Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0056—Peptides, proteins, polyamino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0032—Methine dyes, e.g. cyanine dyes

Definitions

• This invention relates generally to compositions of cyanine
• NIR region near-infrared region are particularly useful because biological tissues are optically transparent in this region.
• B. C. Wilson Optical properties of NIR
• indocyanine green which absorbs and emits in the NIR region
• labeling reagents Sulfoindocyanine succinimidyl esters. Bioconjugate
• hydrophilic peptides typically polyethyleneglycol or oiigosaccharide conjugates have been used, but these resulted in long-
• Patonay A new method for the synthesis of heptamethine cyanine dyes:
• dendrimers which are useful in biomedical applications.
• molecular volume of the dye is substantially smaller than the carrier.
• the invention is directed to compositions, and methods of
• compositions of low molecular weight biomolecule-dye
• the inventive composition comprises cyanine dyes of general formula 1
• W 3 and X 3 may be the same or different and are selected from
• Y 3 is selected
• Z 3 is selected from the
• A is a single or a double bond
• B, C- f , and D- may the same or different and are selected from the group
• B-,, C and D may together form a 6- to 1 2-membered carbocyclic ring or
• a 6- to 12-membered heterocyciic ring optionally containing one or more
• R 1 to R 4 , and R 29 to R 37 are independently selected from the group
• Bm and Dm are independently selected from the
• bioactive peptide group consisting of a bioactive peptide, a protein, a cell, an antibody, an
• antibody fragment a saccharide, a giycopeptide, a peptidomimetic, a
• nonradioactive metal complex and an echogenic agent
• Y 4 is selected
• a 2 is a single or a double bond
• B 2 , C 2 , and D 2 may the same or different and are selected from the group
• B 2 , C 2 , and D 2 may together form a 6- to 1 2-membered carbocyclic ring or
• a 6- to 1 2-membered heterocyciic ring optionally containing one or more
• R 1 to R 4 , and R 45 to R 57 are independently selected from the group
• Bm and Dm are independently selected from the
• bioactive peptide group consisting of a bioactive peptide, a protein, a cell, an antibody, an
• antibody fragment a saccharide, a glycopeptide, a peptidomimetic, a
• nonradioactive metal complex and an echoge ⁇ ic agent
• the inventive composition comprises
• Y 5 is selected
• Z 5 is selected from the
• a 3 is a single or a double bond
• B 3 , C 3 , and D 3 may the same or different and are selected from the group
• B 3 , C 3 , and D 3 may together form a 6- to 1 2-membered carbocyclic ring or
• a 6- to 12-membered heterocyciic ring optionally containing one or more
• R 1 to R 4 , and R 58 to R 66 are independently selected from the group consisting of
• CTC Z O polyhydroxyalkyl, C 5 -C 20 poly hydroxy aryl, C C 10 aminoalkyl, cyano,
• a and c are independently from 1
• inventive composition comprises
• Y 6 is selected
• Z 6 is selected from the
• a 4 is a single or a double bond
• B 4 , C 4 , and D 4 may the same or different and are selected from the group
• B 4 , C 4 , and D 4 may together form a 6- to 1 2-membered carbocyclic ring or
• a 6- to 1 2-membered heterocyciic ring optionally containing one or more
• R 1 to R 4 and R 67 to R 79 are independently selected from the group consisting of
• nonradioactive metal complex and an echogenic agent
• FIG. 1 shows the reaction pathway for the synthesis of bis-
• FIG. 2 shows the reaction pathway for the synthesis of
• FIG. 3 shows the reaction pathway for the synthesis of
• FIG. 4 shows the reaction pathway for the synthesis of non-
• FIG. 5 shows the reaction pathway for the synthesis of long
• FIG. 6 shows the reaction pathway for the synthesis of
• FIGS. 7A-F represent images at 2 minutes and 30 minutes
• ICG indocyanine green
• FIGS. 8A-B show a comparison of the uptake of ICG
• FIG. 8A Cytate 1 (FIG. 8B) in rats with the pancreatic acinar
• FIGS. 9A-B show images of rats with the pancreatic acinar
• FIG. 1 0 is an image of individual organs taken from a rat with
• pancreatic acinar carcinoma (CA20948) about 24 hours after injection
• FIG. 1 1 is an image of bombesinate in an AR42-J tumor-
• FIG. 1 2 is the clearance profile of Cytate 1 from the blood of
• FIG. 1 3 is the clearance profile of Cytate 1 from the blood of
• pancreatic tumor-bearing rat a pancreatic tumor-bearing rat.
• FIG. 14 is the clearance profile of Cytate 2 from the blood of
• FIG. 1 5 is the clearance profile of Cytate 2 from the blood of
• pancreatic tumor-bearing rat a pancreatic tumor-bearing rat.
• FIG. 1 6 is the clearance profile of Cytate 4 from the blood of
• compositions of the present invention comprising
• FIGS. 1 -5 are illustrated in the art and are illustrated in FIGS. 1 -5.
• FIG. 1 illustrates the synthetic scheme for bis-carboxylic acid
• FIG. 2 illustrates the synthetic scheme for tetracarboxylic
• FIG. 3 illustrates the synthetic scheme for polyhydroxy-
• FIG. 4 illustrates the synthetic scheme for non-aggregating
• FIG. 5 illustrates the synthetic scheme for long wavelength-
• the inventive bioconjugates have the
• A is a single or a double bond
• B 1 f C 1 # and D 1 are independently selected from the group consisting of
• heterocyciic ring optionally containing one or more oxygen, nitrogen, or
• a 3 and b 3 are independently from 0 to 3; R 1 to R 4 , and R 29 to
• R 37 are independently selected from the group consisting of hydrogen, C-, -C 10 alkyl, C 5 -C 12 aryl, C C 10 alkoxyl, C r C 10 polyhydroxyalkyl, C 5 -C 12
• Bm and Dm are independently selected from the group consisting of a
• bioactive peptide containing 2 to 30 amino acid units, an antibody, a
• radioactive or nonradioactive metal complex and an echogenic agent
• the inventive bioconjugates have
• Z 4 is selected from the group consisting of
• a 2 is a single or a double bond;
• B 2 , C 2 , and D 2
• a 2 , B 2 , C 2 , and D 2 may together
• heterocyciic ring optionally containing one or more oxygen, nitrogen, or
• a 4 and b 4 are independently from 0 to 3; R 1 to R 4 , and R 45 to
• R 57 are independently selected from the group consisting of hydrogen,
• Bm and Dm are independently selected from the group consisting of a
• bioactive peptide containing 2 to 30 amino acid units, an antibody, a
• radioactive or nonradioactive metal complex and an echogenic agent
• the inventive bioconjugates have the following properties:
• Z 5 is selected from the group consisting of
• a 3 is a single or a double bond; B 3 , C 3 , and D 3
• a 3 , B 3 , C 3 , and D 3 may together
• heterocyciic ring optionally containing one or more oxygen, nitrogen, or
• R 1 to R 4 , and R 58 to R 66 are independently
• Bm and Dm are independently selected from the group consisting of a
• bioactive peptide containing 2 to 30 amino acid units, an antibody, a
• radioactive or nonradioactive metal complex and an echogenic agent
• a and c are independently from 1 to 10
• b and d are independently from
• the inventive bioconjugates have the following properties:
• Z 6 is selected from the group consisting of
• a 4 is a single or a double bond; B 4 , C 4 , and D 4
• R 1 to R 4 , and R 67 to R 79 are independently selected from the
• Bm and Dm are independently selected from the group consisting of a
• bioactive peptide containing 2 to 30 amino acid units, an antibody, a
• radioactive or nonradioactive metal complex and an echogenic agent
• This invention is also related to the method of conjugating
• inventive dyes to peptides or biomolecules by solid phase or solution
• FIG. 6 illustrates the synthetic scheme for
• AA amino acids
• R CONH peptide
• R' R (bis conjugate) or COOH
• This invention is also related to the method of preventing
• a biocompatible organic solvent such as 1 -50% dimethylsulfoxide (DMSO) for example, restored fluorescence by
• the inventive dye-biomolecule conjugates are used for optical and optical
• Dye-biomolecule conjugates are also used for localized
• This may be accomplished by attaching a porphyrin or other
• inventive conjugates can also be used for the detection
• compositions of the invention can be formulated into
• compositions contain an effective amount of the
• Parenteral compositions may be injected directly or mixed with a large volume parenteral composition for
• Such solutions also may contain
• Formulations for enteral administration may vary widely, as is
• Such enteral compositions may optionally include buffers,
• compositions for oral are provided.
• administration may also contain flavoring agents and other ingredients for
• the diagnostic compositions are administered in doses.
• Such doses may vary
• organs or tissues are widely, depending upon the particular dye employed, the organs or tissues
• the imaging equipment being used, and the like.
• compositions may be administered to a patient, typically a
• compositions and methods represent an
• indocyanine dyes with a variety of photophysical and chemical properties.
• the combination also represents an important approach to the use of
• small molecular targeting groups to image tumors by optical methods.
• bromoacetate (1 6.0 g, 70 mmol) was stirred in a 1 00 ml three-neck flask.
• N-bromosuccinimide (6.05 g, 34 mmol) was added portionwise over two
• the hydroxy-indole compound is readily prepared by a known method.
• Example 1 gives the desired product.
• 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
• the octapeptide was prepared by an automated
• fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis using a
• the first peptide cartridge contained
• the peptide was purified by HPLC and analyzed with LC/MS.
• Nle-Gly-Trp-Nle-Asp-Phe-NH 2 SEQ ID NO:6; and D-Asp-Tyr-Nle-Gly-Trp-
• NIe-Asp-Phe-NH 2 (SEQ ID NO:7), where Nle is norleucine.
• a neurotensin analog D-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu
• an activation reagent consisting of a 0.2 M solution of HBTU/HOBt in
• an activation reagent consisting of a 0.2 M solution of HBTU/HOBt in
• the dye-peptide conjugates are sparingly soluble in water
• DSL 6A tumors were induced in male Lewis rats in the left
• the tumors were palpable in approximately 1 4 days.
• the animals were
• Each animal received 500 ⁇ L of a 0.42 mg/mL solution of ICG
• FIGS. 7A-B are tumor images at two minutes (FIG. 7A) and
• Tetracarboxylic acid cyanine dyes were synthesized as
• R R 2 fused phenyl (Formula 2).
• FIGS. 7C-D are images of a rat with an induced
• Example 1 Rat pancreatic acinar carcinoma expressing the SST-2
• flank area and palpable masses were detected nine days post implant.
• FIGS. 7E-F are images of a rat with an induced pancreatic
• FIG. 7E two minutes (FIG. 7E) and 30 minutes (FIG. 7F) post injection.
• Example 2 except that each animal received 500 ⁇ l of a 1 .0 mg/mL
• flank area and palpable masses were detected 24 days post implant.
• FIGS. 8A-B show a comparison of the uptake of ICG and
• ICG indocyanine green
• DSL 6/A and R3327H are not as highly vascularized as
• Octreotate is known to target somatostatin (SST-2)
• Cytate 1 was evaluated in the CA20948 Lewis rat model.
• Cytate 1 was observed in the pancreas, adrenals and tumor tissue, while
• the AR42-J cell line is derived from exocrine rat pancreatic
• acinar carcinoma The acinar carcinoma. It can be grown in continuous culture or maintained in
• CCK cholecystokinin
• EGF epidermal growth factor
• PACAP somatostatin
• SST-2 somatostatin
• FIG. 1 1 is an image of bombesinate in an AR42-J tumor-
• 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.
• Incident laser light delivered from the fiber optic was
• bolus injection typically 0.5 to 2.0
• FIG. 1 2 shows
• FIG. 1 3 shows the clearance
• FIG. 14 shows the clearance profile of Cytate 2 from the
• FIG. 1 5 shows the clearance profile of Cytate 2
• pancreatic tumor CA20948-bearing rat
• FIG. 1 6 shows the clearance profile of Cytate 4 from the

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Indole Compounds (AREA)

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Cited By (9)

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• 2001
  • 2001-01-17 WO PCT/US2001/001468 patent/WO2001052744A1/en not_active Ceased
  • 2001-01-17 AU AU2001232818A patent/AU2001232818A1/en not_active Abandoned
  • 2001-01-17 JP JP2001552799A patent/JP2003529632A/ja active Pending
  • 2001-01-17 EP EP01904882A patent/EP1250091B1/en not_active Expired - Lifetime
  • 2001-01-17 AT AT01904882T patent/ATE392183T1/de not_active IP Right Cessation

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