback in the use of cyanine dye derivatives is the potential for hepatobiliary toxicity resulting from the rapid clearance of these dyes by the liver (G. R. Chemick, et al., Indocyanine green: Observations on its physical properties, plasma decay, and hepatic extraction. J. Clinical Investigation, 1960, 39, 592-600). This is associated with the tendency of cyanine dyes in solution to form aggregates, which could be taken up by Kupffer cells in the liver.
Various attempts to obviate this problem have not been very successful. Typically, hydrophilic peptides, polyethyleneglycol or oligosaccharide conjugates have been used, but these resulted in long-circulating products, which are eventually still cleared by the liver. Another major difficulty with current cyanine and indocyanine dye systems is that they offer a limited scope in the ability to induce large changes in the absorption and emission properties of these dyes. Attempts have been made to incorporate various heteroatoms and cyclic moieties into the polyene chain of these dyes (L. Strekowski, et al., Substitution reactions of a nucleofugal group in hetamethine cyanine dyes. J. Org. Chem., 1992, 57, 4578-4580; N. Narayanan, and G. Patonay, A new method for the synthesis of heptamethine cyanine dyes: Synthesis of new near infrared fluorescent labels. J. Org. Chem., 1995, 60, 2391-2395; U.S. Pat. Nos. 5,732,104; 5,672,333; and 5,709,845), but the resulting dye systems do not show large differences in absorption and emission maxima, especially beyond 830 nm where photoacoustic diagnostic applications are very sensitive. They also possess a prominent hydrophobic core, which enhances liver uptake. Further, most cyanine dyes do not have the capacity to form starburst dendrimers, which are useful in biomedical applications.
For the purpose of tumor detection, many conventional dyes are useful for in vitro applications because of their highly toxic effect on both normal and abnormal tissues. Other dyes lack specificity for particular organs or tissues and, hence, these dyes must be attached to bioactive carriers such as proteins, peptides, carbohydrates, and the like to deliver the dyes to specific regions in the body. Several studies on the use of near infrared dyes and dye-biomolecule conjugates have been published (G. Patonay and M. D. Antoine, Near-Infrared Fluorogenic Labels: New Approach to an Old Problem, Analytical Chemistry, 1991, 63:321 A-327A and references therein; M. Brinkley, A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens, and Cross-Linking Reagents, Perspectives in Bioconjugate Chemistry 1993, pp. 59-70, C. Meares (Ed), ACS Publication, Washington, D.C.; J. Slavik, Fluorescent Probes in Cellular and Molecular Biology, 1994, CRC Press, Inc.; U.S. Pat. No. 5,453,505; WO 98/48846; WO 98/22146; WO 96/17628; WO 98/48838). Of particular interest is the targeting of tumor cells with antibodies or other large protein carriers such as transferrin as delivery vehicles (A. Becker, et al., “Transferrin Mediated Tumor Delivery of Contrast Media for Optical Imaging and Magnetic Resonance Imaging”, Biomedical Optics meeting, Jan. 23-29, 1999, San Jose, Calif.). Such an approach has been widely used in nuclear medicine applications.
Its major advantage is the retention of a carrier's tissue specificity, since the molecular volume of the dye is substantially smaller than the carrier. However, this approach does have some serious limitations in that the diffusion of high molecular weight bioconjugates to tumor cells is highly unfavorable, and is further complicated by the net positive pressure in solid tumors (R. K. Jain, Barriers to Drug Delivery in Solid Tumors, Scientific American 1994, 271:58-65. Furthermore, many dyes in general, and cyanine dyes, in particular, tend to form aggregates in aqueous media that lead to fluorescence quenching.
The invention relates to compounds and compositions, as well as methods of preparing and using such compounds and compositions, of biomolecule-dye conjugates. These conjugates may be utilized, for example, to enhance tumor detection. Conjugates of the present invention may be characterized as preserving the fluorescence efficiency of the dye molecules, not aggregating in solution, forming starburst dendrimers, being capable of absorbing and/or omitting light in the near infrared region (beyond 800 mm), and/or being capable of being rendered tissue-specific.
In one aspect, the present invention relates to a compound of general Formula 1
wherein W1 and X1 may be the same or different and are selected from the group consisting of —CRwRx, —O—, —NRy, —S—, and —Se—; Q2 is a single bond or is selected from the group consisting of —O—, —S—, —Se—, and —NR5; a1 and b1 independently vary from 0 to 5; a and c are independently from 1 to 20; b and d are independently from 1 to 100; Y1 is a constituent selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—N(Ry)—(CH2)b—CONH-Bm, (CH2)a—N(Ry)—(CH2)c—NHCO-Bm, —(CH2)a—NRyRz, —(CH2)a—N(Ry)—CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—N(Ry)—CH2—(CH2OCH2)b—CH2—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(Ry)—(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(Ry)—(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(Ry)—CH2—(CH2OCH2)d—CONH-Bm, —CH2(CH2OCH2)b—CH2NRyRz, and —CH2—(CH2OCH2)b—CH2—N(Ry)—CH2—(CH2OCH2)d—NHCO-Bm; Z1 is a constituent selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—N(Ry)—(CH2)b—CONH-Dm, (CH2)a—N(Ry)—(CH2)c—NHCO-Dm, —(CH2)a—N(Ry)—CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—N(Ry)—CH2—(CH2OCH2)b—CH2—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(Ry)—(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(RF)—(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(Ry)—CH2—(CH2OCH2)d—CONH-Dm, —CH2(CH2OCH2)b—CH2NRyRz, —CH2—(CH2OCH2)b—CH2—N(Ry)—CH2—(CH2OCH2)d—NHCO-Dm, and —(CH2)a—NRyRz; Rw, Rx, Ry, Rz, and R1 to R9 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. At least one of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one preferred family of embodiments, at least one of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin. In another exemplary family of preferred embodiments, at least one of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy.
In another aspect, the present invention relates to a compound of general Formula 2
wherein W2 and X2 may be the same or different and are selected from the group consisting of —CR1R2, —O—, —NR3, —S—, and —Se—; Q2 is a single bond or is selected from the group consisting of —O—, —S—, —Se—, and —NR5; a2 and b2 independently vary from 0 to 5; a and c are independently from 1 to 20; b and d are independently from 1 to 100; Y2 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—N(R3)—(CH2)b—CONH-Bm, (CH2)a—N(R3)—(CH2)C—NHCO-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; Z2 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—N(R3)—(CH2)b—CONH-Dm, (CH2)a—N(R3)—(CH2)c—NHCO-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; R1 to R5, and R16 to R28 are constituents independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. At least one of Y2, Z2, R1, R2, R3, R4, R5, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27 and R28 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one preferred family of embodiments, at least one of Y2, Z2, R1, R2, R3, R4, R5, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27 and R28 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin. In another exemplary family of preferred embodiments, at least one of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy.
In yet another aspect, the invention relates to a compound of general Formula 3
wherein W3 and X3 may be the same or different and are selected from the group consisting of —CR1R2, —O—, —NR3, —S—, and —Se; Y3 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—N(R3)—(CH2)b—CONH-Bm, —(CH2)a—N(R3)—(CH2)c—NHCO-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; Z3 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—N(R3)—(CH2)b—CONH-Dm, (CH2)a—N(R3)—(CH2)c—NHCO-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R13)—CH2—(CH2OCH2)d—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; a and c are independently from 1 to 20; and b and d are independently from 1 to 100; A1 is a single or a double bond; B1, C1, and D1 may the same or different and are selected from the group consisting of —O—, —S—, —Se—, —P—, —CR1R2, —CR1, alkyl, NR3, and —C═O; A1, B1, C1, and D1 may together form a 6- to 12-membered carbocyclic ring or a 6- to 12-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a3 and b3 independently vary from 0 to 5; R1 to R4, and R29 to R37 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. At least one of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33R34, R35, R36, and R37 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one preferred family of embodiments, at least one of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33, R34, R35, R36, and R37 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin. In another exemplary family of preferred embodiments, at least one of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33, R34, R35, R36, and R37 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy.
In still another aspect, the invention relates to a compound of general Formula 4
wherein W4 and X4 may be the same or different and are selected from the group consisting of —CR1R2, —O—, —NR3, —S—, and —Se; Y4 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—N(R3)—(CH2)b—CONH-Bm, (CH2)a—N(R3)—(CH2)c—NHCO-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; Z4 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—N(R3)—(CH2)b—CONH-Dm, (CH2)a—N(R3)—(CH2)c—NHCO-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; a and c are independently from 1 to 20; and b and d are independently from 1 to 100; A2 is a single or a double bond; B2, C2, and D2 may be the same or different and are selected from the group consisting of —O—, —S—, —Se—, —P—, —CR1R2, —CR1, alkyl, NR3, and —C═O; A2, B2, C2, and D2 may together form a 6- to 12-membered carbocyclic ring or a 6- to 12-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a4 and b4 independently vary from 0 to 5; R1 to R4, and R45 to R57 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. At least one of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49, R50, R51, R41, R53, R54, R55, R56 and R57 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one preferred family of embodiments, at least one of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49R50, R51, R52, R53, R54, R55, R56 and R57 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin. In another exemplary family of preferred embodiments, at least one of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49, R50, R51, R52, R53, R54, R55, R56 and R57 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy.
Yet another aspect of the present invention relates to a compound of general Formula 5
wherein W5 and X5 may be the same or different and are selected from the group consisting of —CR1R2, —O—, —NR3, —S—, and —Se; Y5 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)6—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—N(R3)—(CH2)b—CONH-Bm, (CH2)a—N(R3)—(CH2)c—NHCO-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; Z5 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—N(R3)—(CH2)b—CONH-Dm, (CH2)a—N(R3)—(CH2)c—NHCO-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; a and c are independently from 1 to 20; and b and d are independently from 1 to 100; A3 is a single or a double bond; B3, C3, and D3 may be the same or different and are selected from the group consisting of —O—, —S—, —Se—, —P—, —CR1R2, —CR1, alkyl, NR3, and —C═O; A3, B3, C3, and D3 may together form a 6- to 12-membered carbocyclic ring or a 6- to 12-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a5 is independently from 0 to 5; R1 to R4, and R58 to R66 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. At least one of Y5, Z5, R1, R2, R3, R4, R58, R59, R60, R61, R62, R63, R64, R65, and R66 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one preferred family of embodiments, at least one of Y5, Z5, R1, R2, R3, R4, R58, R59, R60, R61, R62, R63, R64, R65, and R66 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin. In another exemplary family of preferred embodiments, at least one of Y5, Z5, R1, R2, R3, R4, R58, R59, R60, R61, R62, R63, R64, R65, and R66 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy.
Still yet another aspect of the invention relates to a compound of general Formula 6
wherein W6 and X6 may be the same or different and are selected from the group consisting of —CR1R2, —O—, —NR3, —S—, and —Se; Y6 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—N(R3)—(CH2)b—CONH-Bm, (CH2)a—N(R3)—(CH2)c—NHCO-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; Z6 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H—(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—N(R3)—(CH2)b—CONH-Dm, (CH2)a—N(R3)—(CH2)c—NHCO-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; a and c are independently from 1 to 20; and b and d are independently from 1 to 100; A4 is a single or a double bond; B4, C4, and D4 may be the same or different and are selected from the group consisting of —O—, —S—, —Se—, —P—, —CR1R2, —CR1, alkyl, NR3, and —C═O; A4, B4, C4, and D4 may together form a 6- to 12-membered carbocyclic ring or a 6- to 12-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a6 is independently from 0 to 5; R1 to R4, and R67 to R79 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, saccharide, peptide, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH or —CH2—(CH2OCH2)b—CO2H; Bm and Dm are independently selected from the group consisting of a peptide, a protein, a cell, an antibody, an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent; At least one of Y6, Z6, R2, R3, R4, R67, R68, R69, R70, R71, R72, R73, R74, R75, R76, R77, R78 and R79 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one preferred family of embodiments, at least one of y, Z6, R1, R2, R3, R4, R67, R68, R69, R70, R71, R72, R73, R74, R75, R76, R77, R78 and R79 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin. In another exemplary family of preferred embodiments, at least one of Y6 Z6, R1, R2, R3, R4, R67, R68, R69, R70, R71, R72, R73, R74, R75, R76, R77R78 and R79 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy.
Yet another aspect of the invention relates to a composition including a pharmaceutically acceptable carrier or excipient and at least one of the compounds of Formulas 1-6 above.
Still another aspect of the invention relates to a method of using a compound of any of Formulas 1-6 above. In this method, an effective amount of the compound is administered to an individual (e.g., patient), and the compound is activated using light.
In some embodiments, a chelate such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), 1,4,7,10 tetraazacyclododecane-tetraacetic acid (DOTA), or their derivatives, may be attached to a compound of any of Formulas 1-6 as one or more R groups. These structures are expected to be highly water soluble.
The invention will be further appreciated in light of the following figures, detailed description, and examples.
The file of U.S. patent application Ser. No. 10/800,531 (filed Mar. 15, 2004), which is hereby incorporated by reference, contains color versions of
Compounds of formulas 1-6 offer significant advantages over conventional, optical dyes known in the art. These inventive compounds form starburst dendrimers which prevent aggregation in solution by preventing intramolecular and intermolecular ordered hydrophobic interactions, and have multiple attachment sites proximal to the dye chromophore for ease of forming bioactive molecules. The presence of rigid and extended chromophore backbone enhances their fluorescence quantum yield and extends their maximum absorption beyond 800 nm. Conjugation of biomolecules to dyes is readily achievable by way of the invention.
The inventive compounds of the present invention exploit the symmetric nature of cyanine and indocyanine dye structures by incorporating one to ten receptor-targeting groups in close proximity to each other, such that the receptor binding can be greatly enhanced due to a cooperative effect. Accordingly, several compounds containing one or more targeting domains have been prepared and tested in vivo for biological activity.
The inventive compounds of formulas 1 to 6 may be useful for various biomedical applications. These include, but are not limited to, tomographic imaging of organs, monitoring of organ functions, coronary angiography, fluorescence endoscopy, detection, imaging, and therapy of tumors, laser guided surgery, photoacoustic methods, and sonofluorescent methods.
Exemplary embodiments to accomplish some of the aforementioned biomedical applications are given below. Compounds of the present invention may be prepared according to methods well known in the art as illustrated in the exemplary synthetic schemes of
Some compounds of the invention are of Formula 1, wherein W1 and X1 may be the same or different and are selected from the group consisting of —CRwRx, —O—, —NR1, —S—, and —Se—; Q2 is a single bond or is selected from the group consisting of —O—, —S—, —Se—, and —NR5; a1 and b1 independently vary from 0 to 5; a and c are independently from 1 to 20; b and d are independently from 1 to 100; Y1 is a constituent selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—N(Ry)—(CH2)b—CONH-Bm, —(CH2)a—N(Ry)—(CH2)c—NHCO-Bm, —(CH2)a—N(Ry)—CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—N(Ry)—CH2—(CH2OCH2)b—CH2—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(Ry)—(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(Ry)—(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(Ry)—CH2—(CH2OCH2)d—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(Ry)—CH2—(CH2OCH2)d—NHCO-Bm, —(CH2)a—NRyRz, and —CH2(CH2OCH2)b—CH2NRyRz; Z1 is a constituent selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—N(Ry)—(CH2)b—CONH-Dm, (CH2)a—N(Ry)—(CH2)c—NHCO-Dm, —(CH2)a—N(Ry)—CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—N(Ry)—CH2—(CH2OCH2)b—CH2—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(Ry)—(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(Ry)—(CH2)8—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(Ry)—CH2—(CH2OCH2)d—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(Ry)—CH2—(CH2OCH2)d—NHCO-Dm, —(CH2)a—NRyRz, and —CH2(CH2OCH2)b—CH2NRyRz; Rw, Rx, Ry, Rz, and R1 to R9 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, mono- or oligosaccharide, peptide with 2 to 30 amino acid units, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide (e.g., containing 2 to 30 amino acid units), a protein, a cell, an antibody, an antibody fragment, a saccharide (e.g., a mono- or oligosaccharide), a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
With regard to compounds of Formula 1, at least one of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4R5, R6, R7, R8 and R9 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm of the compound of Formula 1 is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one family of embodiments, at least one of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin.
In another family of embodiments of the compounds of Formula 1, at least one of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy. For example, in some embodiments, at least two of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 are constituents including Bm or Dm. For instance, one of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 may be constituent including a peptide, and another of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 may be a constituent including a photosensitizer for phototherapy.
In still another family of embodiments of the compounds of Formula 1, at least one of Y1, Z1, Rw, Rx, Ry, Rz, R1, R2, R3, R4, R5, R6, R7, R8 and R9 is a constituent including Bm or Dm. However, in this family of embodiments, Bm and/or Dm is selected from the group consisting of a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
Some compounds of the invention are of Formula 2, wherein W2 and X2 may be the same or different and are selected from the group consisting of —CR1R2, —O—, —NR3, —S—, and —Se—; Q2 is a single bond or is selected from the group consisting of —O—, —S—, —Se—, and —NR5; a2 and b2 independently vary from 0 to 5; a and c are independently from 1 to 20; b and d are independently from 1 to 100; Y2 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—N(R3)—(CH2)b—CONH-Bm, —(CH2)a—N(R3)—(CH2)c—NHCO-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Bm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; Z2 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—N(R3)—(CH2)b—CONH-Dm, (CH2)a—N(R3)—(CH2)c—NHCO-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—N(R3)—CH2—(CH2OCH2)b—CH2—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—CONH-Dm, —CH2—(CH2OCH2)b—CH2—N(R3)—CH2—(CH2OCH2)d—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; R1 to R5, and R16 to R28 are constituents independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, cyano, nitro, halogen, mono- or oligosaccharide, peptide with 2 to 30 amino acid units, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide (e.g., containing 2 to 30 amino acid units), a protein, a cell, an antibody, an antibody fragment, a saccharide (e.g., a mono- or oligosaccharide), a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
With regard to compounds of Formula 2, at least one of Y2, Z2, R1, R2, R3, R4, R5, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27 and R28 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm of the compound is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one family of embodiments, at least one of Y2, Z2, R1, R2, R3, R4, R5, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27 and R28 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin.
In another family of embodiments of the compounds of Formula 2, at least one of Y2, Z2, R1, R2, R3, R4, R5, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27 and R28 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy. For example, in some embodiments, at least two of Y2, Z2, R1, R2, R3, R4, R5, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25R26, R27 and R28 are constituents including Bm or Dm. For instance, one of Y2, Z2, R1, R2, R3, R4, R5, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, and R28 may be constituent including a peptide, anotherof Y2, Z2, R1, R2, R3, R4, R5, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, and R28 may be a constituent including a photosensitizer for phototherapy.
In still another family of embodiments of the compounds of Formula 2, at least one of Y2, Z2, R1, R2, R3, R4, R5, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27 and R28 is a constituent including Bm or Dm. However, in this family of embodiments, Bm and/or Dm is selected from the group consisting of a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
Some compounds of the invention are of Formula 3, wherein W3 and X3 may be the same or different and are selected from the group consisting of —C(CH3)2, —C((CH2)aOH)CH3, —C((CH2)aOH)2, —C((CH2)aCO2H)CH3, —C((CH2)aCO2H)2, —C((CH2)aNH2)CH3, —C((CH2)aNH2)2, C((CH2)aNR3R4)2, —NR3, and —S—; Y3 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—H2NR3R4; Z3 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2), —NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—H2NR3R4; a and c are independently from 1 to 10; and b and d are independently from 1 to 30; A1 is a single or a double bond; B1, C1, and D1 are independently selected from the group consisting of —O—, —S—, NR3, (CH2)a—CR1R2, and —CR1; A1, B1, C1, and D1 may together form a 6- to 10-membered carbocyclic ring or a 6- to 10-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a3 and b3 are independently from 0 to 3; R1 to R4, and R29 to R37 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C12 aryl, C1-C10 alkoxyl, C1-C10 polyhydroxyalkyl, C5-C12 polyhydroxyaryl, C1-C10 aminoalkyl, mono- or oligosaccharide, peptide with 2 to 30 amino acid units, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide (e.g., containing 2 to 30 amino acid units), a protein, a cell, an antibody, an antibody fragment, a saccharide (e.g., a mono- or oligosaccharide), a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
With regard to compounds of Formula 3, at least one of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33, R34, R35, R36, and R37 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm of the compound is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one family of embodiments, at least one of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33, R34, R33, R34, R35, R36, and R37 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin.
In another family of embodiments of the compounds of Formula 3, at least one of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33, R34, R35, R36, and R37 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy. For example, in some embodiments, at least two of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33, R34, R35, R36, and R37 are constituents including Bm or Dm. For instance, one of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33, R3, R35, R36, and R37 may be constituent including a peptide, and another of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33, R34, R35, R36, and R37 may be a constituent including a photosensitizer for phototherapy.
In still another family of embodiments of the compounds of Formula 3, at least one of Y3, Z3, R1, R2, R3, R4, R29, R30, R31, R32, R33, R34, R35, R36, and R37 is a constituent including Bm or Dm. However, in this family of embodiments, Bm and/or Dm is selected from the group consisting of a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
Some compounds of the invention are of Formula 4, wherein W4 and X4 may be the same or different and are selected from the group consisting of —C(CH3)2, —C((CH2)aOH)CH3, —C((CH2)aOH)2, —C((CH2)aCO2H)CH3, —C((CH2)aCO2H)2, —C((CH2)aNH2)CH3, C((CH2)aNH2)2, —C((CH2)aNR3R4)2, —NR3, and —S—; Y4 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—H2NR3R4; Z4 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—H2NR3R4; a and c are independently from 1 to 10; and b and d independently from 1 to 30; A2 is a single or a double bond; B2, C2, and D2 are independently selected from the group consisting of —O—, —S—, NR3, (CH2)a—CR1R2, and —CR1; A2, B2, C2, and D2 may together form a 6- to 10-membered carbocyclic ring or a 6- to 10-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a4 and b4 are independently from 0 to 3; R1 to R4, and R45 to R57 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C12 aryl, C1-C10 alkoxyl, C1-C10 polyhydroxyalkyl, C5-C12 polyhydroxyaryl, C1-C10 aminoalkyl, mono- or oligosaccharide, peptide with 2 to 30 amino acid units, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide (e.g., containing 2 to 30 amino acid units), a protein, a cell, an antibody, an antibody fragment, a saccharide (e.g., a mono- or oligosaccharide), a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
With regard to compounds of Formula 4, at least one of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49, R50, R51, R52, R53, R54, R55, R56 and R57 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm of the compound is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one family of embodiments, at least one of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49, R50, R51, R52, R53, R54, R55, R56 and R57 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin.
In another family of embodiments of the compounds of Formula 4, at least one of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49, R50, R51, R52, R53, R4, R55, R56 and R57 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy. For example, in some embodiments, at least two of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49, R50, R51, R52, R53, R54, R55, R56 and R57 are constituents including Bm or Dm. For instance, one of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49, R50, R51, R52, R53, R54, R55, R56 and R57 may be constituent including a peptide, and another of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49, R50, R51, R52, R53, R54, R55, R56 and R57 may be a constituent including a photosensitizer for phototherapy.
In still another family of embodiments of the compounds of Formula 4, at least one of Y4, Z4, R1, R2, R3, R4, R45, R46, R47, R48, R49, R50, R51, R52, R53, R54, R55, R56 and R57 is a constituent including Bm or Dm. However, in this family of embodiments, Bm and/or Dm is selected from the group consisting of a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
Still a fifth aspect of the invention includes cyanine dyes preferably having the general Formula 5, wherein W5 and X5 may be the same or different and are selected from the group consisting of —C(CH3)2, —C((CH2)aOH)CH3, —C((CH2)aOH)2, —C((CH2)aCO2H)CH3, —C((CH2)aCO2H)2, —((CH2)aNH2)CH3, —C((CH2)aNH2)2, —C((CH2)aNR3R4)2, —NR3, and —S—; Y5 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2), —CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—CH2NR3R4; Z5 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—H2NR3R4; a and care independently from 1 to 10; and b and d are independently from 1 to 30; A3 is a single or a double bond; B3, C3, and D3 are independently selected from the group consisting of —O—, —S—, NR3, (CH2)a—CR1R2, and —CR1; A3, B3, C3, and D3 may together form a 6- to 10-membered carbocyclic ring or a 6- to 10-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; as is from 0 to 3; R1 to R4, and R58 to R66 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C12 aryl, C1-C10 alkoxyl, C1-C10 polyhydroxyalkyl, C5-C12 polyhydroxy aryl, C1-C10 aminoalkyl, mono- or oligosaccharide, peptide with 2 to 30 amino acid units, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide (e.g., containing 2 to 30 amino acid units), a protein, a cell, an antibody, an antibody fragment, a saccharide (e.g., a mono- or oligosaccharide), a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
With regard to compounds of Formula 5, at least one of Y5, Z5, R1, R2, R3, R4, R58, R59, R60, R61, R62, R63, R64, R65, and R66 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm of the compound is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one family of embodiments, at least one of Y5, Z5, R1, R2, R3, R4, R5, R59, R60, R61, R62, R63, R64, R65, and R66 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin.
In another family of embodiments of the compounds of Formula 5, at least one of Y5, Z5, R1, R2, R3, R4, R58, R59, R60, R61, R62, R63, R64, R65, and R62 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy. For example, in some embodiments, at least two of Y5, Z5, R1, R2, R3, R4, R58, R59, R60, R61, R62, R63, R64, R65, and R66 are constituents including Bm or Dm. For instance, one of Y5, Z5, R1, R2, R3, R4, R58, R59, R60, R61, R62, R63, R64, R65, and R66 may be constituent including a peptide, and another of Y5, Z5, R2, R3, R4, R58, R59, R60, R61, R62, R63, R64, R65, and R66 may be a constituent including a photosensitizer for phototherapy.
In still another family of embodiments of the compounds of Formula 5, at least one of Y5, Z5, R1, R2, R3, R4, R58, R59, R60, R61, R62, R63, R64, R65, and R66 is a constituent including Bm or Dm.
However, in this family of embodiments, Bm and/or Dm is selected from the group consisting of a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
Some compounds of the invention are of Formula 6, wherein W6 and X6 may be the same or different and are selected from the group consisting of —C(CH3)2, —C((CH2)aOH)CH3, —C((CH2)aOH)2, —C((CH2)aCO2H)CH3, —C((CH2)aCO2H)2, —C((CH2)aNH2)CH3, C((CH2)aNH2)2, C((CH2)aNR3R4)2, —NR3, and —S—; Y6 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—H2NR3R4; Z6 is selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C20 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl, C1-C20 polyhydroxyalkyl, C5-C20 polyhydroxyaryl, C1-C10 aminoalkyl, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a—CO2H, —(CH2)a—CONH-Dm, —CH2—(CH2OCH2)b—CH2—CONH-Dm, —(CH2)a—NHCO-Dm, —CH2—(CH2OCH2)b—CH2—NHCO-Dm, —(CH2)a—NR3R4, and —CH2(CH2OCH2)b—H2NR3R4; a and c are independently from 1 to 10; and b and d are independently from 1 to 30; A4 is a single or a double bond; B4, C4, and D4 are independently selected from the group consisting of —O—, —S—, NR3, (CH2)a—CR1R2, and —CR1; A4, B4, C4, and D4 may together form a 6- to 10-membered carbocyclic ring or a 6- to 10-membered heterocyclic ring optionally containing one or more oxygen, nitrogen, or sulfur atom; a6 is from 0 to 3; R1 to R4, and R67 to R79 are independently selected from the group consisting of hydrogen, C1-C10 alkyl, C5-C12 aryl, C1-C10 alkoxyl, C1-C10 polyhydroxyalkyl, C5-C12 polyhydroxy aryl, C1-C10 aminoalkyl, mono- or oligosaccharide, peptide with 2 to 30 amino acid units, —CH2(CH2OCH2)b—CH2—OH, —(CH2)a, CO2H, —(CH2)a—CONH-Bm, —CH2—(CH2OCH2)b—CH2—CONH-Bm, —(CH2)a—NHCO-Bm, —CH2—(CH2OCH2)b—CH2—NHCO-Bm, —(CH2)a—OH and —CH2—(CH2OCH2)b—CO2H; and Bm and Dm are independently selected from the group consisting of a peptide (e.g., containing 2 to 30 amino acid units), a protein, a cell, an antibody, an antibody fragment, a saccharide (e.g., a mono- or oligosaccharide), a glycopeptide, a peptidomimetic, a drug, a drug mimic, a hormone, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
With regard to compounds of Formula 6, at least one of Y6, Z6, R1, R2, R3, R4, R67, R68, R69, R70, R71, R72, R73, R74, R75, R76, R77, R78 and R79 is preferably a constituent including Bm or Dm. Further, it is preferred that at least one Bm or Dm of the compound is selected from the group consisting of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. For instance, in one family of embodiments, at least one of Y6, Z6, R1, R2, R3, R4, R67, R68, R69, R70, R72, R73, R74, R75, R76, R77, R78 and R79 is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin.
In another family of embodiments of the compounds of Formula 6, at least one of (6, Z6, R1, R2, R3, R4, R67, R68, R69, R70, R71, R72, R73, R74, R75, R76, R77, R78 and R79 is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy. For example, in some embodiments, at least two of Y6 Z6, R1, R2, R3, R4, R67, R68, R69, R70, R71, R72, R73, R74, R75, R76, R77, R78 and R79 are constituents including Bm or Dm. For instance, one of Y6, Z6, R1, R2, R3, R4, R67, R68, R69, R70, R71, R72, R73, R74, R75, R76, R77, R78 and R79 may be constituent including a peptide, and anotherof Y6 Z6, R1, R2, R3, R4, R67, R68, R69, R70, R7, R72, R73, R74, R75, R76, R77, R78 and R79 may be a constituent including a photosensitizer for phototherapy.
In still another family of embodiments of the compounds of Formula 6, at least one of Y6, Z6, R1, R2, R3, R4, R67, R68, R69, R70, R71, R72, R73, R74, R75, R76, R77, R78 and R79 is a constituent including Bm or Dm. However, in this family of embodiments, Bm and/or Dm is selected from the group consisting of a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
The present invention is also includes method of conjugating dyes to peptides or biomolecules by solid phase or solution synthesis methods. Accordingly, the term “dye” or the like herein shall refer to the compounds representatively illustrated in Formulas 1-6, including described bioconjugates of such compounds. For example, in some preferred bioconjugates of the invention, one or more of the constituents represented by Y groups, Z groups, and R groups of Formulas 1-6 is a constituent including Bm or Dm, wherein Bm and Dm may each be any of a peptide, a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent. In one family of embodiments of Formulas 1-6, one or more of the constituents represented by Y groups, Z groups, and R groups is a constituent including Bm or Dm, and at least one of Bm and Dm is a peptide such as Octreotide, Octreotate, Bombesin, Cholecystokinin, or Neurotensin. In another family of embodiments of Formulas 1-6, one or more of the constituents represented by Y groups, Z groups, and R groups is a constituent including Bm or Dm, and at least one of Bm and Dm is a photosensitizer for phototherapy. For example, one compound of one of Formulas 1-6 has two Y, Z, and/or R group constituents of which a Bm is a part. In this exemplary compound, one Bm is a photosensitizer, and the other Bm is a peptide. In still another family of embodiments of Formulas 1-6, one or more of the constituents represented by Y groups, Z groups, and R groups is a constituent including Bm or Dm, and Bm and/or Dm is selected from the group consisting of a protein, a cell, a metal chelating agent, a radioactive or nonradioactive metal complex, a photosensitizer for phototherapy, and an echogenic agent.
solid support;
presence or absence depends on R′ definition.
This invention is also related to the method of preventing fluorescence quenching. It is known that cyanine dyes generally form aggregates in aqueous media, leading to fluorescence quenching. Where the presence of a hydrophobic core in the dyes leads to fluorescence quenching, the addition of a biocompatible organic solvent, such as 1-50% dimethylsulfoxide (DMSO) for example, restored fluorescence by preventing aggregation and allowed in vivo organ visualization. Large fluorescence enhancement of dyes have been observed under the condition where the dye is encapsulated in, i.e. forms an inclusion complex with, cyclodextrins (W. R. Bergmark et al., Dramatic fluorescence effects for coumarin laser dyes coincluded with organic solvents in cyclodextrins. J. Phys. Chem., 1990, 94, 50208-5022). However, in vivo fluorescence enhancement of dyes coinjected with biocompatible organic solvents has not been previously described. Suitable organic solvent include, but are not limited to dimethylsulfoxide (DMSO), ethyl alcohol, isopropyl alcohol, glycerol, and other biocompatible polyols such as sorbitol, mannitol, xylitol, lactitol, erythritol, polydextrose, sucrose, fructose, maltose, hydrogenated starch hydrolysate (HSH), isomalt (palitinit), polyglycerol, hyperbranched polyglycerol, acetylated polyols, maltodextrine, cyclodextrine, dianhydrosorbitol, starches, polysaccharides, etc. as known to one skilled in the art.
The dye-biomolecule conjugates are used for optical tomographic, endoscopic, photoacoustic, phototherapeutic, and sonofluorescent applications for the detection and treatment of tumors and other abnormalities. The phototherapeutic photosensitizers may include those operating via direct (Type 1) mechanism as described by Rajagopalan et al. (U.S. Pat. No. 6,485,704, and U.S. patent application Ser. Nos. 09/766,347, and 09/898,887, incorporated herein by reference in their entirety), or by photodynamic (PDT or Type II) mechanism as described by Jori et al. (Tumour photosensitizers: approaches to enhance the selectivity and efficiency of photodynamic therapy, Journal of Photochemistry and Photobiology B: Biology 36 (1996) 87-93; Novel Therapeutic Modalities Based on Photosensitized Processes, EPA Newsletter No. 60, (July 1997) 12-18; Far-red-absorbing photosensitizers: their use in the photodynamic therapy of tumours, J. Photochem. Photobiol. A: Chem., 62 (1992) 371-378; and Second Generation Photosensitizers for the Photodynamic Therapy of Tumours, Light in Biology and Medicine, Vol. 2, (1991) 253-266), incorporated herein by reference in their entirety. Type 1 photosensitizers are those moieties that produce reactive intermediates such as free radicals, nitrenes, carbenes, and the like upon photoactivation. These include azides, peroxides, disulfides, sulfenates, and the like. Type II sensitizers are those that produce singlet oxygen species upon photoactivation. These include phthalocyanines, porphyrins, and the like. Incidentally, U.S. Pat. No. 6,217,848 is also herein incorporated by reference in its entirety.
Compounds of the present invention are prepared by the standard bioconjugate chemistry methods known in the art as illustrated in the forthcoming examples. Typically, the coupling between the dyes and the photosensitizers of the present invention is achieved by reacting the carboxyl group in one of the two aforementioned components with the amino group of the other component that results in the formation of the amide bond between the two units. Alternatively, if the two components contain either an amino or a hydroxyl group, the coupling would result in the formation of ester, urea, thiourea, carbamate, or carbonate species. Indeed, in one preferred family of embodiments, the compounds of formulas 1-4 have at least one constituent that includes at least one of Bm and Dm. In one preferred subfamily of such embodiments, at least one of Bm and Dm is a photosensitizer that may be utilized in phototherapy.
Compounds of the invention may be administered (e.g., as a pharmaceutically acceptable composition) for imaging by more than one modality. As one example, a paramagnetic metal ion such as gadolinium or manganese may be included in the chemical formula and the compound may be imaged by optical imaging alone, by magnetic resonance imaging (MR) alone, or by both optical and MR modalities. As another example, a compound may be imaged by optical imaging alone, by nuclear imaging alone, or by both optical and nuclear imaging modalities when a radioactive isotope is included in the chemical formula, such as replacing a halogen atom with a radioactive halogen, and/or including a radioactive metal ion such as Tc99, In111, etc.
It will also be appreciated that compounds of the invention (e.g., in the form of a pharmaceutically acceptable composition) may be administered with other contrast agents or media used to enhance an image from a non-optical modality. These include agents for enhancing an image obtained by modalities including but not limited to MR, ultrasound (US), x-ray, positron emission tomography (PET), computed tomography (CT), single photon emission computed tomography (SPECT), etc. Both optical and non-optical agents may be formulated as a single composition (that is, one composition containing one, two or more components, for example, an optical agent and a MR agent), or may be formulated as separate compositions. The inventive optical imaging contrast agent and the non-optical contrast agent are administered in doses effective to achieve the desired enhancement, diagnosis, therapy, etc., as known to one skilled in the art. The inventive compounds, either alone or combined with a contrast agent, may be administered to a patient, typically a warm-blooded animal, systemically or locally to the organ or tissue to be imaged. The patient is then imaged by optical imaging and/or by another modality. As one example of this embodiment, the inventive compounds may be added to contrast media compositions. As another example, the inventive compounds may be co-administered with contrast media, either simultaneously or within the same diagnostic and/or therapeutic procedure (for example, administering the inventive compound and administering a contrast agent then performing optical imaging followed by another imaging modality, or administering the inventive compound and administering a contrast agent then performing another imaging modality followed by optical imaging, or administering the inventive compound and optical imaging, then administering a contrast agent and MR, US, CT, etc. imaging, or administering a contrast agent and imaging by MR, US, CT, etc., then administering the inventive compound and optical imaging, or administering the inventive compound and a contrast agent, and simultaneously imaging by an optical modality and MR, US, CT, etc.). As another example, an optical imaging agent may be added as an additive or excipient for a non-optical imaging modality. In this embodiment, the optically active component, such as the dyes disclosed herein, could be added as a buffering agent to control pH or as a chelating agent to improve formulation stability, etc. in CT contrast media, MR contrast media, x-ray contrast media, US contrast media, etc. The CT, MR, x-ray, US contrast media would then also function as an optical imaging agent. The information obtained from the modality using the non-optical contrast agent is useful in combination with the image obtained using the optical contrast agent.
Compounds of the invention may be used for localized therapy. This may be accomplished by attaching a porphyrin or photodynamic therapy agent to a bioconjugate, shining light of appropriate wavelength for detection and treatment of the abnormality.
Compounds of the invention can be used for the detection of the presence of tumors and other abnormalities by monitoring the blood clearance profile of the compounds, for laser assisted guided surgery for the detection of small micrometastases of, e.g., somatostatin subtype 2 (SST-2) positive tumors, upon laparoscopy, and for diagnosis of atherosclerotic plaques and blood clots.
Compounds of the invention can be formulated into diagnostic and therapeutic compositions for enteral or parenteral administration. These compositions contain an effective amount of the compound along with conventional pharmaceutical carriers and excipients appropriate for the type of administration contemplated. For example, parenteral formulations advantageously contain at least one inventive compound in a sterile aqueous solution or suspension. Parenteral compositions may be injected directly or mixed with a large volume parenteral composition for systemic administration. Such solutions also may contain pharmaceutically acceptable buffers and, optionally, electrolytes such as sodium chloride.
Formulations for enteral administration may vary widely, as is well known in the art. In general, such formulations are liquids, which include an effective amount of the inventive agent in aqueous solution or suspension. Such enteral compositions may optionally include buffers, surfactants, thixotropic agents, and the like. Compositions for oral administration may contain flavoring agents and other ingredients for enhancing their organoleptic qualities.
In one embodiment, compositions of the invention may be formulated as micelles, liposomes, microcapsules, microparticles, nanocapsules, or nanoparticles. These formulations may enhance delivery, localization, target specificity, administration, etc. of the agents. Preparation and loading of these are well known in the art.
As one example, liposomes may be prepared from dipalmitoyl phosphatidylcholine (DPPC) or egg phosphatidylcholine (PC) because this lipid has a low heat transition. Liposomes are made using standard procedures as known to one skilled in the art (e.g., Braun-Falco et al., (Eds.), Griesbach Conference, Liposome Dermatics, Springer-Verlag, Berlin (1992), pp. 69-81; 91-117 which is expressly incorporated by reference herein). Polycaprolactone, poly(glycolic) acid, poly(lactic) acid, polyanhydride or lipids may be formulated as microspheres. As an illustrative example, the optical agent may be mixed with polyvinyl alcohol (PVA), the mixture then dried and coated with ethylene vinyl acetate, then cooled again with PVA. In a liposome, the optical agent may be within one or both lipid bilayers, in the aqueous between the bilayers, or with the center or core. Liposomes may be modified with other molecules and lipids to form a cationic liposome. Liposomes may also be modified with lipids to render their surface more hydrophilic which increases their circulation time in the bloodstream. The thus-modified liposome has been termed a “stealth” liposome, or a long-lived liposome, as described in U.S. Pat. No. 6,258,378, and in Stealth Liposomes. Lasic and Martin (Eds.) 1995 CRC Press, London, which are expressly incorporated by reference herein. Encapsulation methods include detergent dialysis, freeze drying, film forming, injection, as known to one skilled in the art and disclosed in, for example, U.S. Pat. No. 6,406,713 which is expressly incorporated by reference herein in its entirety.
The agent formulated in liposomes, microcapsules, etc. may be administered by any of the routes previously described. In a formulation applied topically, the optical agent is slowly released over time. In an injectable formulation, the liposome, capsule, etc., circulates in the bloodstream and is delivered to the desired site.
Diagnostic compositions of the invention may be administered in doses effective to achieve the desired enhancement. Such doses may vary widely, depending upon the particular compound employed, the organs or tissues to be imaged, the imaging equipment being used, and the like. The diagnostic compositions of the invention are used in the conventional manner. The compositions may be administered to a patient, typically a warm-blooded animal, either systemically or locally to the organ or tissue to be imaged, and the patient then subjected to the imaging procedure.
Compounds, compositions and methods of the present invention represent an important approach to the synthesis and use of novel cyanine and indocyanine dyes with a variety of photophysical and chemical properties. Various areas of the invention also represent an important approach to the use of small molecular targeting groups to image tumors by optical methods. The invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the scope of the invention in any manner.
(
A mixture of 1,1,2-trimethyl-[1H]-benz[e]indole (9.1 g, 43.58 mmoles) and 3-bromopropanoic acid (10.0 g, 65.37 mmoles) in 1,2-dichlorobenzene (40 mL) was heated at 110° C. for 12 hours. The solution was cooled to room temperature and the red residue obtained was filtered and washed with acetonitrile:diethyl ether (1:1) mixture. The solid obtained was dried under vacuum to give 10 g (64%) of light brown powder. A portion of this solid (6.0 g; 16.56 mmoles), glutaconaldehyde dianil monohydrochloride (2.36 g, 8.28 mmoles) and sodium acetate trihydrate (2.93 g, 21.53 mmoles) in ethanol (150 mL) were refluxed for 90 minutes. After evaporating the solvent, 40 mL of a 2 N aqueous HCl was added to the residue. The mixture was centrifuged and the supernatant was decanted. This procedure was repeated until the supernatant became nearly colorless. About 5 mL of water:acetonitrile (3:2) mixture was added to the solid residue and lyophilized to obtain 2 g of dark green flakes. The purity of the compound was established with 1H-NMR and liquid chromatography-mass spectroscopy (LC-MS).
(
A mixture of 1,1,2-trimethyl-[1H]-benz[e]indole (20 g, 95.6 mmoles) and 6-bromohexanoic acid (28.1 g, 144.1 mmoles) in 1,2-dichlorobenzene (250 mL) was heated at 110° C. for 12 hours. The green solution was cooled to room temperature and the brown solid precipitate formed was collected by filtration. After washing the solid with 1,2-dichlorobenzene and diethyl ether, the brown powder obtained (24 g, 64%) was dried under vacuum at room temperature. A portion of this solid (4.0 g; 9.8 mmoles), glutaconaldehyde dianil monohydrochloride (1.4 g, 5 mmoles) and sodium acetate trihydrate (1.8 g, 12.9 mmoles) in ethanol (80 mL) were refluxed for 1 hour. After evaporating the solvent, 20 mL of a 2 N aqueous HCl was added to the residue. The mixture was centrifuged and the supernatant was decanted. This procedure was repeated until the supernatant became nearly colorless. About 5 mL of water:acetonitrile (3:2) mixture was added to the solid residue and lyophilized to obtain about 2 g of dark green flakes. The purity of the compound was established with 1H-NMR and LC-MS.
(
This compound was prepared as described in Example 1 except that 1,1,2-trimethylindole was used as the starting material.
(
This compound was prepared as described in Example 1 except that □-bromohexaoxyethyleneglycolpropiolic acid was used in place of bromopropanoic acid and the reaction was carried out in 1,2-dimethoxypropane.
(
A solution of 50 ml of dimethylformamide and benzyl bromoacetate (16.0 g, 70 mmol) was stirred in a 100-mL three-neck flask. Solid potassium bicarbonate (7.8 g, 78 mmol) was added. The flask was purged with argon and cooled to 0° C. with an ice bath. To the stirring mixture was added dropwise a solution of ethanolamine (1.9 g, 31 mmol) and 4 ml of dimethylformamide over 5 minutes. After the addition was complete the mixture was stirred for 1 hour at 0° C. The ice bath was removed and the mixture stirred at room temperature overnight. The reaction mixture was partitioned between 100 ml of methylene chloride and 100 ml of saturated sodium bicarbonate solution. The layers were separated and the methylene chloride layer was again washed with 100 ml of saturated sodium bicarbonate solution. The combined aqueous layers were extracted twice with 25 ml of methylene chloride. The combined methylene chloride layers were washed with 100 ml of brine, and dried over magnesium sulfate. The methylene chloride was removed with aspirator vacuum at about 35° C., and the remaining dimethylformamide was removed with vacuum at about 45° C. The crude material was left on a vacuum line overnight at room temperature.
The crude material was then dissolved in 100 ml of methylene chloride at room temperature. Triphenylphosphine (8.91 g, 34 mmol) was added and dissolved with stirring. An argon purge was started and the mixture was cooled to 0° C. with an ice bath. The N-bromosuccinimide (6.05 g, 34 mmol) was added portionwise over two minutes. The mixture was stirred for 1.5 hours at 0° C. The methylene chloride was removed with vacuum and gave purple oil. This oil was triturated with 200 ml of ether with constant manual stirring. During this time the oil became very thick. The ether solution was decanted and the oil was triturated with 100 ml of ether. The ether solution was decanted and the oil was again triturated with a 100 ml portion of ether. The ether was decanted and the combined ether solution was allowed to stand for about two hours to allow the triphenylphosphine oxide to crystallize. The ether solution was decanted from the crystals and the solid was washed with 100 ml of ether. The volume of the combined ether abstracts was reduced with vacuum until a volume of about 25 ml was obtained. This was allowed to stand over night at 0° C. Ether (10 ml) was added to the cold mixture, which was mixed to suspend the solid. The mixture was percolated through a column of 45 g of silica gel and eluted with ether; 75 ml fractions were collected. The fractions that contained product, as determined by thin layer chromatography, were pooled and the ether was removed with vacuum. This yielded 10.1 g of crude product. The material was flash chromatographed on silica gel with hexane, changing to 9:1 hexane:ether. The product-containing fractions were pooled and the solvents removed with vacuum. This yielded 7.4 g (57% yield) of pure product.
A mixture of 10% palladium on carbon (1 g) and a solution of the benzyl ester (10 g) in 150 ml of methanol was hydrogenolyzed at 25 psi for two hours. The mixture was filtered over celite and the residue was washed with methanol. The solvent was evaporated to give viscous oil in quantitative yield.
Reaction of the bromide with 1,1,2-trimethyl-[1H]-benz[e]indole was carried out as described in Example 1.
The hydroxy-indole compound is readily prepared by a known method (P. L. Southwick, et al., One pot Fischer synthesis of (2,3,3-trimethyl-3-H-indol-5-yl)-acetic acid derivatives as intermediates for fluorescent biolabels. Org. Prep. Proced. Int. Briefs, 1988, 20(3), 279-284). Reaction of p-carboxymethylphenylhydrazine hydrochloride (30 mmol, 1 equiv.) and 1,1-bis(hydroxymethyl)propanone (45 mmole, 1.5 equiv.) in acetic acid (50 mL) at room temperature for 30 minutes and at reflux for one minute gives (3,3-dihydroxymethyl-2-methyl-3-H-indol-5-yl)-acetic acid as a solid residue. The reaction of 3-bromopropyl-N,N-bis(carboxymethyl)amine, which was prepared as described in Example 5, with the intermediate indole and subsequent reaction of the indole intermediate with glutaconaldehyde dianil monohydrochloride (see Example 1) gives the desired product.
The intermediate 2-chloro-1-formyl-3-hydroxymethylenecyclohexane was prepared as described in the literature (G. A. Reynolds and K. H. Drexhage, Stable heptamethine pyrylium dyes that absorb in the infrared. J. Org. Chem., 1977, 42(5), 885-888). Equal volumes (40 mL each) of dimethylformamide (DMF) and dichloromethane were mixed and the solution was cooled to −10° C. in acetone-dry ice bath. Under argon atmosphere, phosphorus oxychloride (40 mL) in dichloromethane was added dropwise to the cool DMF solution, followed by the addition of 10 g of cyclohexanone. The resulting solution was allowed to warm up to room temperature and refluxed for six hours. After cooling to room temperature, the mixture was poured into ice-cold water and stored at 4° C. for twelve hours. About 8 g of yellow powder was obtained after filtration. Condensation of the cyclic dialdehyde with the indole intermediate is carried out as described in Example 1. Further functionalization of the dye with bis isopropylidene acetal protected monosaccharide was accomplished by the method described in the literature (J. H. Flanagan, et al., Near infrared heavy-atom-modified fluorescent dyes for base-calling in DNA-sequencing application using temporal discrimination. Anal. Chem., 1998, 70(13), 2676-2684).
These dyes are prepared as described in Example 7. These dyes absorb in the infrared region. The typical example shown in
The procedure described below is for the synthesis of Octreotate. The amino acid sequence of Octreotate is: D-Phe-Cys′-Tyr-D-Trp-Lys-Thr-Cys′-Thr (SEQ ID NO:1), wherein Cys' indicates the presence of an intramolecular disulfide bond between two cysteine amino acids. Other peptides of this invention were prepared by a similar procedure with slight modifications in some cases.
The octapeptide was prepared by an automated fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis using a commercial peptide synthesizer from Applied Biosystems (Model 432A SYNERGY Peptide Synthesizer). The first peptide cartridge contained Wang resin pre-loaded with Fmoc-Thr on 25-μmole scale. Subsequent cartridges contained Fmoc-protected amino acids with side chain protecting groups for the following amino acids: Cys(Acm), Thr(t-Bu), Lys(Boc), Trp(Boc) and Tyr(t-Bu). The amino acid cartridges were placed on the peptide synthesizer and the product was synthesized from the C- to the N-terminal position. The coupling reaction was carried out with 75 μmoles of the protected amino acids in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt). The Fmoc protecting group was removed with 20% piperidine in dimethylformamide. After the synthesis was complete, the thiol group was cyclized with thallium trifluoroacetate and the product was cleaved from the solid support with a cleavage mixture containing trifluoroacetic acid (85%):water (5%):phenol (5%):thioanisole (5%) for 6 hours. The peptide was precipitated with t-butyl methyl ether and lyophilized with water:acetonitrile (2:3) mixture. The peptide was purified by HPLC and analyzed with LC/MS.
Octreotide, D-Phe-Cys′-Tyr-D-Trp-Lys-Thr-Cys′-Thr-OH (SEQ ID NO:2), wherein Cys′ indicates the presence of an intramolecular disulfide bond between two cysteine amino acids, was prepared by the same procedure.
Bombesin analogs were prepared by the same procedure except that cyclization with thallium trifluoroacetate was not needed. Side-chain deprotection and cleavage from the resin was carried out with 50 μL each of ethanedithiol, thioanisole and water, and 850 μL of trifluoroacetic acid. Two analogues were prepared: Gly-Ser-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (SEQ ID NO:3) and Gly-Asp-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (SEQ ID NO:4).
Cholecystokinin octapeptide analogs were prepared as described for Octreotate without the cyclization step. Three analogs were prepared: Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 (SEQ ID NO:5); Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2 (SEQ ID NO:6); and D-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2 (SEQ ID NO:7) wherein Nle is norleucine.
A neurotensin analog, D-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu (SEQ ID NO:8), was prepared as described for Octreotate without the cyclization step.
The method described below is for the synthesis of Octreotate-cyanine dye conjugates, but a similar procedure is used for the synthesis of other peptide-dye conjugates.
Octreotate was prepared as described in Example 9 but the peptide was not cleaved from the solid support and the N-terminal Fmoc group of Phe was retained. The thiol group was cyclized with thallium trifluoroacetate and the Phe was deprotected to liberate the free amine. Bisethylcarboxymethylindocyanine dye (53 mg, 75 umoles) was added to an activation reagent consisting of a 0.2 M solution of HBTU/HOBt in DMSO (375 μL), and 0.2 M solution of diisopropylethylamine in DMSO (375 μL). The activation was complete in about 30 minutes and the resin-bound peptide (25 μmoles) was added to the dye. The coupling reaction was carried out at room temperature for three hours. The mixture was filtered and the solid residue was washed with DMF, acetonitrile and THF. After drying the green residue, the peptide was cleaved from the resin and the side chain protecting groups were removed with a mixture of 85% trifluoroacetic acid, 2.5% water, 2.5% thioanisole and 2.5% phenol. The resin was filtered and cold t-butyl methyl ether (MTBE) was used to precipitate the dye-peptide conjugate, which was dissolved in acetonitrile:water (2:3) mixture and lyophilized. The product was purified by HPLC to give the monoOctreotate-Bismethylcarboxymethylindocyanine dye (Cytate 1, 80%) and the bis Octreotate-Bismethylcarboxymethylindocyanine dye (Cytate 2, 20%). The monoOctreotate conjugate is obtained almost exclusively (>95%) over the bis conjugate by reducing the reaction time to two hours. However, this also leads to incomplete reaction, and the free Octreotate must be carefully separated from the dye conjugate in order to avoid saturation of the receptors by the non-dye conjugated peptide.
Octreotate-bispentylcarboxymethylindocyanine dye was prepared as described above with some modifications. Bispentylcarboxymethylindocyanine dye (60 mg, 75 μmoles) was added to an activation reagent consisting of a 0.2 M solution of HBTU/HOBt in DMSO (400 μL), and 0.2 M solution of diisopropylethylamine in DMSO (400 μL). The activation was complete in about 30 minutes and the resin-bound peptide (25 μmoles) was added to the dye. The reaction was carried out at room temperature for three hours. The mixture was filtered and the solid residue was washed with DMF, acetonitrile and THF. After drying the green residue, the peptide was cleaved from the resin and the side chain protecting groups were removed with a mixture of 85% trifluoroacetic acid, 2.5% water, 2.5% thioanisole and 2.5% phenol.
The resin was filtered and cold t-butyl methyl ether (MTBE) was used to precipitate the dye-peptide conjugate, which was dissolved in an acetonitrile:water (2:3) mixture and lyophilized. The product was purified by HPLC to give Octreotate-1,1,2-trimethyl-[1H]-benz[e]indole propanoic acid conjugate (10%), monoOctreotate-bispentylcarboxymethylindocyanine dye (Cytate 3, 60%) and bis Octreotate-bispentylcarboxymethylindocyanine dye (Cytate 4, 30%).
The dye-peptide conjugates are sparingly soluble in water and require the addition of solubilizing agents or co-solvents. Addition of 1-20% aqueous ethanol to the conjugates partially quenched the fluorescence intensity in vitro and the fluorescence was completely quenched in vivo (the conjugate was not detected by the charged coupled device (CCD) camera). Addition of 1-50% of DMSO either re-established or increased the fluorescence intensity of the conjugates in vitro and in vivo. The dye fluorescence remained intense for over one week. The DMSO formulations were well tolerated by experimental animals used for this invention.
A non-invasive in vivo fluorescence imaging apparatus was employed to assess the efficacy of contrast agents developed for tumor detection in animal models. A LaserMax Inc. laser diode of nominal wavelength 780 nm and nominal power of 40 mW was used. The detector was a Princeton Instruments model RTE/CCD-1317-K/2 CCD camera with a Rodenstock 10 mm F2 lens (stock #542.032.002.20) attached. An 830 nm interference lens (CV1 Laser Corp., part # F10-830-4-2) was mounted in front of the CCD input lens such that only emitted fluorescent light from the contrast agent was imaged. Typically, an image of the animal was taken pre-injection of contrast agent. This image was subsequently subtracted (pixel by pixel) from the post injection images. However, the background subtraction was never done once the animal had been removed from the sample area and returned at a later time for images taken several hours post injection.
DSL 6A tumors were induced in male Lewis rats in the left flank area by the introduction of material from a solid (donor) implant and the tumors were palpable in approximately 14 days. The animals were anesthetized with xylazine; ketamine; acepromazine 1.5:1.5:0.5 at 0.8 mL/kg via intramuscular injection. The area of the tumor (left flank) was shaved to expose tumor and surrounding surface area. A 21 gauge butterfly equipped with a stopcock and two syringes containing heparinized saline was placed into the later tail vein of the rat. Patency of the vein was checked prior to administration of the ICG via the butterfly apparatus. Each animal received 500 mL of a 0.42 mg/mL solution of ICG in water.
The Figures are false color images of fluorescent intensity measured at the indicated times, with images constrained to the tumor and a small surrounding area. As is shown, the dye intensity in the tumor is considerably diminished 30 minutes post-ICG injection.
The imaging apparatus and the procedure used are described as in Example 12. Prostrate tumors (Dunning R3327-H) were induced in young male Copenhagen rats in the left flank area from a solid implant. These tumors grow very slowly and palpable masses were present 4-5 months post implant.
The Figures are false color images of fluorescent intensity measured at the indicated times, with images constrained to the tumor and a small surrounding area. As is shown, the dye intensity in the tumor is considerably diminished 30 minutes post-ICG injection.
The imaging apparatus and the procedure used are described in Example 12. Rat pancreatic acinar carcinoma expressing the SST-2 receptor (CA20948) was induced by solid implant technique in the left flank area, and palpable masses were detected nine days post implant. The images obtained at 2 and 30 minutes post injection are shown in
The Figures are false color images of fluorescent intensity measured at the indicated times, with images constrained to the tumor and a small surrounding area. As is shown, the dye intensity in the tumor is considerably diminished and almost absent 30 minutes post-ICG injection.
The imaging apparatus and the procedure used are described in Example 12 except that each animal received 500 μl of a 1.0 mg/mL solution of Cytate 1 solution of 25% dimethylsulfoxide in water.
Rat pancreatic acinar carcinoma expressing the SST-2 receptor (CA20948) were induced by solid implant technique in the left flank area, and palpable masses were detected 24 days post implant. Images were obtained at various times post injection. Uptake into the tumor was seen at two minutes but was not maximal until about five minutes.
Using indocyanine green (ICG), three different tumor lines were imaged optically using a CCD camera apparatus. Two of the lines, DSL 6/A (pancreatic) and Dunning R3327H (prostate) indicated slow perfusion of the agent over time into the tumor and reasonable images were obtained for each. The third line, CA20948 (pancreatic), indicated only a slight but transient perfusion that was absent after only 30 minutes post injection. This indicated no non-specific localization of ICG into this line compared to the other two tumor lines, suggesting a different vascular architecture for this type of tumor (see
Octreotate is known to target somatostatin (SST-2) receptors, hence, cyano-Octreotates (Cytate 1 and Cytate 2) was prepared. Cytate 1 was evaluated in the CA20948 Lewis rat model. Using the CCD camera apparatus, localization of this dye was observed in the tumor (indicated by arrow) at 45 minutes post injection (
Tumor visualization was easily observed (indicated by arrow) showing specificity of this agent for the SST-2 receptors present in the CA20948 tumor line.
Individual organs were removed at about 24 hours post Cytate 1 administration and imaged. As shown in
The AR42-J cell line is derived from exocrine rat pancreatic acinar carcinoma. It can be grown in continuous culture or maintained in vivo in athymic nude mice, SCID mice, or in Lewis rats. This cell line is particularly attractive for in vitro receptor assays, as it is known to express a variety of hormone receptors including cholecystokinin (CCK), epidermal growth factor (EGF), pituitary adenylate cyclase activating peptide (PACAP), somatostatin (SST-2) and bombesin.
In this model, male Lewis rats were implanted with solid tumor material in a similar manner as described for the CA20948 rat model. Palpable masses were present seven days post implant, and imaging studies were conducted on animals at 10-12 days post implant when the mass had achieved about 2-2.5 g.
A laser of appropriate wavelength for excitation of the dye chromophore was directed into one end of a fiber optic bundle and the other end was positioned a few millimeters from the ear of a rat. A second fiber optic bundle was also positioned near the same ear to detect the emitted fluorescent light and the other end was directed into the optics and electronics for data collection. An interference filter (IF) in the collection optics train was used to select emitted fluorescent light of the appropriate wavelength for the dye chromophore.
Sprague-Dawley or Fischer 344 rats were used in these studies. The animals were anesthetized with urethane administered via intraperitoneal injection at a dose of 1.35 g/kg body weight. After the animals had achieved the desired plane of anesthesia, a 21 gauge butterfly with 12″ tubing was placed in the lateral tail vein of each animal and flushed with heparinized saline. The animals were placed onto a heating pad and kept warm throughout the entire study. The lobe of the left ear was affixed to a glass microscope slide to reduce movement and vibration.
Incident laser light delivered from the fiber optic was centered on the affixed ear. Data acquisition was then initiated, and a background reading of fluorescence was obtained prior to administration of the test agent. For Cytates 1 or 2, the peptide-dye conjugate was administered to the animal through a bolus injection, typically 0.5 to 2.0 ml, in the lateral tail vein. This procedure was repeated with several dye-peptide conjugates in normal and tumor bearing rats. Representative profiles as a method to monitor blood clearance of the peptide-dye conjugate in normal and tumor bearing animals are shown in
In rats treated with Cytates 1 or 2, the fluorescence signal rapidly increased to a peak value. The signal then decayed as a function of time as the conjugate cleared from the blood stream.
It should be understood that the embodiments of the present invention shown and described in the specification are only specific embodiments of inventors who are skilled in the art and are not limiting in any way. Therefore, various changes, modifications, or alterations to those embodiments may be made or resorted to without departing from the spirit of the invention in the scope of the following claims. The references cited are expressly incorporated by reference herein in their entirety.
This application is a continuation-in-part of: co-pending U.S. patent application Ser. No. 11/146,377 filed 6 Jun. 2005, which is a divisional of co-pending U.S. patent application Ser. No. 11/075,792 filed 9 May 2005, which is a continuation-in-part of co-pending U.S. patent application Ser. No. 10/800,531 filed 15 Mar. 2004, which is a continuation-in-part of U.S. Pat. No. 6,706,254 filed 23 May 2001, which is a continuation-in-part of U.S. Pat. No. 6,395,257 filed 18 Jan. 2000;co-pending U.S. patent application Ser. No. 09/757,332 filed 9 Jan. 2001, which is a continuation-in-part of U.S. Pat. No. 6,180,086 filed 18 Jan. 2000;co-pending U.S. patent application Ser. No. 10/654,033 filed 3 Sep. 2003, which is a continuation-in-part of U.S. Pat. No. 6,641,798 filed 23 May 2001, which is a continuation-in-part of U.S. Pat. No. 6,264,920 filed 10 Aug. 2000, which is a divisional of U.S. Pat. No. 6,180,087 filed 18 Jan. 2000; ANDco-pending U.S. patent application Ser. No. 11/126,137 filed 10 May 2005, which is a continuation of U.S. Pat. No. 6,939,532 filed 9 Jan. 2001, which is a continuation-in-part of U.S. Pat. No. 6,183,726 filed 18 Jan. 2000;each of which is expressly incorporated by reference herein it is entirety.
Number | Date | Country | |
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Parent | 11075792 | Mar 2005 | US |
Child | 11146377 | US |
Number | Date | Country | |
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Parent | 11146377 | Jun 2005 | US |
Child | 11371572 | US | |
Parent | 10800531 | Mar 2004 | US |
Child | 11075792 | US | |
Parent | 09864011 | May 2001 | US |
Child | 10800531 | US | |
Parent | 09484322 | Jan 2000 | US |
Child | 09864011 | US |