Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0493—Steroids, e.g. cholesterol, testosterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/57572—Gastrin releasing peptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
  • C07K7/086—Bombesin; Related peptides

Definitions

• This invention relates to novel gastrin releasing peptide (GRP) compounds which are useful as diagnostic imaging agents or radiotherapeutic agents.
• GRP compounds are labeled with radionuclides or labels detectable by in vivo light imaging and include the use of novel linkers between the label and the targeting peptide, which provides for improved pharmacokinetics.
• radiopharmaceuticals e.g., diagnostic imaging agents, radiotherapeutic agents
• diagnostic imaging agents e.g., diagnostic imaging agents, radiotherapeutic agents
• radiotherapeutic agents e.g., diagnostic imaging agents, radiotherapeutic agents
• These newer radiopharmaceutical agents typically consist of a targeting agent connected to a metal chelator, which can be chelated to (e.g., complexed with) a diagnostic metal radionuclide such as, for example, technetium or indium, or a therapeutic metal radionuclide such as, for example, lutetium, yttrium, or rhenium.
• a diagnostic metal radionuclide such as, for example, technetium or indium
• a therapeutic metal radionuclide such as, for example, lutetium, yttrium, or rhenium.
• the role of the metal chelator is to hold (i.e., chelate) the metal radionuclide as the radiopharmaceutical agent is delivered to the desired site.
• a metal chelator which does not bind strongly to the metal radionuclide would render the radiopharmaceutical agent ineffective for its desired use since the metal radionuclide would therefore not reach its desired site.
• metal chelators such as that reported in U.S. Pat. No. 5,662,885 to Pollak et. al., hereby incorporated by reference, which exhibited strong binding affinity for metal radionuclides and the ability to conjugate with the targeting agent.
• the concept of using a "spacer" to create a physical separation between the metal chelator and the targeting agent was further introduced, for example in U.S. Pat. 5,976,495 to Pollak et. al., hereby incorporated by reference.
• the targeting agent by virtue of its affinity for certain binding sites, is to direct the diagnostic agent, such as a radiopharmaceutical agent containing the metal radionuclide, to the desired site for detection or treatment.
• the targeting agent may include a protein, a peptide, or other macromolecule which exhibits a specific affinity for a given receptor.
• Other known targeting agents include monoclonal antibodies (MAbs), antibody fragments (F ab 's and (F at e's), and receptor-avid peptides.
• MAbs monoclonal antibodies
• F ab 's and (F at e's) include receptor-avid peptides.
• GRP gastrin releasing peptide
• GRP-R gastrin releasing peptide receptors
• BBN bombesin
• tetradecapeptide 14 amino acid peptide isolated from frog skin
• Bombesin and GRP analogues may take the form of agonists or antagonists. Binding of GRP or BBN agonists to the GRP receptor increases the rate of cell division of these cancer cells and such agonists are internalized by the cell, while binding of GRP or BBN antagonists generally does not result in either internalization by the cell or increased rates of cell division. Such antagonists are designed to competitively inhibit endogenous GRP binding to GRP receptors and reduce the rate of cancer cell proliferation. See, e.g., Hoffken, K.; Peptides in Oncology II, Somatostatin Analogues and Bombesin Antagonists (1993), pp. 87-112.
• BBN or GRP analogues that are antagonists.
• Davis et al. Metabolic Stability and Tumor Inhibition of Bombesin/GRP Receptor Antagonists, Peptides, vol. 13, pp. 401- 407, 1992.
• the drug In designing an effective compound for use as a diagnostic or therapeutic agent for cancer, it is important that the drug have appropriate in vivo targeting and pharmacokinetic properties. For example, it is preferable that for a radiopharmaceutical, the radiolabeled peptide have high specific uptake by the cancer cells (e.g., via GRP receptors). In addition, it is also preferred that once the radionuclide localizes at a cancer site, it remains there for a desired amount of time to deliver a highly localized radiation dose to the site.
• radiolabeled peptides that are cleared efficiently from normal tissues is also an important factor for radiopharmaceutical agents.
• biomolecules e.g., MAb, F ab or peptides
• metallic radionuclides via a chelate conjugation
• a large percentage of the metallic radionuclide in some chemical form can become "trapped" in either the kidney or liver parenchyma (i.e., is not excreted into the urine or bile).
• the compounds include a chemical moiety capable of complexing a medically useful metal ion or radionuclide (metal chelator) attached to a GRP receptor targeting peptide by a linker or spacer group.
• metal chelator metal chelator
• these compounds include an optical label (e.g. a photolabel or other label detectable by light imaging, optoacoustical imaging or photoluminescence) attached to a GRP receptor targeting peptide by a linker or spacer group.
• compounds of the present invention may have the formula: M-N-O-P-G wherein M is the metal chelator (in the form complexed with a metal radionuclide or not), or the optical label, N-O-P is the linker, and G is the GRP receptor targeting peptide.
• the metal chelator M may be any of the metal chelators known in the art for complexing with a medically useful metal ion or radionuclide.
• Preferred chelators include DTPA, DOTA, DO3 A, HP-DO3A, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM, or peptide chelators, such as, for example, those discussed herein.
• the metal chelator may or may not be complexed with a metal radionuclide, and may include an optional spacer such as a single amino acid.
• Preferred metal radionuclides for scintigraphy or radiotherapy include 99m Tc, 5I Cr, 67 Ga, 68 Ga, 47 Sc, 51 Cr, ]67 Tm, 14I Ce, 1 H In, I6 ⁇ Yb, 175 Yb, 140 La, 90 Y, 88 Y, 153 Sm, ,66 Ho, 165 Dy, ,66 Dy, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, ]86 Re, ,88 Re, 203 Pb, 2 ⁇ Bi, 2]2 Bi, 213 Bi, 2,4 Bi, 225 Ac, ,05 Rh, ]09 Pd, H7m Sn, I49 Pm, 161 Tb, 377 Lu, 198 Au and I99 Au.
• the preferred radionuclides include 64 Cu, 67 Ga, 68 Ga, 99m Tc, and , n In, with 99m Tc, and ⁇ ⁇ In being particularly preferred.
• the preferred radionuclides include 64 Cu, 90 Y, 105 Rh, m In, U7m Sn, 149 Pm, 153 Sm, !6! Tb, ,66 Dy, 166 Ho, ,75 Yb, 177 Lu, ,86/, 88 R e , and )99 Au, with 177 Lu and 90 Y being particularly preferred.
• a most preferred chelator used in compounds of the invention is 1 -substituted 4,7,10-tricarboxymethyl 1,4,7,10 tetraazacyclododecane triacetic acid (DO3A).
• DO3A 1,4,7,10 tetraazacyclododecane triacetic acid
• Preferred labels include, without limitation, optical dyes, including organic chromophores or fluorophores, such as cyanine dyes light absorbing compounds, light reflecting and scattering compounds, and bioluminescent molecules.
• optical dyes including organic chromophores or fluorophores, such as cyanine dyes light absorbing compounds, light reflecting and scattering compounds, and bioluminescent molecules.
• the linker N-O-P contains at least one non-alpha amino acid.
• the linker N-O-P contains at least one substituted bile acid.
• the linker N-O-P contains at least one non- alpha amino acid with a cyclic group.
• the GRP receptor targeting peptide may be GRP, bombesin or any derivatives or analogues thereof.
• the GRP receptor targeting peptide is a GRP or bombesin analogue which acts as an agonist.
• the GRP receptor targeting peptide is a bombesin agonist binding moiety disclosed in U.S. Pat. 6,200,546 and US 2002/0054855, incorporated herein by reference.
• a single or multi-vial kit that contains all of the components needed to prepare the diagnostic or therapeutic agents of the invention is provided in an exemplary embodiment of the present invention.
• a novel method of radiotherapy using the compounds of the invention is also provided, as is a novel method for preparing a radiotherapeutic agent comprising the step of adding to an injectable therapeutic medium a substance comprising a compound of the invention.
• FIG. 1A is a graphical representation of a series of chemical reactions for the synthesis of intermediate C ((3 ⁇ , 5/)-3-( H-Fluoren-9-ylmethoxy)aminocholan-24-oic acid), from A (Methy]-(3/3, 5 ?)-3-aminocholan-24-ate) and B ((3 ⁇ , 5/J)-3-aminocholan-24-oic acid), as described in Example I.
• FIG. IB is a graphical representation of the sequential reaction for the synthesis of N-[(3 ⁇ ,5 ⁇ )-3-[[[[[[4,7,l 0-Tris(carboxymethyl)-1 ,4,7,10-tetraazacyclododec-l - yl]acetyl]amino] acetyljamino] cholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl- glycyl-L-histidyl-L-leucyl-L-methioninamide (L62), as described in Example I.
• FIG. 2A is a graphical representation of the sequential reaction for the synthesis of N-[4-[[[[[[4,7,10-Tris(carboxymethyl)-l, 4,7,10-tetraazacyclododec-l- yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-trypto ⁇ hyl-L-alanyl-L-valyl-glycyl- L-histidyl-L-leucyl-L-methioninamide (L70), as described in Example II.
• FIG. 2B is a general graphical representation of the sequential reaction for the synthesis of N-[4-[2-[[[4,7,l 0-Tris(carboxymethyl)-1 ,4,7,10-tetraazacyclododec-l - yl]acetyl]amino]ethoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L- histidyl-L-leucyl-L-methioninamide (L73), N-[3-[[[[[4,7,l 0-Tris(carboxymethyl)-1 ,4,7,10- tetraazacyclododec-l-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl- L-valyl-glycyl-L-L
• FIG. 2C is a chemical structure of the linker used in the synthesis reaction of
• FIG. 2B for synthesis of N-[4-[2-[[[4,7,10-Tris(carboxymethyl)-l, 4,7,10-tetraazacyclododec- l-yl]acetyl]amino]ethoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L- histidyl-L-leucyl-L-methioninamide (L73), as described in Example II.
• FIG. 2D is a chemical structure of the linker used in the synthesis reaction of
• FIG. 2B for synthesis ofN-[3-[[[[[4,7,10-Tris(carboxymethyl)-l,4,7,10-tetraazacyclododec-l- yl]acetyl]amino]methyl]benzoyl]-L-glutaminy]-L-tryptophyl-L-alany]-L-va]yl-glycyl-L- histidyl-L-leucyl-L-methioninamide (L115), as described in Example II.
• FIG. 2E is a chemical structure of the linker used in the synthesis reaction of
• FIG. 2B for synthesis of N-[4-[[[[4,7,10-Tris(carboxymethyl)-l, 4,7,10-tetraazacyclododec-l- yl]acetyl]amino]methyl]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L- histidyl-L-leucyl-L-methioninamide (L116), as described in Example II.
• FIG. 2F is a graphical representation of the sequential reaction for the synthesis of N-[[4,7,l 0-Tris(carboxymethyl)-1 ,4,7, 10-tetraazacyclododec-l -yl]acetyl]glycyl- 4-piperidinecarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl- L-methioninamide (L74), as described in Example II.
• FIG. 3A is a graphical representation of a series of chemical reactions for the synthesis of intermediate (3 ⁇ ,5 ⁇ )-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-l 2- oxocholan-24-oic acid (C), as described in Example III.
• FIG. 3B is a graphical representation of the sequential reaction for the synthesis of N-[(3 ⁇ ,5 ⁇ )-3-[[[[[[4,7,10-Tris(carboxymethyl)-l,4,7,10-tetraazacyclododec-l- yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-glutaminyl-L-try ⁇ tophyl-L- alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L67), as described in Example III.
• FIG. 3C is a chemical structure of (3 ⁇ ,5 ⁇ )-3-Amino-12-oxocholan-24-oic acid (B), as described in Example III.
• FIG. 3D is a chemical structure of (3 ⁇ ,5 ⁇ )-3-[[(9H-Fluoren-9- ylmethoxy)amino]acetyl]amino-12-oxocholan-24-oic acid (C), as described in Example III.
• FIG 3E is a chemical structure of ⁇ -[(3 ⁇ ,5 ⁇ )-3-[[[[[[4,7,l 0-
• FIG. 4 A is a graphical representation of a sequence of reactions to obtain intermediates (3 ⁇ ,5 ⁇ ,12a)-3-[[(9 ⁇ -Fluoren-9-ylmethoxy)amino]acetyl]amino-12- hydroxycholan-24-oic acid (3a) and (3 ⁇ ,5 ⁇ ,7a,12a)-3-[[(9H-Fluoren-9- ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid (3b), as described in Example IN. [0038] FIG.
• 4B is a graphical representation of the sequential reaction for the synthesis of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-l,4,7,10-tetraazacyclododec- l-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L- tryptophyl-L-alanyl-L-valyl-g]ycyl-L-histidyl-L-]eucyl-L-methioninamide (L63), as described in Example IN.
• FIG. 4C is a graphical representation of the sequential reaction for the synthesis of N-[(3 ⁇ ,5 ⁇ ,7a,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-l, 4,7,10-tetraazacyclo dodec-l-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl- L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L64), as described in Example IN.
• FIG. 4D is a chemical structure of (3 ⁇ ,5 ⁇ ,7a,12a)-3-amino-7,12- dihydroxycholan-24-oic acid (2b), as described in Example IN.
• FIG. 4E is a chemical structure of (3 ⁇ ,5 ⁇ ,12a)-3-[[(9H-Fluoren-9- ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oic acid (3a), as described in Example IV;
• FIG. 4F is a chemical structure of (3 ⁇ ,5 ⁇ ,7a,12a)-3-[[(9H-Fluoren-9- ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid (3b), as described in Example IN.
• FIG. 4G is a chemical structure ofN-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[[[[4,7,10- Tris(carboxymethyl)-l, 4,7,10-tetraazacyclododec-l-yl]acetyl]amino]acetyl]amino]-12- hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl- L-leucyl-L-methioninamide (L63), as described in Example IN.
• FIG. 4H is a chemical structure ofN-[(3 ⁇ ,5 ⁇ ,7a,12a)-3-[[[[[[4,7,10-
• FIG. 5 A is a general graphical representation of the sequential reaction for the synthesis of 4-[[[[4,7, 10-Tris(carboxym ethyl)- 1 ,4,7, 10-tetraazacyclododec-l - yl]acetyl]amino]methyl]benzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L- histidyl-L-leucyl-L-methioninamide (L71); and J , r ⁇ r ⁇ -4-[[[[4,7,10-tris(carboxymethyl)-
• FIG. 5B is a chemical structure of the linker used in compound L71 as shown in Fig. 5A and as described in Example V.
• FIG. 5C is a chemical structure of the linker used in compound L72 as shown in Fig. 5A and as described in Example V.
• FIG. 5D is a chemical structure of Rink amide resin functionalised with bombesin[7-14] (B), as described in Example N.
• FIG. 5E is a chemical structure of 7>a/w-4-[[[(9H-fluoren-9- ylmethoxy)carbonyl]amino]methyl]cyclohexanecarboxylic acid (D), as described in Example
• FIG. 6A is a graphical representation of a sequence of reactions for the synthesis of intermediate linker 2-[[[9H-Fluoren-9- ylmethoxy)carbonyl]amino]methyl]benzoic acid (E), as described in Example NI.
• FIG. 6B is a graphical representation of a sequence of reactions for the synthesis of intermediate linker 4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3- nitrobenzoic acid ( ⁇ ), as described in Example NI.
• FIG. 6C is a graphical representation of the synthesis of N-[2-[[[[4,7, 10-
• FIG. 6D is a graphical representation of the synthesis of N-[4-[[[[4,7,l 0-
• FIG. 7A is a graphical representation of a sequence of reactions for the synthesis of intermediate linker [4-[[[9H-Fluoren-9- ylmethoxy)carbonyl]amino]methyl]phenoxy] acetic acid (E), as described in Example NIL
• FIG. 7B is a graphical representation of the synthesis of N-[[4-[[[[4,7,l 0- Tris(carboxym ethyl)- 1 ,4,7, 10-tetraazacyclododec- 1 - yl]acetyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl- L-histidyl-L-leucyl-L-methioninamide (L124), as described in Example VII.
• FIG. 7C is a chemical structure of N-[[4-[[[[4,7,l O-Tris(carbo ⁇ ymethyl)- 1, 4,7,10-tetraazacyclododec-l-yl]acetyl]amino]methy]]phenoxy]acetyl]-L-glutaminyl-L- tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L124), as described in Example VII.
• FIG. 8A is a graphical representation of a sequence of reactions for the synthesis of intermediate 4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3- methoxybenzoic acid (E), as described in Example VIII.
• FIG. 8B is a graphical representation of the synthesis of N-[4-[[[[4,7,l 0-
• FIG. 8C is a chemical structure of N-[4-[[[[4,7,l O-Tris(carboxymethyl)- l,4,7,10-tetraazacyclododec-l-yl]acetyl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L- tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L125), as described in Example NIII.
• FIG. 9A is a graphical representation of a reaction for the synthesis of 3-
• FIG. 9B is a graphical representation of a reaction for the synthesis of 6-
• FIG. 9C is a graphical representation of a reaction for the synthesis of 4- [[[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]methylamino]benzoic acid ⁇ (D), as described in Example IX.
• FIG. 9D is a graphical representation of a reaction for the synthesis of N-[4-
• FIG. 10A is a graphical representation of a reaction for the synthesis of 7-
• FIG. 10B is a graphical representation of a reaction for the synthesis of N-[4-
• FIG. 11 A is a graphical representation of a reaction for the synthesis of NN-
• FIG. IIB is a graphical representation of a reaction for the synthesis of NN- Dimethylglycyl-L-serinyl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-(3 ⁇ ,5 ⁇ ,7a,12a)-3- amino-7,12-dihydroxy-24-oxocholan-24-yl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl- glycyl-L-histidyl-L-leucyl-L-methioninamide, (L239), as described in Example XL
• FIG. 12 A is a graphical representation of a reaction for the synthesis of 4-
• FIG. 12B is a graphical representation of a reaction for the synthesis of 4-[[
• FIG. 12C is a graphical representation of a reaction for the synthesis of 4- [[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methylbenzoic acid (E), as described in Example XII.
• FIG. 12D is a chemical structure of N-[4-[[[[4,7,l O-Tris(carboxymethyl)-
• FIG. 12E is a chemical structure of compound N-[4-[[[[4,7,l 0-
• FIG. 12F is a chemical structure of ⁇ -[4-[[[[4,7,10-Tris(carboxymethyl)- 1 ,4,7, 1 Otetraazacyclododec-1 -yl] acetyl]glycyl]amino]3-methylbenzoyl]L-glutaminyl-L- tryptophyl-1-alanyl-L-valyl-glycyl-L-histidyl-leucyl-L-methioninamide (L242), as described in Example XII.
• FIG. 13A is a graphical representation of a reaction for the synthesis of 4-
• FIG. 13B is a graphical representation of a reaction for the synthesis of N-[4-
• FIG. 13C is a chemical structure of compound L244, as described in Example
• FIG. 14A and FIG. 14B are graphical representations of the binding and competition curves described in Example XLIII.
• FIG. 15A is a graphical representation of the results of radiotherapy experiments described in Example LN.
• FIG. 15B is a graphical representation of the results of other radiotherapy experiments described in Example LN.
• FIG. 16 is a chemical structure of ⁇ -[4-[[[[4,7,l O-Tris(carboxymethyl)- 1,4,7,10 tetraazacyclododec-1-yl] acetyl]glycyl]amino]-L-Lysinyl-(3,6,9)-trioxaundecane- 1,1 1 -dicarboxylic acid-3,7-dideoxy-3-aminocholic acid)-L-arginyl-L-glutaminyl-L- triptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L65).
• FIG. 17 is a chemical structure ofN-[2-S-[[[[[12a-Hydroxy-17a-(l-methyl-3- carboxypropyl)etiocholan-3 ⁇ -carbamoylmethoxyethoxyethoxyacetyl]-amino -6-[4,7, 10- tris(carboxymethyl)-l, 4,7,10-tetraazacyclododec-l-yl]acetyl]amino]acetyl]amino] hexanoyl- L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L66).
• FIG. 18A is a chemical structure of N-[4-[[[[[[4,7,l O-Tris(carboxymethyl)-
• FIG. 18B is a chemical structure N-[4-[[[[[4,7,10-Tris(carboxymethyl)-
• 18C is a chemical structure N-[4-[[4,7,10-Tris(carboxymethyl)-l,4,7,10- tetraazacyclododec-l-y]]-2-hydroxy-3-propoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L- alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L144).
• FIG. 18D is a chemical structure N-[(3 ⁇ ,5 ⁇ ,7 «,72 ⁇ )-3-[[[[[[4,7, 10-
• FIG. 18E is a chemical structure of N-[4-[[[[[[4,7,l O-Tris(carboxymethyl)-
• FIG. 19 dicloses chemical structures of intermediates which may be used to prepare compounds L64 and L70 as described in Example LNI.
• FIG. 20 is a graphical representation of the preparation of L64 using segment coupling as described in Example LNI.
• FIG. 21 is a graphical representation of the preparation of (lR)-l-(Bis ⁇ 2- [bis(carboxymethyl)amino] ethyl ⁇ amino)pro ⁇ ane-3-carboxylic acid- 1 -carboxyl-glycyl-4- aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L- methioninamide (L201).
• FIG. 22A is a graphical representation of chemical structure of chemical intermediates used to prepare L202.
• FIG. 22B is a graphical representation of the preparation of N-[(3 ⁇ ,5 ⁇ ,12a)-3-
• FIG. 23A is a graphical representation of chemical structure of chemical intermediates used to prepare L203.
• FIG. 23B is a graphical representation of the preparation of N-[(3 ⁇ ,5 ⁇ ,12a)-3-
• FIG. 24 is a graphical representation of the preparation of N-[(3 ⁇ ,5 ⁇ ,12a)-3-
• FIG. 25 is a graphical representation of the preparation of N-[(3 ⁇ ,5 ⁇ ,12a)-3- [[[4,7,10-Tris(carboxymethyl)-1, 4,7,10-tetraazacyclododec-l-yl]acetyl]amino]-4- aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L- leucyl-L-methioninamide (L205).
• FIG. 26A is a graphical representation of chemical structures of chemical intermediates used to prepare L206.
• FIG. 26B is a graphical representation of the preparation of N-[(3 ⁇ ,5 ⁇ ,12a)-3-
• FIG. 27A is a graphical representation of chemical structures of chemical intermediates used to prepare L207.
• FIG. 27B is a graphical representation of the preparation of N-[(3 ⁇ ,5 ⁇ ,12a)-3-
• FIG. 28 is a graphical representation of the preparation of N-[(3 ⁇ ,5 ⁇ ,12a)-3-
• FIG.29A is a graphical representation of chemical structures of chemical intermediates used to prepare L209.
• FIG. 29B is a graphical representation of the preparation of L209.
• FIG.30A is a graphical representation of chemical structures of chemical intermediates used to prepare L210.
• FIG. 30B is a chemical structure of L210.
• FIG. 31 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10-
• FIG. 32 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxymethyl)- 1 ,4,7, 10-tetraazacyclododec-l -yl]acetyl]amino]-glycyl-4- aminobenzoyl-L-glutamyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L- methioninamide L212.
• FIG. 33 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxymethyl)-l,4,7,10-tetraazacyclododec-l-yl]acetyl]amino]-glycyl-4- aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L- methionine carboxylate L213.
• FIG. 34 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxymethyl)-l, 4,7,10-tetraazacyclododec-l -yl] acetyl] amino] -glycyl -4- aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L- histidyl-L-leucyl-L-methioninamide L214. [00109] FIG.
• FIG.36 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxymethyl)-l, 4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4- aminobenzoyl-L-glutaminyl-L-arginyl-L-leucyl-glycyl-L-asparginyl-L-glutaminyl-L- tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L215.
• FIG.36 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10-
• FIG. 37 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxymethyl)-l ,4,7,10-tetraazacyclododec-l -yl]acetyl]amino]-glycyl-4- aminobenzoyl-L-glutaminyl-L-lysyl-L-tyrosinyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl- L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L217.
• FIG.38 is a chemical structure of L218.
• FIG. 39 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxym ethyl)- 1, 4,7,10-tetraazacyclododec-l -yl] acetyl] amino] -gly cyl -4- aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L- histidyl-L-leucyl-aminopentyl, L219.
• FIG.40 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxym ethyl)- 1 ,4,7,10-tetraazacyclododec-l -yl]acetyl]amino]-glycyl-4- aminobenzoyl-L-glutaminyl-L-tryptophyl-L-serinyl-L-valyl-D-alanyl-L-histidyl-L-leucyl-L- methioninamide, L220.
• FIG. 41 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxymethyl)- 1 ,4,7, 10-tetraazacyclododec- 1 -yl] acetyl]amino]-glycyl-4- aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L- histidyl-L-leucyl-L-leucinamide, L221.
• FIG. 42 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxym ethyl)- 1, 4,7,10-tetraazacyclododec-l-yl]acetyl]amino]-glycyl-4- aminobenzoyl-D-tyrosinyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-L- histidyl-L-phenylalanyl-L-norleucinarnide, L222.
• FIG. 43 is a chemical structure ofN-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10-
• FIG. 44 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxymethyl)-l , 4,7,10-tetraazacyclododec-l-yl]acetyl]amino]-glycyl-4- aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-glycyl-L-histidyl-L-phenylalanyl-L- leucinamide, L224.
• FIG. 45 is a chemical structure ofN-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxymethyl)-l ,4,7,10-tetraazacyclododec-l -yl]acetyl]amino]-glycyl-4- aminobenzoyl-L-leucyl-L-tryptophyl-L-alanyl-L-valinyl-glycyl-L-serinyl-L-phenylalanyl-L- methioninamide, L225.
• FIG. 46 is a chemical structure ofN-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10- Tris(carboxymethyl)-l, 4,7,10-tetraazacyclododec-l-yl]acetyl]amino]-glycyl-4- aminobenzoyl-L-histidyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L- methioninamide, L226.
• FIG. 47 is a chemical structure ofN-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,10-
• FIG. 48 is a chemical structure of N-[(3 ⁇ ,5 ⁇ ,12a)-3-[[[4,7,l 0- Tris(carboxymethyl)-l,4,7,10-tetraazacyclododec-l-yl]acetyl]amino]-glycyl-4- aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-phenylalanyl- L-methioninamide, L228.
• FIG. 49A is a graphical representation of a reaction for the synthesis of (3 ⁇ ,5 ⁇ ,7a,12a)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-dihydroxycholan-24-oic acid (B) as described in Example LNII.
• FIG. 49B is a graphical representation of a reaction for the synthesis of ⁇ -[3 ⁇ ,5 ⁇ ,7a,12a)-3-[[[2-[2-[[[4,7,10-Tris(carboxymethyl)-l,4,7,10-tetraazacyclododec-l- yl] acetyl] amino] ethoxy] ethoxy] acetyl] amino] -7,12-dihy droxy-24-oxocholan-24-yl] -L- glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L69), as described in Example LNII.
• FIG. 50 is a graphical representation of a reaction for the synthesis of N-[4-[2- Hydroxy-3-[4,7,10-tris(carboxymethyl)-l, 4,7,10-tetraazacyclododec-l -yl]propoxy]benzoyl]- L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L144), as described in Example LVIII.
• FIG. 51 is a chemical structure of L300. 00127] FIG.
• FIG. 52 is a TOCSY spectrum of Lu-L70 in DMSO-d 6 at 25°C. 00128]
• FIG. 53 is a COSY spectrum of Lu-L70 in DMSO-d 6 at 25°C. 00129]
• FIG. 54 is a NOESY spectrum of Lu-L70 in DMSO-d 6 at 25°C. 00130]
• FIG. 55 is a gHSQC spectrum of Lu-L70 in DMSO-d 6 at 25°C.
• FIG. 56 is a gHMBC spectrum of Lu-L70 in DMSO-d 6 at 25 °C. 00132]
• FIG. 57 is a gHSQCTOCSY spectrum of Lu-L70 in DMSO-d 6 at 25°C.
• FIG. 58 is a Regular 1H-NMR (bottom) and selective homo-decoupling of the water peak at 3.5 ppm of Lu-L70 in DMSO-d 6 at 15 °C.
• FIG. 59 is a TOCSY Spectrum of ,75 Lu-DO3 A-monoamide-Aoc-QWAVGHLM- NH 2 in DMSO-d 6 at 25 °C.
• FIG. 60 is a chemical structure of L70.
• FIG. 61 is a chemical structure of 175 Lu-DO3A-monoamide-Aoc-QWAVGHLM- NH 2 .
• FIG. 62 is a chemical structure of I75 Lu-L70 with a bound water molecule.
• FIG. 63 is a chemical structure of L301.
• the compounds include an optical label or a chemical moiety capable of complexing a medically useful metal ion or radionuclide (metal chelator) attached to a GRP receptor targeting peptide by a linker or spacer group.
• metal chelator metal ion or radionuclide
• compounds of the present invention may have the formula: M-N-O-P-G wherein M is the metal chelator (in the form complexed with a metal radionuclide or not), or an optical label, N-O-P is the linker, and G is the GRP receptor targeting peptide.
• M is the metal chelator (in the form complexed with a metal radionuclide or not), or an optical label
• N-O-P is the linker
• G is the GRP receptor targeting peptide.
• linkers of the present invention may have the formula: N-O-P wherein each of N, O and P are defined throughout the specification.
• Compounds meeting the criteria defined herein were discovered to have improved pharmacokinetic properties compared to other GRP receptor targeting peptide conjugates known in the art. For example, compounds containing the linkers of the present invention were retained in the bloodstream longer, and thus had a longer half life than prior known compounds.
• metal chelator refers to a molecule that forms a complex with a metal atom, wherein said complex is stable under physiological conditions. That is, the metal will remain complexed to the chelator backbone in vivo.
• a metal chelator is a molecule that complexes to a radionuclide metal to form a metal complex that is stable under physiological conditions and which also has at least one reactive functional group for conjugation with the linker N-O-P.
• the metal chelator M may be any of the metal chelators known in the art for complexing a medically useful metal ion or radionuclide.
• the metal chelator may or may not be complexed with a metal radionuclide.
• the metal chelator can include an optional spacer such as, for example, a single amino acid (e.g., Gly) which does not complex with the metal, but which creates a physical separation between the metal chelator and the linker.
• the metal chelators of the mvention may include, for example, linear, macrocyclic, terpyridine, and N 3 S, N 2 S 2 , orN 4 chelators (.see also, U.S. 5,367,080, U.S. 5,364,613, U.S. 5,021,556, U.S. 5,075,099, U.S. 5,886,142, the disclosures of which are incorporated by reference in their entirety), and other chelators known in the art including, but not limited to, HYNIC, DTP A, EDTA, DOTA, TETA, and bisamino bisthiol (BAT) chelators (see also U.S. 5,720,934).
• N 4 chelators are described in U.S. Patent Nos. 6,143,274; 6,093,382; 5,608,110; 5,665,329; 5,656,254; and 5,688,487, the disclosures of which are incorporated by reference in their entirety.
• Certain N 3 S chelators are described in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Patent Nos. 5,662,885; 5,976,495; and 5,780,006, the disclosures of which are incorporated by reference in their entirety.
• the chelator may also include derivatives of the chelating ligand mercapto- acetyl-glycyl-glycyl-glycine (MAG3), which contains an N 3 S, and N 2 S 2 systems such as MAMA (monoamidemonoaminedithiols), DADS (N 2 S diaminedithiols), CODADS and the like.
• MAG3 chelating ligand mercapto- acetyl-glycyl-glycyl-glycine
• MAMA monoamidemonoaminedithiols
• DADS N 2 S diaminedithiols
• CODADS CODADS
• the metal chelator may also include complexes containing ligand atoms that are not donated to the metal in a tetradentate array.
• complexes containing ligand atoms that are not donated to the metal in a tetradentate array include the boronic acid adducts of technetium and rhenium dioximes, such as those described in U.S. Patent Nos. 5,183,653; 5,387,409; and 5,118,797, the disclosures of which are incorporated by reference in their entirety.
• Examples of preferred chelators include, but are not limited to, diethylenetriamine pentaacetic acid (DTP A), 1,4,7, 10-tetraazacyclotetradecane-l,4,7,10-tetraacetic acid (DOTA), 1 -substituted 1 ,4,7,-tricarboxymethyl 1,4,7,10-tetraazacyclododecane triacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), 4-carbonylmethyl-l 0-phosponomethyl- 1,4,7,10-Tetraazacyclododecane-l,7-diacetic acid (Cm4pml0d2a); and 1,4,8,11- tetraazacyclotetradecane- 1,4,8,11-tetraacetic acid (TETA).
• DTP A diethylenetriamine pentaacetic acid
• DOTA 1,4,7, 10-tetraazacyclotetradecane-l,4,7,10-
• Additional chelating ligands are ethylenebis-(2-hydroxy-phenylglycine) (EHPG), and derivatives thereof, including 5-C1-EHPG, 5-Br-EHPG, 5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof, including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl-DTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivatives thereof; the class of macrocyclic compounds which contain at least 3 carbon atoms, more preferably at least 6, and at least two heteroatoms (O and/or N), which macrocyclic compounds can consist of one ring, or two or three rings joined together at the hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA,
• Particularly preferred metal chelators include those of Formula 1, 2 and 3 (for ⁇ n In and radioactive lanthanides, such as, for example I77 Lu, 90 Y, I53 Sm, and 166 Ho) and those of Formula 4, 5 and 6 (for radioactive 99 Tc, 186 Re, and I 88 Re) set forth below.
• These and other metal chelating groups are described in U.S. Patent Nos. 6,093,382 and 5,608,110, which are incorporated by reference in their entirety. Additionally, the chelating group of formula 3 is described in, for example, U.S. Patent No. 6,143,274; the chelating group of formula 5 is described in, for example, U.S. Patent Nos.
• spacers which do not actually complex with the metal radionuclide such as an extra single amino acid Gly may be attached to these metal chelators (e.g., N,N-dimethylGly-Ser-Cys- Gly; N,N-dimethylGly-Thr-Cys-Gly; N,N-diethylGly-Ser-Cys-Gly; N,N-dibenzylGly-Ser- Cys-Gly).
• metal chelators e.g., N,N-dimethylGly-Ser-Cys- Gly; N,N-dimethylGly-Thr-Cys-Gly; N,N-diethylGly-Ser-Cys-Gly; N,N-dibenzylGly-Ser- Cys-Gly.
• Other useful metal chelators such as all of those disclosed in U.S. Pat. No.
• sulfur protecting groups such as Acm (acetamidomethyl), trityl or other known alkyl, aryl, acyl, alkanoyl, aryloyl, mercaptoacyl and organothiol groups may be attached to the cysteine amino acid of these metal chelators.
• R is alkyl, preferably methyl.
• X is either CH or O;
• Y is C J -C J O branched or unbranched alkyl; aryl, aryloxy, arylamino, arylaminoacyl; arylalkyl - where the alkyl group or groups attached to the aryl group are C ⁇ -C] 0 branched or unbranched alkyl groups, C J -C J O branched or unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or polyhydroxy- polyalkoxyalkyl groups;
• the metal chelator includes cyclic or acyclic polyaminocarboxylic acids such as DOTA (1,4,7,10-tetraazacyclododecane -1,4,7,10- tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DTPA-bismethylamide, DTPA- bismorpholineamide, Cm4pml 0d2a (1 ,4-carbonylm ethyl- 10-phosponomethyl- 1 ,4,7,10- Tetraazacyclododecane-l,7-diacetic acid), DO3A N-[[4,7,10-Tris(carboxymethyl)- 1,4,7, 10- tetraazacyclododec-1-yl] acetyl, HP-DO3A, DO3A-monoamide and derivatives thereof.
• DOTA 1,4,7,10-tetraazacyclododecane -1,4,7,10- te
• Preferred metal radionuclides for scintigraphy or radiotherapy include 99m Tc, 51 Cr, 67 Ga, 68 Ga, 47 Sc, 51 Cr, ,67 Tm, ]4I Ce, ⁇ n In, 168 Yb, ,75 Yb, I40 La, 90 Y, 88 Y, ] 53 Sm, ]66 Ho, 165 Dy, 166 Dy, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, )86 Re, 188 Re, 203 Pb, 2, 1 Bi, 2I2 Bi, 213 Bi, 214 Bi, ,05 Rh, ]09 Pd, 117m Sn, 149 Pm, ,61 Tb, 177 Lu, 198 Au and I99 Au and oxides or nitrides thereof.
• the choice of metal will be determined based on the desired therapeutic or diagnostic application.
• the preferred radionuclides include 64 Cu, 67 Ga, 68 Ga, 99m Tc, and ⁇ 'in, with 99m Tc and n 'in being especially preferred.
• the preferred radionuclides include 64 Cu, 90 Y, ]05 Rh, ] I I In, 117ra Sn, ]49 Pm, 153 Sm, 16, Tb, 166 Dy, I 66 Ho, ]75 Yb, 177 Lu, , 86 !88 Re, and ,99 Au, with ⁇ 7 Lu and 90 Y being particularly preferred.
• 99m Tc is particularly useful and is a preferred for diagnostic radionuclide because of its low cost, availability, imaging properties, and high specific activity.
• This isotope has a single photon energy of 140 keV and a radioactive half-life of about 6 hours, and is readily available from a 99 Mo- 99m Tc generator.
• the 99m Tc labeled peptide can be used to diagnose and monitor therapeutic 177 Of) progress in primary tumors and metastases.
• Peptides labeled with Lu, Y or other therapeutic radionuclides can be used to provide radiotherapy for primary tumors and metastasis related to cancers of the prostate, breast, lung, etc. IB. Optical Labels
• the compounds of the invention may be conjugated with photolabels, such as optical dyes, including organic chromophores or fluorophores, having extensive delocalized ring systems and having absorption or emission maxima in the range of 400-1500 nm.
• the compounds of the invention may alternatively be derivatized with a bioluminescent molecule.
• the preferred range of absorption maxima for photolabels is between 600 and 1000 nm to minimize interference with the signal from hemoglobin.
• photoabsorption labels have large molar absorptivities, e.g. > 10 5 cmf'M "1 , while fluorescent optical dyes will have high quantum yields.
• optical dyes include, but are not limited to those described in WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO 98/47538, and references cited therein.
• the photolabels may be covalently linked directly to compounds of the invention, such as, for example, compounds comprised of GRP receptor targeting peptides and linkers of the invention.
• indocyanine green which absorbs and emits in the NIR region has been used for monitoring cardiac output, hepatic functions, and liver blood flow and its functionalized derivatives have been used to conjugate biomolecules for diagnostic purposes
• the linker N-O-P contains at least one non- alpha amino acid.
• N is 0 (where 0 means it is absent), an alpha or non-alpha amino acid or other linking group
• O is an alpha or non-alpha amino acid
• P is 0, an alpha or non-alpha amino acid or other linking group, wherein at least one of N, O or P is a non-alpha amino acid.
• N Gly
• O a non-alpha amino acid
• P 0.
• Alpha amino acids are well known in the art, and include naturally occurring and synthetic amino acids.
• Non-alpha amino acids are also known in the art and include those which are naturally occurring or synthetic.
• HPLC method refers to the 10 minute time for the HPLC gradient.
• HPLC RT refers to the retention time of the compound in the HPLC.
• MS refers to mass spectra where molecular weight is calculated from mass/unit charge (m/e).
• 4 IC 50 refers to the concentration of compound to inhibit 50% binding of iodinated bombesin to a GRP receptor on cells.
• 2B Linkers Containing At Least One Substituted Bile Acid
• the linker N-O-P contains at least one substituted bile acid.
• N is 0 (where 0 means it is absent), an alpha amino acid, a substituted bile acid or other linking group; O is an alpha amino acid or a substituted bile acid; and P is 0, an alpha amino acid, a substituted bile acid or other linking group, wherein at least one of N, O or P is a substituted acid.
• Bile acids are found in bile (a secretion of the liver) and are steroids having a hydroxyl group and a five carbon atom side chain terminating in a carboxyl group.
• substituted bile acids at least one atom such as a hydrogen atom of the bile acid is substituted with another atom, molecule or chemical group.
• substituted bile acids include those having a 3 -amino, 24-carboxyl function optionally substituted at positions 7 and 12 with hydrogen, hydroxyl or keto functionality.
• Other useful substituted bile acids in the present invention include substituted cholic acids and derivatives thereof.
• Specific substituted cholic acid derivatives include: (3 ⁇ ,5 ⁇ )-3-aminocholan-24-oic acid; (3 ⁇ ,5 ⁇ ,12a)-3-amino-12-hydroxycholan-24-oic acid; (3 ⁇ ,5 ⁇ ,7a,12a)-3-amino-7,12-dihydroxycholan-24-oic acid; Lys-(3,6,9)-trioxaundecane-l ,11 -dicarbonyl-3,7-dideoxy-3- aminocholic acid); (3 ⁇ ,5 ⁇ ,7a)-3-amino-7-hydroxy-12-oxocholan-24-oic acid; and (3 ⁇ ,5 ⁇ ,7a)-3-amino-7-hydroxycholan-24-oic acid.
• HPLC method refers to the 10 minute time for the HPLC gradient.
• HPLC RT refers to the retention time of the compound in the HPLC.
• MS refers to mass spectra where molecular weight is calculated from mass/unit charge (m/e).
• IC 5 o refers to the concentration of compound to inhibit 50% binding of iodinated bombesin to a GRP receptor on cells.
• 2C Linkers Containing At Least One Non- Alpha Amino Acid With A Cyclic Group
• the linker N-O-P contains at least one non-alpha amino acid with a cyclic group.
• N is 0 (where 0 means it is absent), an alpha amino acid, a non-alpha amino acid with a cyclic group or other linking group;
• O is an alpha amino acid or a non-alpha amino acid with a cyclic group; and
• P is 0, an alpha amino acid, a non-alpha amino acid with a cyclic group, or other linking group, wherein at least one of N, O or P is a non-alpha amino acid with a cyclic group.
• Non-alpha amino acids with a cyclic group include substituted phenyl, biphenyl, cyclohexyl or other amine and carboxyl containing cyclic aliphatic or heterocyclic moieties.
• Examples of such include: 4-aminobenzoic acid (hereinafter referred to as "Abz4 in the specification") 3-aminobenzoic acid 4-aminomethyl benzoic acid 8-aminooctanoic acid trans-4-aminomethylcyclohexane carboxylic acid 4-(2-aminoethoxy)benzoic acid isonipecotic acid 2-aminomethylbenzoic acid 4-amino-3-nitrobenzoic acid 4-(3-carboxymethyl-2-keto-l-benzimidazolyl-piperidine 6-(piperazin-l -yl)-4-(3H)-quinazolinone-3-acetic acid (2S,5S)-5-amino-l,2,4,5,6,7-hex
• HPLC method refers to the 10 minute time for the HPLC gradient.
• HPLC RT refers to the retention time of the compound in the HPLC.
• MS refers to mass spectra where molecular weight is calculated from mass/unit charge (m/e).
• IC 5 0 refers to the concentration of compound to inhibit 50% binding of iodinated bombesin to a GRP receptor on cells.
• linking groups which may be used within the linker N-O-P include a chemical group that serves to couple the GRP receptor targeting peptide to the metal chelator or optical label while not adversely affecting either the targeting function of the GRP receptor targeting peptide or the metal complexing function of the metal chelator or the detectabihty of the optical label.
• Suitable other linking groups include peptides (i.e., amino acids linked together) alone, a non-peptide group (e.g., hydrocarbon chain) or a combination of an amino acid sequence and a non-peptide spacer.
• linking groups for use within the linker N-O-P include L-glutamine and hydrocarbon chains, or a combination thereof.
• other linking groups for use withm the linker N-O-P include a pure peptide linking group consisting of a series of amino acids (e.g., diglycine, triglycine, gly-gly-glu, gly-ser-gly, etc.), in which the total number of atoms between the N- terminal residue of the GRP receptor targeting peptide and the metal chelator or the optical label in the polymeric chain is ⁇ 12 atoms.
• R is an activated COOH group
• linking groups for use within the linker N-O-P may be formed from linker precursors having electrophiles or nucleophiles as set forth below: LP1 : a linker precursor having on at least two locations of the linker the same electrophile El or the same nucleophile Nul ; LP2: a linker precursor having an electrophile El and on another location of the linker a different electrophile E2; LP3: a linker precursor having a nucleophile Nul and on another location of the linker a different nucleophile Nu2; or LP4: a linker precursor having one end functionalized with an electrophile El and the other with a nucleophile Nul .
• the preferred nucleophiles Nul/Nu2 include-OH, -NH, -NR, -SH, -HN-NH 2 , - RN-NH 2 , and -RN-NHR', in which R' and R are independently selected from the definitions for R given above, but for R' is not H.
• Y can be selected from the following groups:
• the GRP receptor targeting peptide (i.e., G in the formula M-N-O-P-G) is any peptide, equivalent, derivative or analogue thereof which has a binding affinity for the GRP receptor family.
• the GRP receptor targeting peptide may take the form of an agonist or an antagonist.
• a GRP receptor targeting peptide agonist is known to "activate" the cell following binding with high affinity and may be internalized by the cell.
• GRP receptor targeting peptide antagonists are known to bind only to the GRP receptor on the cell without being internalized by the cell and without “activating" the cell.
• the GRP receptor targeting peptide is an agonist.
• the GRP agonist is a bombesin (BBN) analogue and/or a derivative thereof.
• the BBN derivative or analog thereof preferably contains either the same primary structure of the BBN binding region (i.e., BBN(7-14) [SEQ ID NO:l]) or similar primary structures, with specific amino acid substitutions that will specifically bind to GRP receptors with better or similar binding affinities as BBN alone (i.e., Kd ⁇ 25nM).
• Suitable compounds include peptides, peptidomimetics and analogues and derivatives thereof.
• the presence of L-methionine (Met) at position BBN- 14 will generally confer agonistic properties while the absence of this residue at BBN- 14 generally confers antagonistic properties [Hoffken, 1994].
• Analogues of BBN receptor targeting peptides include molecules that target the GRP receptors with avidity that is greater than or equal to BBN, as well as muteins, retropeptides and retro-inverso-peptides of GRP or BBN.
• these analogues may also contain modifications which include substitutions, and/or deletions and/or additions of one or several amino acids, insofar that these modifications do not negatively alter the biological activity of the peptides described therein. These substitutions may be carried out by replacing one or more amino acids by their synonymous amino acids.
• Synonymous amino acids within a group are defined as amino acids that have sufficient physicochemical properties to allow substitution between members of a group in order to preserve the biological function of the molecule.
• Deletions or insertions of amino acids may also be introduced into the defined sequences provided they do not alter the biological functions of said sequences. Preferentially such insertions or deletions should be limited to 1, 2, 3, 4 or 5 amino acids and should not remove or physically disturb or displace amino acids which are critical to the functional conformation.
• Muteins of the GRP receptor targeting peptides described herein may have a sequence homologous to the sequence disclosed in the present specification in which amino acid substitutions, deletions, or insertions are present at one or more amino acid positions. Muteins may have a biological activity that is at least 40%, preferably at least 50%, more preferably 60-70%, most preferably 80-90% of the peptides described herein.
• Analogues of GRP receptor targeting peptides also include peptidomimetics or pseudopeptides incorporating changes to the amide bonds of the peptide backbone, including thioamides, methylene amines, and E-olefins. Also peptides based on the structure of GRP, BBN or their peptide analogues with amino acids replaced by N- substituted hydrazine carbonyl compounds (also known as aza amino acids) are included in the term analogues as used herein.
• the GRP receptor targeting peptide can be prepared by various methods depending upon the selected chelator.
• the peptide can generally be most conveniently prepared by techniques generally established and known in the art of peptide synthesis, such as the solid-phase peptide synthesis (SPPS) approach.
• SPPS solid-phase peptide synthesis
• SPPS involves the stepwise addition of amino acid residues to a growing peptide chain that is linked to an insoluble support or matrix, such as polystyrene.
• the C-terminal residue of the peptide is first anchored to a commercially available support with its amino group protected with an N-protecting agent such as a t-butyloxycarbonyl group (Boc) or a fluorenylmethoxycarbonyl (Fmoc) group.
• an N-protecting agent such as a t-butyloxycarbonyl group (Boc) or a fluorenylmethoxycarbonyl (Fmoc) group.
• the amino protecting group is removed with suitable deprotecting agents such as TFA in the case of Boc or piperidine for Fmoc and the next amino acid residue (in N-protected form) is added with a coupling agent such as N,N'- dicyclohexylcarbodiimide (DCC), orN,N'-diisopropylcarbodiimide (DIG) or 2-(lH- benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU).
• DCC N,N'- dicyclohexylcarbodiimide
• DIG N,N'-diisopropylcarbodiimide
• HBTU 2-(lH- benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate
• a suitable reagent such as trifluoroacetic acid (TFA) or hydrogen flu
• the linker may then be coupled to form a conjugate by reacting the free amino group of the Trp residue of the GRP receptor targeting peptide with an appropriate functional group of the linker.
• the entire construct of chelator, linker and targeting moiety discussed above may also be assembled on resin and then cleaved by agency of suitable reagents such as trifluoroacetic acid or HF, as well.
• Bombesin (7-14) is subject to proteolytic cleavage in vitro and in vivo, which shortens the half-life of the peptide. It is well known in the literature that the amide bond of the backbone of the polypeptide may be substituted and retain activity, while resisting proteolytic cleavage. For example, to reduce or eliminate undesired proteolysis, or other degradation pathways that diminish serum stability, resulting in reduced or abolished bioactivity, or to restrict or increase conformational flexibility, it is common to substitute amide bonds within the backbone of the peptides with functionality that mimics the existing conformation or alters the conformation in the manner desired. Such modifications may produce increased binding affinity or improved pharmacokinetic behavior.
• the hydrolysis can also be prevented by incorporation of a D-amino acid of one of the amino acids of the labile amide bond, or by alpha-methyl aminoacid derivatives.
• Backbone amide bonds have also been replaced by heterocycles such as oxazoles, pyrrolidinones, imidazoles, as well as ketomethylenes and fluoroolefins.
• incorporación of the metal within the radiopharmaceutical conjugates can be achieved by various methods commonly known in the art of coordination chemistry.
• the metal is m Tc, a preferred radionuclide for diagnostic imaging, the following general procedure can be used to form a technetium complex.
• a peptide-chelator conjugate solution is formed by initially dissolving the conjugate in water, dilute acid, or in an aqueous solution of an alcohol such as ethanol. The solution is then optionally degassed to remove dissolved oxygen.
• a thiol protecting group such as Acm (acetamidomethyl), trityl or other thiol protecting group may optionally be used to protect the thiol from oxidation.
• the thiol protecting group(s) are removed with a suitable reagent, for example with sodium hydroxide, and are then neutralized with an organic acid such as acetic acid (pH 6.0-6.5).
• the thiol protecting group can be removed in situ during technetium chelation.
• the labeling step sodium pertechnetate obtained from a molybdenum generator is added to a solution of the conjugate with a sufficient amount of a reducing agent, such as stannous chloride, to reduce technetium and is either allowed to stand at room temperature or is heated.
• a reducing agent such as stannous chloride
• the labeled conjugate can be separated from the contaminants 99m TcO ⁇ and colloidal 99m TcO 2 chromatographically, for example with a C-l 8 Sep Pak cartridge [Millipore Corporation, Waters Chromatography Division, 34 Maple Street, Milford, Massachusetts 01757] or by HPLC using methods known to those skilled in the art.
• the labeling can be accomplished by a transchelation reaction.
• the technetium source is a solution of technetium that is reduced and complexed with labile ligands prior to reaction with the selected chelator, thus facilitating ligand exchange with the selected chelator.
• suitable ligands for transchelation includes tartrate, citrate, gluconate, and heptagluconate.
• the conjugate can be labeled using the techniques described above, or alternatively, the chelator itself may be labeled and subsequently coupled to the peptide to form the conjugate; a process referred to as the "prelabeled chelate" method.
• Re and Tc are both in row NIIB of the Periodic Table and they are chemical congeners.
• compounds of the present invention can be used to treat and/or detect any pathology involving overexpression of GRP receptors (or ⁇ MB receptors) by procedures established in the art of radiodiagnostics, radiotherapeutics and optical imaging.
• optical dyes include, but are not limited to those described in WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841 , WO 96/23524, WO 98/47538, and references cited therein, hereby incorporated by reference in their entirety.
• GRP-R expression is highly upregulated in a variety of human tumors. See e.g., WO 99/62563.
• compounds of the invention may be widely useful in treating and diagnosing cancers, including prostate cancer (primary and metastatic), breast cancer (primary and metastatic), colon cancer, gastric cancer, pancreatic cancer, non small cell lung cancer, small cell lung cancer, gastrinomas, melanomas, glioblastomas, neuroblastomas, uterus leiomyosarcoma tumors, prostatic intraepithelial neoplasias [PIN], and ovarian cancer. Additionally, compounds of the invention may be useful to distinguish between conditions in which GRP receptors are upregulated and those in which they are not (e.g. chronic pancreatitis and ductal pancreatic carcinoma, respectively
• the compounds of the invention which, as explained in more detail in the Examples, show greater specificity and higher uptake in tumors in vivo than compounds without the novel linkers disclosed herein, exhibit an improved ability to target GRP receptor-expressing tumors and thus to image or deliver radiotherapy to these tissues. Indeed, as shown in the Examples, radiotherapy is more effective (and survival time increased) using compounds of the invention.
• the diagnostic application of these compounds can be as a first line diagnostic screen for the presence of neoplastic cells using scintigraphic, optical, sonoluminescence or photoacoustic imaging, as an agent for targeting neoplastic tissue using hand-held radiation detection instrumentation in the field of radioimmuno guided surgery (RIGS), as a means to obtain dosimetry data prior to administration of the matched pair radiotherapeutic compound, and as a means to assess GRP receptor population as a function of treatment over time.
• the therapeutic application of these compounds can be defined as an agent that will be used as a first line therapy in the treatment of cancer, as combination therapy where these agents could be utilized in conjunction with adjuvant chemotherapy, and/or as a matched pair therapeutic agent.
• the matched pair concept refers to a single unmetallated compound which can serve as both a diagnostic and a therapeutic agent depending on the radiometal that has been selected for binding to the appropriate chelate. If the chelator cannot accommodate the desired metals, appropriate substitutions can be made to accommodate the different metal while maintaining the pharmacology such that the behavior of the diagnostic compound in vivo can be used to predict the behavior of the radiotherapeutic compound.
• any suitable chemotherapeutic may be used, including for example, antineoplastic agents, such as platinum compounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate, adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, a, L -PAM or phennylalanine mustard), mercaptopurine, mitotane.
• antineoplastic agents such as platinum compounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate, adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside, arabinosyl adenine, mercap
• the therapeutic may be monoclonal antibody, such as a monoclonal antibody capable of binding to melanoma antigen.
• a conjugate labeled with a radionuclide metal such as 99m Tc
• a radionuclide metal such as 99m Tc
• a pharmaceutically acceptable carrier and/or solution such as salt solutions like isotonic saline.
• Radiolabeled scintigraphic imaging agents provided by the present invention are provided having a suitable amount of radioactivity.
• 99 Tc radioactive complexes it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 millicurie (mCi) to 100 mCi per mL.
• the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 30 mCi.
• the solution to be injected at unit dosage is from about 0.01 L to about 10 mL.
• the amount of labeled conjugate appropriate for administration is dependent upon the distribution profile of the chosen conjugate in the sense that a rapidly cleared conjugate may need to be administered in higher doses than one that clears less rapidly. In vivo distribution and localization can be tracked by standard scintigraphic techniques at an appropriate time subsequent to administration; typically between thirty minutes and 180 minutes depending upon the rate of accumulation at the target site with respect to the rate of clearance at non-target tissue.
• a gamma camera calibrated for the gamma ray energy of the nuclide incorporated in the imaging agent can be used to image areas of uptake of the agent and quantify the amount of radioactivity present in the site. Imaging of the site in vivo can take place in a few minutes. However, imaging can take place, if desired, hours or even longer, after the radiolabeled peptide is injected into a patient. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 hour to permit the taking of scintiphotos.
• the compounds of the present invention can be administered to a patient alone or as part of a composition that contains other components such as excipients, diluents, radical scavengers, stabilizers, and carriers, all of which are well-known in the art.
• the compounds can be administered to patients either intravenously or intraperitoneally.
• the compounds made in accordance with the present invention form stable, well-defined 99m Tc or Re labeled compounds. Similar compounds of the invention can also be made by using appropriate chelator frameworks for the respective radiometals, to form stable, well-defined products labeled with 153 Sm, 90 Y, 166 Ho, 105 Rh, 199 Au, 149 Pm, , 77 Lu, ] 11 In or other radiometals.
• the radiolabeled GRP receptor targeting peptides selectively bind to neoplastic cells expressing GRP receptors, and if an agonist is used, become internalized, and are retained in the tumor cells for extended time periods.
• the radioactive material that does not reach (i.e., does not bind) the cancer cells is preferentially excreted efficiently into the urine with minimal retention of the radiometal in the kidneys. 6.
• a number of optical parameters may be employed to determine the location of a target with in vivo light imaging after injection of the subject with an optically-labeled compound of the invention.
• Optical parameters to be detected in the preparation of an image may include transmitted radiation, absorption, fluorescent or phosphorescent emission, light reflection, changes in absorbance amplitude or maxima, and elastically scattered radiation.
• biological tissue is relatively translucent to light in the near infrared (NIR) wavelength range of 650-1000 nm. NIR radiation can penetrate tissue up to several centimeters, permitting the use of compounds of the present invention to image target-containing tissue in vivo.
• NIR near infrared
• the use of visible and near- infrared (NIR) light in clinical practice is growing rapidly.
• Compounds absorbing or emitting in the visible, NIR, or long-wavelength (UN-A, >350 nm) region of the electromagnetic spectrum are potentially useful for optical tomographic imaging, endoscopic visualization, and phototherapy.
• the compounds of the invention may be conjugated with photolabels, such as optical dyes, including organic chromophores or fluorophores, having extensive delocalized ring systems and having absorption or emission maxima in the range of 400-1500 nm.
• the compounds of the invention may alternatively be derivatized with a bioluminescent molecule.
• the preferred range of absorption maxima for photolabels is between 600 and 1000 nm to minimize interference with the signal from hemoglobin.
• photoabsorption labels have large molar absorptivities, e.g., > 10 5 cirf'M "1 , while fluorescent optical dyes will have high quantum yields.
• optical dyes include, but are not limited to those described in US 6,641 ,798, WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO 98/47538, and references cited therein, all hereby incorporated by reference in their entirety.
• the photolabels may be covalently linked directly to compounds of the invention, such as, for example, compounds comprised of GRP receptor targeting peptides and linkers of the invention.
• indocyanine green which absorbs and emits in the NIR region has been used for monitoring cardiac output, hepatic functions, and liver blood flow
• 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, H. Tanigami, H. Ueyama, T. Mashimo, and I. Yoshiya 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, S. Shaldon, L. Chiandussi, et al., The use of Indocyanine green in the measurement of hepatic blood flow and as a test of hepatic function. Clin. Sci.
• the patient is scanned with one or more light sources (e.g., a laser) in the wavelength range appropriate for the photolabel employed in the agent.
• the light used may be monochromatic or polychromatic and continuous or pulsed. Transmitted, scattered, or reflected light is detected via a photodetector tuned to one or multiple wavelengths to determine the location of target-containing tissue (e.g., tissue containing GRP) in the subject. Changes in the optical parameter may be monitored over time to detect accumulation of the optically-labeled reagent at the target site (e.g., the tumor or other site with GRP receptors). Standard image processing and detecting devices may be used in conjunction with the optical imaging reagents of the present invention.
• optical imaging reagents described above may also be used for acousto- optical or sonoluminescent imaging performed with optically-labeled imaging agents (see U.S. 5,171,298, WO 98/57666, and references therein).
• acousto-optical imaging ultrasound radiation is applied to the subject and affects the optical parameters of the transmitted, emitted, or reflected light.
• sonoluminescent imaging the applied ultrasound actually generates the light detected. Suitable imaging methods using such techniques are described in WO 98/57666.
• Radioisotope therapy involves the administration of a radiolabeled compound in sufficient quantity to damage or destroy the targeted tissue. After administration of the compound (by e.g., intravenous, subcutaneous, or intraperitonal injection), the radiolabeled pharmaceutical localizes preferentially at the disease site (in this instance, tumor tissue or other tissue that expresses the pertinent GRP receptor). Once localized, the radiolabeled compound then damages or destroys the diseased tissue with the energy that is released during the radioactive decay of the isotope that is administered. As discussed herein, the invention also encompasses use of radiotherapy in combination with adjuvant chemotherapy (or in combination with any other appropriate therapeutic agent).
• the design of a successful radiotherapeutic involves several critical factors: 1. selection of an appropriate targeting group to deliver the radioactivity to the disease site; 2. selection of an appropriate radionuclide that releases sufficient energy to damage that disease site, without substantially damaging adjacent normal tissues; and 3. selection of an appropriate combination of the targeting group and the radionuclide without adversely affecting the ability of this conjugate to localize at the disease site.
• this often involves a chelating group that coordinates tightly to the radionuclide, combined with a linker that couples said chelate to the targeting group, and that affects the overall biodistribution of the compound to maximize uptake in target tissues and minimize uptake in normal, non-target organs.
• Radiotherapeutic agents may contain a chelated 3+ metal ion from the class of elements known as the lanthanides (elements of atomic number 57-71) and their analogs (i.e. M 3+ metals such as yttrium and indium).
• Typical radioactive metals in this class include the isotopes 90-Yttrium, I l l-Indium, 149-Promethium, 153-Samarium, 166-Dysprosium, 166- Holmium, 175-Ytterbium, and -Lutetium.
• chelating ligands encapsulate the radiometal by binding to it via multiple nitrogen and oxygen atoms, thus preventing the release of free (unbound) radiometal into the body. This is important, as in vivo dissociation of 3 + radiometals from their chelate can result in uptake of the radiometal in the liver, bone and spleen [Brechbiel MW, Gansow OA, "Backbone-substituted DTPA ligands for 90 Y radioimmunotherapy", Bioconj. Chem.
• any of the chelators for therapeutic radionuclides disclosed herein may be used.
• forms of the DOTA chelate [Tweedle MF, Gaughan GT, Hagan JT, "1 -Substituted- 1,4,7-triscarboxymethyl- 1,4,7, 10- tetraazacyclododecane and analogs.” US Patent 4,885,363, Dec.
• DOTA-type macrocycles to targeting groups through a linker (e.g., by activation of one of the carboxylates of the DOTA to form an active ester, which is then reacted with an amino group on the linker to form a stable amide bond), are known to those skilled in the art. (See, e.g., Tweedle et al. US Patent 4,885,363).
• Coupling can also be performed on DOTA-type macrocycles that are modified on the backbone of the polyaza ring.
• the selection of a proper nuclide for use in a particular radiotherapeutic application depends on many factors, including: a. Physical half-life - This should be long enough to allow synthesis and purification of the radiotherapeutic construct from radiometal and conjugate, and delivery of said construct to the site of injection, without significant radioactive decay prior to injection.
• the radionuclide should have a physical half-life between about 0.5 and 8 days. b.
• Radionuclides that are particle emitters are particularly useful, as they emit highly energetic particles that deposit their energy over short distances, thereby producing highly localized damage.
• Beta emitting radionuclides are particularly preferred, as the energy from beta particle emissions from these isotopes is deposited within 5 to about 150 cell diameters.
• Radiotherapeutic agents prepared from these nuclides are capable of killing diseased cells that are relatively close to their site of localization, but cannot travel long distances to damage adjacent normal tissue such as bone marrow. c. Specific activity (i.e.
• Radionuclides that have high specific activity are particularly preferred.
• the specific activity of a radionuclide is determined by its method of production, the particular target that is used to produce it, and the properties of the isotope in question.
• Many of the lanfhanides and lanthanoids include radioisotopes that have nuclear properties that make them suitable for use as radiotherapeutic agents, as they emit beta particles. Some of these are listed in the table below.
• Radiotherapeutic derivatives of the invention containing beta-emitting isotopes of rhenium ( 186 -Re and 188 -Re) are also particularly preferred.
• Dosages And Additives [00211] Proper dose schedules for the compounds of the present invention are known to those skilled in the art. The compounds can be administered using many methods which include, but are not limited to, a single or multiple IN or IP injections.
• Radiopharmaceuticals For radiopharmaceuticals, one administers a quantity of radioactivity that is sufficient to permit imaging or, in the case of radiotherapy, to cause damage or ablation of the targeted GRP-R bearing tissue, but not so much that substantive damage is caused to non-target (normal tissue).
• the quantity and dose required for scintigraphic imaging is discussed supra.
• the quantity and dose required for radiotherapy is also different for different constructs, depending on the energy and half-life of the isotope used, the degree of uptake and clearance of the agent from the body and the mass of the tumor. In general, doses can range from a single dose of about 30-50 mCi to a cumulative dose of up to about 3 Curies.
• dosages sufficient to achieve the desired image enhancement are known to those skilled in the art and may vary widely depending on the dye or other compound used, the organ or tissue to be imaged, the imaging equipment used, etc.
• compositions of the invention can include physiologically acceptable buffers, and can require radiation stabilizers to prevent radiolytic damage to the compound prior to injection.
• Radiation stabilizers are known to those skilled in the art, and may include, for example, para-aminobenzoic acid, ascorbic acid, gentistic acid and the like.
• a single, or multi-vial kit that contains all of the components needed to prepare the diagnostic or therapeutic agents of this invention is an integral part of this invention.
• such kits will often include all necessary ingredients except the radionuclide.
• a single- vial kit for preparing a radiopharmaceutical of the invention preferably contains a chelator/linker/targeting peptide conjugate of the formula M- N-O-P-G, a source of stannous salt (if reduction is required, e.g., when using technetium), or other pharmaceutically acceptable reducing agent, and is appropriately buffered with pharmaceutically acceptable acid or base to adjust the pH to a value of about 3 to about 9.
• kit contents be in lyophilized form.
• Such a single vial kit may optionally contain labile or exchange ligands such as glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and can also contain reaction modifiers such as diethylenetriamine-pentaacetic acid (DPT A), ethylenediamine tetraacetic acid (EDTA), or a, ⁇ , or ⁇ -cyclodextrin that serve to improve the radiochemical purity and stability of the final product.
• DPT A diethylenetriamine-pentaacetic acid
• EDTA ethylenediamine tetraacetic acid
• a, ⁇ , or ⁇ -cyclodextrin that serve to improve the radiochemical purity and stability of the final product.
• a multi-vial kit may also contain stabilizers, bulking agents such as mannitol, that are designed to aid in the freeze-drying process, and other additives known to those skilled in the art.
• a multi-vial kit preferably contains the same general components but employs more than one vial in reconstituting the radiopharmaceutical.
• one vial may contain all of the ingredients that are required to form a labile Tc(N) complex on addition of pertechnetate (e.g. the stannous source or other reducing agent).
• Pertechnetate is added to this vial, and after waiting an appropriate period of time, the contents of this vial are added to a second vial that contains the chelator and targeting peptide, as well as buffers appropriate to adjust the pH to its optimal value. After a reaction time of about 5 to 60 minutes, the complexes of the present invention are formed. It is advantageous that the contents of both vials of this multi-vial kit be lyophilized. As above, reaction modifiers, exchange ligands, stabilizers, bulking agents, etc. may be present in either or both vials.
• the compounds of the present invention can be prepared by various methods depending upon the selected chelator.
• the peptide portion of the compound can be most conveniently prepared by techniques generally established and known in the art of peptide synthesis, such as the solid-phase peptide synthesis (SPPS) approach. Because it is amenable to solid phase synthesis, employing alternating FMOC protection and deprotection is the preferred method of making short peptides. Recombinant DNA technology is preferred for producing proteins and long fragments thereof.
• SPPS solid-phase peptide synthesis
• Solid-phase peptide synthesis involves the stepwise addition of amino acid residues to a growing peptide chain that is linked to an insoluble support or matrix, such as polystyrene.
• the C-terminal residue of the peptide is first anchored to a commercially available support with its amino group protected with an N-protecting agent such as a t- butyloxycarbonyl group (Boc) or a fluorenylmethoxycarbonyl (Fmoc) group.
• Boc t- butyloxycarbonyl group
• Fmoc fluorenylmethoxycarbonyl
• the amino protecting group is removed with suitable deprotecting agents such as TFA in the case of Boc or piperidine for Fmoc and the next amino acid residue (in N-protected form) is added with a coupling agent such as diisopropylcarbodiimide (DIC).
• DIC diisopropylcarbodiimide
• the reagents are washed from the support.
• the peptide is cleaved from the support with a suitable reagent such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF).
• TFA trifluoroacetic acid
• HF hydrogen fluoride
• the compounds of the invention may also be prepared by the process known in the art as segment coupling or fragment condensation (Barlos, K. and Gatos, D.; 2002 "Convergent Peptide Synthesis” in Fmoc Solid Phase Synthesis - A Practical Approach; Eds. Chan, W.C. and White, P.D.; Oxford University Press, New York; Chap. 9, pp. 215-228).
• segments of the peptide usually in side-chain protected form are prepared separately by either solution phase synthesis or solid phase synthesis or a combination of the two methods.
• the choice of segments is crucial and is made using a division strategy that can provide a manageable number of segments whose C-terminal residues and N-terminal residues are projected to provide the cleanest coupling in peptide synthesis.
• the C-terminal residues of the best segments are either devoid of chiral alpha carbons (glycine or other moieties achiral at the carbon oc to the carboxyl group to be activated in the coupling step) or are compromised of amino acids whose propensity to racemization during activation and coupling is lowest of the possible choices.
• the choice of N-terminal amino acid for each segment is based on the ease of coupling of an activated acyl intermediate to the amino group.
• each of the segments is chosen based on the synthetic accessibility of the required intermediates and the relative ease of manipulation and purification of the resulting products (if needed).
• the segments are then coupled together, both in solution, or one on solid phase and the other in solution to prepare the final structure in fully or partially protected form. [00220]
• the protected target compound is then subjected to removal of protecting groups, purified and isolated to give the final desired compound.
• each segment can be purified separately, allowing the removal of side products such as deletion sequences resulting from incomplete couplings or those derived from reactions such as side-chain amide dehydration during coupling steps, or internal cyclization of side-chains (such as that of Gin) to the alpha amino group during deprotection of Fmoc groups.
• side products such as deletion sequences resulting from incomplete couplings or those derived from reactions such as side-chain amide dehydration during coupling steps, or internal cyclization of side-chains (such as that of Gin) to the alpha amino group during deprotection of Fmoc groups.
• side products would all be present in the final product of a conventional resin-based 'straight through' peptide chain assembly whereas removal of these materials can be performed, if needed, at many stages in a segment coupling strategy.
• segment coupling strategy Another important advantage of the segment coupling strategy is that different solvents, reagents and conditions can be applied to optimize the synthesis of each of the segments to high purity and yield resulting in improved purity and yield of the final product. Other advantages realized are decreased consumption of reagents and lower costs.
• Peptides were prepared using an Advanced ChemTech 496 ⁇ MOS synthesizer, an Advanced ChemTech 357 FBS synthesizer and/or by manual peptide synthesis. However the protocols for iterative deprotection and chain extension employed were the same for all. D. Automated synthesis with the Symphony instrument (made by Rainin) [00225] The synthesis was run with Symphony Software (Version 3) supplied by
• Analytical HPLC was performed using a Shimadzu-LC-lOA dual pump gradient analytical LC system employing Shimadzu-ClassNP software version 4.1 for system control, data acquisition, and post run processing.
• Mass spectra were acquired on a Hewlett- Packard Series 1100 MSD mass spectrometer interfaced with a Hewlett-Packard Series 1100 dual pump gradient HPLC system fitted with an Agilent Technologies 1100 series autosampler fitted for either direct flow injection or injection onto a Waters Associates XTerra MS C18 column (4.6 mm x 50 mm, 5 ⁇ particle, 12 ⁇ A pore).
• the instrument was driven by a HP Kayak workstation using 'MSD convinced workstation using 'MSD convinced software for sample submission and HP Chemstation software for instrument control and data acquisition.
• the samples were introduced via direct injection using a 5 ⁇ L injection of sample solution at a concentration of 1 mg/mL and analyzed using positive ion electrospray to obtain m/e and m/z (multiply charged) ions for confirmation of structure.
• 1H- ⁇ MR spectra were obtained on a Narian Innova spectrometer at 500 MHz.
• 3 C- ⁇ MR spectra were obtained on the same instrument at 125.73 MHz.
• a solution of Reagent B (2 mL; 88:5:5:2 - TFA:phenol:water:TIPS) was added to the resin and the reaction block or individual vessel was shaken for 4.5h at ambient temperature. The resulting solution containing the deprotected peptide was drained into a vial. This procedure was repeated two more times with 1 mL of Reagent B. The combined filtrate was concentrated under reduced pressure using a Genevac HT-12 series II centrifugal concentrator. The residue in each vial was then triturated with 2 mL of Et 2 O and the supernatant was decanted. This procedure was repeated twice to provide the peptides as colorless solids.
• the crude peptides were dissolved in water/acetonitrile and purified using either a Waters XTerra MS Cl 8 preparative HPLC column (50 mm x 19 mm, 5 micron particle size, 120A pore size) or a Waters- YMC C18 ODS column (250 mm x 30 mm i.d., 10 micron particle size. 120 A pore size).
• the product-containing fractions were collected and analyzed by HPLC. The fractions with >95% purity were pooled and the peptides isolated by lyophilization.
• Fmoc-L-methionine N- ⁇ -Fmoc-L-leucine, N- ⁇ -Fmoc-N m -trityl-L-histidine, N- ⁇ -Fmoc- glycine, N- ⁇ -Fmoc-L-valine, N- ⁇ -Fmoc-L-alanine, N- ⁇ -Fmoc-N n -Boc-L-tryptophan.
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 7 mL). N-a-Fmoc-glycine (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol) and DMA (7 mL) were added to the resin. The mixture was shaken for 3 h at room temperature, the solution was emptied and the resin washed with DMA (5 x 7 mL).
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min.
• the solution was emptied and the resin washed with DMA (5 x 7 mL) followed by addition of 1,4,7,10- tetraazacyclododecane- 1,4,7,10-tetraacetic acid tris( 1,1 -dimethyl ethyl) ester adduct with ⁇ aCl (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) to the resin.
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL).
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for 20 min then the solution was emptied and the resin washed with DMA (5 7 mL).
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied and fresh 50% morpholine in DMA (7 L) was added. The suspension was stirred for 20 min then the solution was emptied and the resin was washed with DMA (5 x 7 mL).
• Resin D (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for 20 min then the solution was emptied and the resin was washed with DMA (5 x 7 mL).
• the resin was shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture was shaken for 20 min. The solution was emptied and the resin was washed with DMA (5 x 7 mL).
• Tributylamine (4.8 L; 20.2 mmol) was added dropwise to a solution of N-a- Fmoc-glycine (6.0 g; 20.2 mmol) in THF (120 mL) stirred at 0°C.
• Isobutyl chloroformate (2.6 mL; 20.2 mmol) was subsequently added and, after 10 min, a suspension of tributylamine (3.9 mL; 16.8 mmol) and (3 ⁇ ,5 ⁇ ,12a)-3-amino- 12-hydroxycholan-24-oic acid 2a (6.6 g; 16.8 mmol) in DMF (120 mL) was added dropwise, over 1 h, into the cooled solution.
• Tributylamine (3.2 mL; 13.5 mmol) was added dropwise to a solution of N-a- Fmoc-glycine (4.0 g; 13.5 mmol) in THF (80 mL) stirred at 0°C.
• N-Hydroxysuccinimide (1.70 g, 14.77 mmol) and DIC (1.87 g, 14.77 mmol) were added sequentially to a stirred solution of Fmoc-Gly-OH (4.0 g, 13.45 mmol) in dichloromethane (15 mL); the resulting mixture was stirred at room temperature for 4 h.
• the N,N'-diisopropylurea formed was removed by filtration and the solid was washed with ether (20 mL). The volatiles were removed and the solid Fmoc-Gly-succinimidyl ester formed was washed with ether (3 x 20 mL).
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL).
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture was shaken for 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for 20 min, the solution was emptied and the resin was washed with DMA (5 7 mL).
• the resin was shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture was shaken for 20 min. The solution was emptied and the resin was washed with DMA (5 x 7 mL).
• the mixture was shaken for 3 h at room temperature, the solution was emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture was shaken for 20 min. The solution was emptied and the resin was washed with DMA (5 x 7 mL).
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for 20 min then the solution was emptied and the resin washed with
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for 20 min. The solution was emptied and the resin washed with DMA (5 7 mL).
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied and fresh 50% morpholine in DMA (7 L) was added. The suspension was stirred for 20 min then the solution was emptied and the resin was washed with DMA (5 x 7 L).
• the resin was then shaken with 50% morpholine in DMA (6 mL) for 10 min, the solution was emptied, fresh 50% morpholine in DMA (6 mL) was added and the mixture was shaken for 20 min. The solution was emptied and the resin was washed with DMA (5 x 7 mL).
• ⁇ aCl 750 mg; 1.19 mmol
• ⁇ OBt 182 mg; 1.19 mmol
• DIEA 404 ⁇ L; 2.36 mmol
• DIC 185 ⁇ L; 1.19 mmol
• DMA 6 mL
• Triphenylphosphine (6.06 g; 23 mmol) was added to a solution of (4- azidomethyl)-3-methoxybenzoic acid methyl ester B (5 g; 22 mmol) in THF (50 mL): hydrogen evolution and formation of a white solid was observed. The mixture was stirred under nitrogen at room temperature. After 24 h more triphenylphosphine (0.6 g; 2.3 mmol) was added. After 24 h the azide was consumed and H 2 O (10 mL) was added. After 4 h the white solid disappeared. The mixture was heated at 45 °C for 3 h and was stirred overnight at room temperature.
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
• DOTA tri-t-butyl ester adduct with ⁇ aCl 2 (0.79 g; 1.2 mmol) (5), ⁇ OBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin.
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution filtered and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was filtered and the resin washed with DMA (5 x 7 mL).
• DOTA tri-t-butyl ester adduct with NaCI (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin.
• the mixture was shaken for 24 h at room temperature, filtered and the resin washed with DMA (5 x 7 mL), CH2C12 (5 x 7 mL) and vacuum dried.
• the resin was shaken in a flask with Reagent B (25 mL) for 4.5 h.
• Resin A 500 mg; 0.3 mmol was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and fresh 50 % morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL).
• Fmoc-4-aminobenzoic acid (290 mg; 0.80 mmol), HOBt (120 mg; 0.80 mmol), DIC (130 ⁇ L; 0.80 mmol) and DMA (5 mL) were added to the resin, the mixture shaken for 3 h at room temperature, emptied and the resin washed with DMA (5 x 5 mL). The resin was then shaken with 50% morpholine in DMA (5 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (5 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 5 mL).
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 7 mL).
• Fmoc-glycine (0.36 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) and N- ethyldiisopropylamine (0.40 mL; 2.4 mmol) were stirred for 15 min in DMA (7 mL) then the solution was added to the resin, the mixture shaken for 2 h at room temperature, emptied and the resin washed with DMA (5 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min.
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
• N-a-Fmoc-S-acetamidomethyl-L-cysteine (0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the mixture was shaken for 3 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL).
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
• N-a-Fmoc-O-t-butyl-L-serine (0.46 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), and DMA (7 mL) were added to the resin, the mixture was shaken for 3 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL).
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL). N,N-Dimethyl glycine (0.12 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) and N-ethyldiisopropylamine (0.40 mL; 2.4 mmol) were stirred for 15 min in DMA (7 mL) then the solution was added to the resin.
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL).
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
• N-a-Fmoc-S-acetamidomethyl-L-cysteine (0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the mixture was shaken for 3 h at room temperature, emptied and the resin washed with DMA (5 x 7 L).
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 L) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
• N-a-Fmoc-O-t-butyl-L-serine (0.46 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), and DMA (7 mL) were added to the resin, the mixture was shaken for 3 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL).
• the resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min. The solution was emptied and the resin washed with DMA (5 x 7 mL). NN-Dimethylglycine (0.12 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) and N-ethyldiisopropylamine (0.40 L; 2.4 mmol) were stirred for 15 min in DMA (7 mL) then the solution was added to the resin.
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL).
• DMA 5 x 7 mL
• CH 2 C1 2 5 x 7 mL
• Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was emptied and the resin washed with DMA (5 x 7 mL).
• Trifluoroacetic acid ethyl ester (50 g; 0.35 mol) was dropped into a solution of diethylenetriamine (18 g; 0.175 mol) in THF (180 mL) at 0°C in 1 h. After 20 h at room temperature, the mixture was evaporated to an oily residue (54 g). T he oil was crystallized from Et 2 O (50 mL), filtered, washed with cooled Et 2 O (2 x 30 mL) and dried to obtain A as a white solid (46 g; 0.156 mol). Yield 89%. B .
• DOTA tri- t-butyl ester (112 mg; 0.178 mmol) HATU (70 mg; 0.178 mmol) and DIEA (60 ⁇ L; 0.356 mmol) were added to a solution of F (50 mg; 0.0445 mmol) in DMA (3 mL) and CH 2 C1 2 (2 mL) and stirred for 24 h at room temperature.
• the crude was evaporated to reduced volume (1 mL) and shaken with Reagent B (25 mL) for 4.5 h. After evaporation of the solvent, the residue was treated with Et 2 O (20 mL) to give a precipitate.
• Fmoc-Gly-OH (10.0 equiv.) was treated with HATU (10.0 equiv.) and DIEA (20.0 equiv.) in NMP (10 mL of NMP was used for one gram of the amino acid by weight) and the solution was stirred for 10-15 min at RT before transferring to the vessel containing the amine loaded resin. The volume of the solution was made to 15.0 ml for every gram of the resin. The coupling was continued for 20h at RT and the resin was drained of all the reactants. This procedure was repeated one more time and then washed with NMP before moving on to the next step. C.
• D03A monoamide [00319] 8.0 equivalents of DOTA mono acid was dissolved in NMP and treated with 8.0 equivalents of HBTU and 16.0 equivalents of DIEA. This solution was stirred for 15 min at RT and then transferred to the amine on the resin and the coupling was continued for 24h at RT. The resin was then drained, washed and then the peptide was cleaved and purified. D. Cleavage of the crude peptides from the resin and purification:
• the resin was suspended in Reagent B (15.0 ml/g) and shaken for 4h at RT. The resin was then drained and washed with 2 5 mL of Reagent B again and combined with the previous filtrate. The filtrate was then concentrated under reduced pressure to a paste/liquid at RT and triturated with 25.0 L of anhydrous ether (for every gram of the resin used). The suspension was then centrifuged and the ether layer was decanted. This procedure was repeated two more times and the colorless precipitate after ether wash was purified by preparative HPLC.
• Fmoc-6-aminonicotinic acid 1 was prepared as described in the literature ("Synthesis of diacylhydrazine compiounds for therapeutic use.” Hoelzemann, G.; Goodman, S. (Merck Patent G.m.b.H.., Germany). Ger.Offen. 2000, 16 pp.
• Boc-Glutamic acid (5.0 g, 20.2 mol) was dissolved in THF (50.0 mL) and cooled to 0°C in an ice bath.
• HATU (15.61 g, 41.0 mmol) was added followed by DIEA (6.5 g, 50.0 mmol).
• DIEA 6.5 g, 50.0 mmol
• the reaction mixture was stirred at 0°C for 30 min.
• Benzyl ester of glycine [8.45 g, 50 mmol, generated from neutralizing benzyl glycine hydrochloride with sodium carbonate and by extraction with DCM and solvent removal] was added in THF (25.0 mL).
• THF 25.0 mL
• the reaction mixture was allowed to come to RT and stirred for 20h at RT. All the volatiles were removed under reduced pressure.
• H-Glu(G-OBnVG-OBn [00339] The fully protected glutamic acid derivative (1.7 g, 3.2 mmol) B from above was dissolved in DCM/TFA (4:1, 20 mL) and stirred until the starting material disappeared on TLC (2 h). The reaction mixture was poured into ice cold saturated sodium bicarbonate solution (200 mL) and the organic layer was separated and the aqueous layer was extracted with 2 x 50 mL of DCM and combined with the organic layer. The DCM layer was washed with saturated sodium bicarbonate (2 x 100 mL), water (2 x 100 mL) and dried (sodium sulfate).
• Example XXIV - Figure 31 Synthesis of L211 [00347] Prepared from 0.2 g Of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.08 mmol) using standard protocols.
• Fmoc-Q(Trt)-W(Boc)-A-N-G-H(Trt)-L-M-resin A (0.2g, 0.08 mmol) was used.
• Fmoc-Lys(ivDde) was employed for the introduction of lysine.
• the protecting group of the lysine was removed using 10% hydrazine in DMF (2 x 10 L; 10 min each and then washed). The rest of the amino acids were then introduced using procedures described in the "general" section to complete the required peptide sequence.
• L218 in FIG. 38 as obtained in a yield of 40.0 mg (23.2%).
• Example XXXII- Figure 39 Synthesis of L219 [00355] 4-Sulfamylbutyryl AM ⁇ ovagel resin was used (1.1 mmol/g; 0.5 g; 0.55 mmol). The first amino acid was loaded on to this resin at -20° C for 20h. The rest of the sequence was completed utilizing normal coupling procedures. After washing, the resin was alkylated with 20.0 eq. of iodoacetonitrile and 10.0 equivalents of DIEA for 20h. The resin was then drained of the liquids and washed and then cleaved with 2.0 eq. of pentylamine in 5.0 mL of THF for 20h.
• the solution was degassed by passing nitrogen through the solution and then treated with solid methyl -3 -bromobenzoate, or methyl -4- bromobenzoate (1.35 g, 6.3 mmol) followed by the Pd (0) catalyst ⁇ [(C 6 H 5 ) 3 P] 4 Pd; 0.023g, 0.003 mmol ⁇ .
• the reaction mixture was kept at reflux under nitrogen until the starting bromobenzoate was consumed as determined by TLC analysis (2-3 h).
• the reaction mixture was then diluted with 250 mL of water and extracted with ethyl acetate (3 x 50 L). The organic layers were combined and washed with saturated sodium bicarbonate solution (2 x 50 mL) and dried ( ⁇ a 2 SO 4 ). The solvent was removed under reduced pressure and the residue was chromatographed over flash silica gel (100 g). Elution with 40% ethyl acetate in hexanes yielded the product either as a solid or oil. Yield:
• the 4,4'- isomer was crystallized from hexane/ether and the 3,3 '-isomer was triturated with isopropyl ether to remove all the impurities; the product was homogeneous as determined on TLC analysis and further purification was not required.
• Methyl-4-azidomethyl-4-biphenylcaroxylate 0.245 g (46%); m. p. - 106-108° C.
• Tributylamine (3.2 L); 13.5 mmol) was added dropwise to a solution of Fmoc-glycine (4.0 g, 13.5 mmol) in THF (80 mL) stirred at 0° C.
• Isobutylchloroformate (1.7 mL; 13.5 mmol) was subsequently added and, after 10 min, a suspension of tributylamine (2.6 mL; 11.2 mmol) and (3 ⁇ , 5 ⁇ , la, 12 ⁇ )-3-amino-7,12-dihydroxycholan-24-oic acid (4.5 g; 11.2 mmol) in DMF (80 mL) was added dropwise, over 1 h, into the cooled solution.
• Example XLIII In vitro Binding Assay for GRP Receptors in PC3 Cell Lines - FIGS. 14 A-B [00369] To identify potential lead compounds, an in vitro assay that identifies compounds with high affinity for GRP-R was used. Since the PC3 cell line, derived from human prostate cancer, is known to exhibit high expression of GRP-R on the cell surface, a radio ligand binding assay in a 96-well plate format was developed and validated to measure the binding of I25 I-BBN to GRP-R positive PC3 cells and the ability of the compounds of the invention to inhibit this binding.
• This assay was used to measure the IC 50 for RP527 ligand, DO3A- monoamide-Aoc-QWAVGHLM-NH 2 (controls) and compounds of the invention which inhibit the binding of ,25 I-BBN to GRP-R.
• RP527 N,N-dimethylglycine-Ser-Cys(Acm)- Gly-5-aminopentanoic acid-BBN (7-14) [SEQ. ID.NO: 1], which has MS - 1442.6 and IC50 - 0.84).
• the Radioligand Binding Plate Assay was validated for BBN and BBN analogues (including commercially available BBN and LI) and also using 99m Tc RP527 as the radioligand.
• PC3 human prostate cancer cell line
• ATCC American Type Culture Collection
• RPMI 1640 tissue culture flasks
• This growth medium was supplemented with 10% heat inactivated FBS (Hyclone, SH30070.03), 10 mM HEPES (GibcoBRL, 15630-080), and antibiotic/antimycotic (GibcoBRL, 15240-062) for a final concentration of penicillin-streptomycin (100 units/mL), and fungizone (0.25 ⁇ g/mL).
• Binding buffer [00372] RPMI 1640 (ATCC) supplemented with 20mM HEPES, 0.1 % BSA (w/v), 0.5 mM PMSF (AEBSF), bacitracin (50 mg/500 ml), pH 7.4.
• 125 I-BBN carrier free, 2200 Ci/mmole was obtained from Perkin-EImer.
• a 96-well plate assay was used to determine the IC 50 of various compounds of the invention to inhibit binding of ]25 I-BBN to human GRP-R. The following general procedure was followed:
• PC3 cells human prostate cancer cell line
• PC3 cells for assay were plated at a concentration of 2.0x10 4 /well either in 96- well white /clear bottomed microtiter plates (Falcon Optilux-I) or 96 well black/clear collagen I cellware plates (Beckton Dickinson Biocoat). Plates were used for binding studies on day 1 or 2 post-plating. The plates were checked for confluency (>90% confluent) prior to assay.
• FIGS. 14A-B Competition binding curves for RP527 (control) and L70, a compound of the invention can be found in FIGS. 14A-B. These data show that the IC50 of the RP527 control is 2.5nM and that of L70, a compound of this invention is 5nM. The IC50 of the DO3A- monoamide-Aoc-QWAVGHLM-NH 2 control was 5nM. IC50 values for those compounds of the invention tested can be found in Tables 1-3, supra, and show that they are comparable to that of the controls and thus would be expected to have sufficient affinity for the receptor to allow uptake by receptor bearing cells in vivo. C. Internalization & Efflux assay:
• the cells were collected to determine the amount of internalized 125 I-BBN, and all samples were analyzed in the gamma counter. Data for the internalization assay was normalized by comparing counts obtained at the various time points with the counts obtained at the final time point (T40 min).
• Example XLIV Preparation of Tc-labeled GRP compounds.
• Peptide solutions of compounds of the invention identified in Table 6 were prepared at a concentration of 1 mg/mL in 0.1% aqueous TFA.
• a stannous chloride solution was prepared by dissolving SnCl 2 « 2H O (20 mg/mL) in 1 N HCL
• Stannous gluconate solutions containing 20 ⁇ g of SnCl »2H 2 O/100 ⁇ L were prepared by adding an aliquot of the SnCl 2 solution (10 ⁇ L) to a sodium gluconate solution prepared by dissolving 13 mg of sodium gluconate in water.
• a hydroxypropyl gamma cyclodextrin [HP- ⁇ -CD] solution was prepared by dissolving 50 mg of HP- ⁇ -CD in 1 mL of water.
• the 99m Tc labeled compounds identified below were prepared by mixing 20 ⁇ L of solution of the unlabeled compounds (20 ⁇ g), 50 ⁇ L of HP- ⁇ -CD solution, 100 ⁇ L of Sn- gluconate solution and 20 to 50 ⁇ L of 99m Tc pertechnetate (5 to 8 mCi, Syncor). The final volume was around 200 ⁇ L and final pH was 4.5 - 5. The reaction mixture was heated at 100°C for 15 to 20 min. and then analyzed by reversed phase HPLC to determine radiochemical purity (RCP).
• RCP radiochemical purity
• 99mTc complex of L2 was N,N'-dimefhylglycyl-Ser-Cys (Acm)-Gly-RJQWAVGHLM-NH 2 .
• J refers to 8-amino-3,6-dioxaoctanoic acid and "a” refers to D-alanine.
• This compound was synthesized by incubating 10 ⁇ g L64 ligand (10 ⁇ L of a 1 mg/mL solution in water), 100 ⁇ L ammonium acetate buffer (0.2M, pH 5.2) and ⁇ l-2 mCi of 177 LuCl 3 in 0.05N HC1 (MURR) at 90°C for 15 min. Free 177 Lu was scavenged by adding 20 ⁇ L of a 1 % Na 2 EDTA*2H 2 O (Aldrich) solution in water. The resulting radiochemical purity (RCP) was ⁇ 95%.
• the radiolabeled product was separated from unlabeled ligand and other impurities by HPLC, using a YMC Basic C8 column [4.6 x 150 mm], a column temperature of 30°C and a flow rate of 1 mL/min, with a gradient of 68%A/32%B to 66%A/34%B over 30 min., where A is citrate buffer (0.02M, pH 3.0), and B is 80% CH 3 CN/20% CH 3 OH.
• the isolated compound had an RCP of -100% and an HPLC retention time of 23.4 minutes.
• Samples for biodistribution and cell binding studies were prepared by collecting the desired HPLC peak into 1000 ⁇ L of citrate buffer (0.05 M, pH 5.3, containing 1% ascorbic acid, and 0.1% HSA). The organic eluent in the collected eluate was removed by centrifugal concentration for 30 min.
• the purified sample was diluted with cell-binding media to a concentration of 1.5 ⁇ Ci/mL within 30 minutes of the in vitro study.
• the sample was diluted with citrate buffer (0.05 M, pH 5.3, containing 1% sodium ascorbic acid and 0.1 % HSA) to a final concentration of 50 ⁇ Ci/mL within 30 minutes of the in vivo study.
• citrate buffer 0.05 M, pH 5.3, containing 1% sodium ascorbic acid and 0.1 % HSA
• This compound was synthesized by incubating 70 ⁇ g L64 ligand (70 ⁇ L of a 1 mg/mL solution in water), 200 ⁇ L ammonium acetate buffer (0.2M, pH 5.2) and -30 - 40 mCi of 177 LuCl 3 in 0.05N HCl (MURR) at 85°C for 10 min. After cooling to room temperature, free 177 Lu was scavenged by adding 20 ⁇ L of a 2% Na 2 EDTA*2H 2 O (Aldrich) solution in water. The resulting radiochemical purity (RCP) was -95%.
• MURR 0.05N HCl
• the radiolabeled product was separated from unlabeled ligand and other impurities by HPLC, using a 300VHP Anion Exchange column (7.5 x 50 mm) (Vydac) that was sequentially eluted at a flow rate of 1 mL/min with water, 50% acetonitrile/water and then 1 g/L aqueous ammonium acetate solution.
• the desired compound was eluted from the column with 50% CH 3 CN and mixed with -1 mL of citrate buffer (0.05 M, pH 5.3) containing 5% ascorbic acid, 0.2% HSA, and 0.9% (v:v) benzyl alcohol.
• This compound was synthesized by incubating 10 ⁇ g L64 ligand (5 ⁇ L of a 2 mg/mL solution in 0.01 N HCl), 60 ⁇ L ethanol, 1.12 mCi of ⁇ ⁇ InCl 3 in 0.05N HCl (80 ⁇ L) and 155 ⁇ L sodium acetate buffer (0.5M, pH 4.5) at 85°C for 30 min. Free n 'in was scavenged by adding 20 ⁇ L of a 1% Na 2 EDTA.2H 2 O (Aldrich) solution in water. The resulting radiochemical purity (RCP) was 87%.
• the radiolabeled product was separated from unlabeled ligand and other impurities by HPLC, using a Vydac Cl 8 column, [4.6 x 250 mm], a column temperature of 50°C and a flow rate of 1.5 mL/min. with a gradient of 75%A/25%B to 65%A/35%B over 20 min where A is 0.1% TFA in water, B is 0.085% TFA in acetonitrile. With this system, the retention time for ⁇ n In-L64 is 15.7 min. The isolated compound had an RCP of 96.7%.
• Example XL VIII - Preparation of Lu-DO3A-monoamide-Aoc-OWAVGHLM-NH 2 (Control) [00385] A stock solution of peptide was prepared by dissolving DO3A-monoamide-Aoc- QWAVGHLM-NH 2 ligand (prepared as described in US Application Publication No. 2002/0054855 and WO 02/87637, both incorporated by reference) in 0.01 N HCl to a concentration of 1 mg/mL. 177 Lu- DO3 A-monoamide- Aoc-QW AN GHLM-NH 2 was prepared by mixing the following reagents in the order shown.
• Aoc-QWAVGHLM-NH 2 was formed in 97.1% yield (RCP) and had a retention time of -16.1 min on this system.
• This compound was prepared as described for 177 Lu- DO3A-monoamide-Aoc- QWANGHLM-NH 2 .
• the compound was analyzed by HPLC using a C 18 column (VYDAC Cat # 218TP54) that was eluted at flow rate of 1 mL/min with a gradient of 30-34% B over 20 min (where solvent is A. 0.1%TFA/H 2 O and B is 0.1%TFA/CH 3 CN).
• the I77 Lu-L63 that formed had an RCP of 97.8% and a retention time of -14.2 min on this system.
• Example L - Preparation of 177 Lu-L70 for cell binding and biodistribution studies [00388] This compound was prepared following the procedures described above, but substituting L70 (the ligand of Example II). Purification was performed using a YMC Basic C8 column (4.6 x 150 mm), a column temperature of 30°C and a flow rate of 1 mL/min. with a gradient of 80%A 20%B to 75%A/25%B over 40 min., where A is citrate buffer (0.02M, pH 4.5), and B is 80% CH 3 CN/20% CH 3 OH. The isolated compound had an RCP of -100% and an HPLC retention time of 25.4 min.
• Example LI - Preparation of 177 Lu- L70 for radiotherapy studies [00389] This compound was prepared as described above for L64.
• Radiolabeled product was separated from unlabeled ligand and other impurities by HPLC, using a Waters XTerra Cl 8 cartridge linked to a Vydac strong anion exchange column [7.5 x 50 mm], a column temperature of 30°C and a flow rate of 1 mL/min. with the gradient listed in the Table below, where A is 0.1 mM NaOH in water, pH 10.0, B is 1 g/L ammonium acetate in water, pH 6.7 and C is acetonitrile.
• the sample was diluted with citrate buffer (0.05 M, pH 5.3, containing 5% sodium ascorbic acid and 0.2% HSA) to a final concentration of 10 ⁇ Ci/mL within 30 minutes of the in vivo study.
• citrate buffer 0.05 M, pH 5.3, containing 5% sodium ascorbic acid and 0.2% HSA
• GRP-R GRP -preferring receptor
• NMB-R neuromedin-B preferring receptor
• BB3-R bombesin receptor subtype 3
• BB4-R bombesin receptor subtype 4
• Human ileal carcinoid tissue was used as a source for NMB-R, human prostate carcinoma for GRP-R and human bronchial carcinoid for BB3-R subtype receptors.
• receptor autoradiography was also performed with other bombesin radioligands, such as 125 I-Tyr 4 -bombesin or a compound known as the Universal ligand, ,25 I-[DTyr 6 , ⁇ Ala 11 , Phe 13 , Nle 14 ]-BBN(6-14), which binds to all three subsets of GRP-R, on adjacent tissue sections.
• GRP-R-expressing tumors such as prostatic, mammary and renal cell carcinomas, identified as such with established radioligands, were also visualized in vitro with 177 Lu-L70. Due to a better sensitivity, selected tumors with low levels of GRP-R could be identified with 177 Lu-L70, but not with 125 I-Tyr 4 -BBN, as shown in Table 8a. All NMB-R-expressing tumors identified with established radioligands were also visualized with Lu-L70. Conversely, none of the BB3 tumors were detected with Lu- L70.
• GRP-R pancreatic tissues that are pathologically altered (chronic pancreatitis)
• 177 Lu-L70 identifies these GRP-R in chronic pancreatitis with greater sensitivity than 125 I-Tyr 4 -BBN.
• a few colon carcinomas showed a low density of hetero geneously distributed GRP receptors 177 measured with Lu-L70 (Table 8a).
• the smooth muscles of the colon express GRP-R and were detected in vitro with 177 Lu-L70 as well as with the established bombesin ligands.
• Lu-L70 The sensitivity of 177 Lu-L70 seems better than that of 125 I- Tyr 4 -BBN or the 125 I-labeled universal bombesin analog. Therefore, a few tumors expressing 1 7 a low density of GRP-R can be readily identified with Lu-L70, while they are not positive with I-Tyr -BBN. The binding characteristics of Lu-L70 could also be confirmed in non-neoplastic tissues. While the mouse pancreas, as control, was shown to express a very high density of GRP-R, the normal human pancreatic acini were devoid of GRP-R.
• GRP-R in conditions of chronic pancreatitis GRP-R could be identified in acini, as reported previously in Fleischmann et al., "Bombesin Receptors in Distinct Tissue Compartments of Human Pancreatic Diseases," Lab. Invest. 80:1807-1817 (2000) and tissue, 1 7 17 ⁇ A again with better sensitivity by using Lu-L70 than by using I-Tyr -BBN.
• the BB 3 -expressing islets were not detected with 177 Lu-L70, while they were strongly labeled with the universal ligand, as reported previously in Fleischmann et al., "Bombesin Receptors in Distinct Tissue Compartments of Human Pancreatic Diseases," Lab. Invest. 80:1807-1817 (2000). While a minority of colon carcinomas had GRP-R, usually in very low density and heterogeneously distributed, the normal colonic smooth muscles expressed a high density of GRP-R.
• Radiotherapy studies were performed using the PC3 tumor-bearing nude mouse model.
• mice were administered 100 ⁇ L of 177 Lu-labeled compound of the invention at 30 mCi/kg, i.v, or s.c under sterile conditions. The subjects were housed in a barrier environment for the duration of the study.
• Compounds L64 and L70 can be prepared employing the collection of intermediates generally represented by A-D (FIG. 19), which themselves are prepared by standard methods known in the art of solid and solution phase peptide synthesis (Synthetic Peptides - A User's Guide 1992, Grant, G., Ed. WH. Freeman Co., NY, Chap 3 and Chap 4 pp. 77 - 258; Chan, W.C. and White, P.D. Basic Procedures in Fmoc Solid Phase Peptide Synthesis - A Practical Approach 2002, Chan, W.C. and White, P.D. Eds Oxford University Press, New York, Chap. 3 pp 41 - 76; Barlos, K and Gatos, G.
• the mixture was shaken for 3 h at room temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution filtered, fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for another 20 min.
• Resin A (0.4 g; 0.24 mmol) was shaken in a solid phase peptide synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution filtered and fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred for another 20 min then the solution was filtered and the resin washed with DMA (5 x 7 mL).
• L300 (0.033 g, 17%) was obtained after preparative column chromatography. The retention time was 6.66 minutes. The molecular formula is C 72 H 9 ⁇ 9 O] 8 . The calculated molecular weight is 1518.71; 1519.6 observed. The sequence is DO3 A-Gly-Abz4-Gln-Trp-Ala-Nal-Gly-His- Phe-Leu- ⁇ H 2 . The structure of L300 is shown in Figure 51.
• C6 cells were obtained from ATCC (CCL-107) and cultured in F12K media (ATCC) supplemented with 2 mM L-glutamine, 1.5 g/L Sodium bicarbonate, 15% horse serum and 2.5% FBS.
• Cells for the assays were plated at a concentration of 9.6 x 10 4 / well in 48 well poly-lysine coated plates (Beckton Dickinson Biocoat).
• PC3 were obtained from ATCC (CRL-1435) and cultured in RPMI 1640 (ATCC) supplemented with 2 mM L-glutamine, 1.5 g/L Sodium bicarbonate, 10 M HEPES and 10% FBS.
• RPMI 1640 (ATCC) containing 25 mM HEPES, 0.2% BSA fraction V, 1.0 mMAEBSF (CAS # 3087-99-7) and 0.1 % Bacitracin (CAS # 1405-87-4), pH 7.4.
• Custom made 125 I-[Tyr°]NMB, > 2.0 Ci/ ⁇ mole (Amersham Life Science) [ 125 I-NMB] and commercially available 125 I-[Tyr 4 ]BBN, >2.0 Ci/ ⁇ mole (Perkin Elmer Life Science) [ 125 I-BBN] were used as radioligands.
• Cholic acid containing linkers (3-aminocholic acid in L64, 3-amino-12- hydroxycholanic in L63 and 3-amino-12-ketocholanic in L67 conferred the peptides with dual affinity for both GRP-R and ⁇ MB-R.
• Cycloalkyl and aromatic substituted alanine containing linkers (3-cyclohexylalanine in DO3 A-monoamide -Cha-Cha-QWANGHLM- ⁇ H 2 , 1-Naphthylalanine in DO3 A-monoamide -Cha-Nal 1 -QWAVGHLM-NH 2 , 4- Benzoylphenylalanine in DO3 A-monoamide -Nall-Bpa4-QWANGHLM-NH 2 and Biphenylalanine in DO3 A-monoamide -Nai 1 -Bip-QWANGHLM-NH 2 ) imparted the peptides with selective affinity towards GRP-R.
• a linker containing only 4-(2- Aminoethylpiperazine)-l also contributed to the peptides with GRP-R selectivity (L89).
• G-4-amino benzoic acid linker conferred the compound with an affinity to GRP-R in addition to its inherent NMB-R affinity (L227 vs GNLWATGHFM-NH 2 ). Shifting the position of Gly around the linker altered the affinity of L70 from its dual affinity to a selective affinity to NMB-R (L204). 3-methoxy substitution in 4-aminobenzoic acid in L70 (as in L240) changed the dual affinity to a selective affinity to GRP-R.
• These compounds provide information on the combined presence of these two receptor subtypes in vitro and in vivo, that can be used for diagnostic purposes. Targeting both receptors may increase the sensitivity of the examination at the expense of specificity. When these compounds are radiolabeled with a therapeutic radionuclide, therapy can be performed on tissues containing both receptors, which may increase the dose delivered to the desired tissues.
• peptides modified in the targeting portion were made and assayed for competitive binding to GRP-R in human prostate cancer (PC3) cells. All these peptides have a common linker conjugated to a metal chelating moiety (DOTA-Gly-Abz4-).
• the binding data (IC 50 nM) are given below in Table 13.
• PC3 cell lines were obtained from ATCC (CRL-1435) and cultured in RPMI 1640 (ATCC) supplemented with 2 mM L-glutamine, 1.5 g/L Sodium bicarbonate, 10 mM HEPES and 10% FBS. Cultures were maintained in a humidified atmosphere containing 5% CO 2 /95% air at 37 °C.
• PC3 cells for the assays were plated at a concentration of 2.0 x 10 4 cells/ well in a 96-well white/clear bottom plates (Falcon Optilux-I). Plates were used for the assays on day 2 of the post-plating.
• RPMI 1640 (ATCC) containing 25 mM HEPES, 0.2% BSA fraction N, 1.0 mM AEBSF (CAS # 3087-99-7) and 0.1 % Bacitracin (CAS # 1405-87-4), pH 7.4.
• 125 I-Tyr -Bombesin [ 1 , 2 Z 5 3 I ⁇ -BB ⁇ ]
• 125 I-BBN (Cat # NEX258) was obtained from PerkinElmer Life Sciences.
• Radioactivity was detected using either a LKB CompuGamma counter or a microplate scintillation counter.
• the bound radioactivity of 125 I-BBN was plotted against the inhibition concentrations of the competitors, and the concentration at which 125 I-BBN binding was inhibited by 50% (IC 50 ) was obtained from the binding curve.
• IC 50 concentration at which 125 I-BBN binding was inhibited by 50%
• Results/Conclusions Analysis of the binding results of various peptides modified in the targeting portion indicated the following: [00432] Neuromedin analogs (GNLWATGHFM-NH 2 , yGNLWATGHFM-NH 2 ) are unable to compete for the GRP-R except when conjugated to DO3A-monoamide-G-Abz4 (L227). They are, however, effective NMB competitors. This is similar to the requirement for derivatisation of the amino end of the bombesin sequence as reflected in QWAVGHLM-NH 2 , DO3A-monoamide-QWAVGHLM-NH 2 & L70. Replacement of the histidine (L225) reduces competition at the GRP-R.
• a proposed chemical structure of 5 Lu-L70 with a water molecule can be seen in FIG. 62.
• a water molecule occupies a ninth coordination site by capping the square plane described by the coordinated oxygens. This has other precedents. Coordination of water at the ninth site of Lu in Na[Lu(DOTA)H 2 O)] « 4H 2 O was observed in an x-ray structure, as shown by Aime et al, Inorg. Chim. Acta 1996, 246, 423-429, which is incorporated by reference.

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