WO2005049096A2 - Chelateurs a retention sanguine amelioree - Google Patents

Chelateurs a retention sanguine amelioree Download PDF

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WO2005049096A2
WO2005049096A2 PCT/US2004/037769 US2004037769W WO2005049096A2 WO 2005049096 A2 WO2005049096 A2 WO 2005049096A2 US 2004037769 W US2004037769 W US 2004037769W WO 2005049096 A2 WO2005049096 A2 WO 2005049096A2
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meo
pharmaceutical composition
dota
blood retention
bombesin
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PCT/US2004/037769
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WO2005049096A3 (fr
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Phillip S. Athey
Garry E. Kiefer
Jaime Simon
R. Keith Frank
Christopher Adair
Gyongyi Gulyas
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Dow Global Technologies Inc.
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    • 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
    • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
    • A61K51/065—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules conjugates with carriers being macromolecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. 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
    • A61K51/0497—Organic compounds conjugates with a carrier being an organic compounds
    • 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

Definitions

  • This invention relates to functionalized chelants that enhance the blood retention of biotargeting molecules conjugated therewith.
  • radionuclides complexed with suitable chelants, as well as their conjugates, that is, such complexes covalently attached to a biotargeting molecule, for example, a protein or peptide, for diagnosis and/or therapeutic treatment of cancer in mammals is known.
  • Tissue specificity is provided either by the chelant itself, for example, bone agents like methyleneamine diphosphonic acid (MDP) or EDTMP, or by a biotargeting molecule, for example, a monoclonal antibody.
  • MDP methyleneamine diphosphonic acid
  • EDTMP EDTMP
  • the radionuclide provides potent cytotoxicity and/or imagable gamma photons. See, for example, U.S. Patent Nos.
  • Chelants are often used to carry radionuclides to different organs and tissues.
  • a gamma emitting radionuclide When a gamma emitting radionuclide is used, it is typically for diagnostic purposes.
  • 99m Tc-MDP is used to diagnose bone cancer.
  • This radiopharmaceutical is administered intravenously and it can very quickly localizes in bone or is eliminated from the body via the kidneys. In fact greater than 50% of the radioactivity is eliminated via the kidneys.
  • a relatively small amount of radioactivity i.e. about 15 mCi, is typically injected for bone scans.
  • the combination of low amount of radioactivity and the use of a pure gamma-emitting radionuclide results in a low radiation dose to the kidneys.
  • therapeutic radiopharmaceuticals have a very different intent. In this case the objective is to destroy undesirable tissue(s).
  • the radionuclides used in therapy are typically particle-emitters, e.g. beta or alpha emitters. In these cases, high amounts of energy are deposited in a relatively small volume. In addition, significantly higher amounts of radioactivity are used. It is not unusual to inject 75 mCi up to several curies of radiation of therapeutic bone agents such as Sm-EDTMP or 166 Ho-DOTMP.
  • Radiopharmaceuticals based on bioconjugates from small biotargeting molecules such as peptides or monoclonal antibody fragments have more rapid renal clearance than those constructed from full monoclonal antibodies, e.g. IgG. These small bioconjugates also have kidney toxicity problems. For example, in therapy studies using radiometal- labeled Fab fragments, the kidney is the first dose-limiting organ [see Eur. J. Nucl. Med., 25(2), 201-12, 1998]. Additionally, radiopharmaceuticals that exhibit rapid renal clearance can have less than optimal uptake in target tissues.
  • the present invention is a pharmaceutical composition that exhibits enhanced blood retention, the composition comprising: a biotargeting molecule; and a chelating agent conjugated to the biotargeting molecule; wherein the composition exhibits a half-life in the bloodstream of from 15 minutes to 24 hours.
  • the present invention is directed to the use of functionalized chelants to modify the pharmacokinetics of radiopharmaceuticals. Compared with non-functionalized chelants, chelants functionalized with bulky and/or lipophilic groups can slow down the renal clearance and enhance the blood retention of metals complexed therewith. This enhanced blood retention leads to increased uptake of metal in target tissue.
  • these functionalized chelants when conjugated to a biotargeting molecule such as a protein or antibody fragment, can enhance the blood retention of said bioconjugate thus increasing their uptake in target tissues (e.g., tumor).
  • Figures 1 a-c are graphs illustrating the blood retention for several embodiments of the present invention.
  • Figures 2 a-b are graphs illustrating the blood retention for an embodiment of the present invention.
  • Figure 3 is a graph illustrating the blood retention for several embodiments of the present invention.
  • Figures 4 a-c are graphs illustrating the biodistribution results for several embodiments of the present invention.
  • Figures 5 a-c are graphs illustrating biodistribution results for several embodiments of the present invention.
  • Figure 6 is a graph illustrating the blood retention of an embodiment of the present invention.
  • chelant refers to compounds that have the ability to sequester metal ions. Such compounds are of great utility for therapeutic and diagnostic applications when they are, for example, complexed to radioactive metal ions. These types of complexes have been used to carry radioactive metals to bone to treat pain associated with metastatic bone cancer or to ablate the bone marrow in preparation for a bone marrow transplant [see, for example, US patents 4,898,724 and 4,976,950].
  • the term ' ⁇ functional chelant refers to compounds which have the dual functionality of sequestering metal ions plus the ability to covalently bind a biotargeting molecule having specificity for tumor cell epitopes or antigens.
  • biotargeting molecule refers to any protein, antibody, antibody fragment, hormone, peptide, growth factor, antigen, hapten or any other carrier which functions in this invention to recognize a specific biological target site.
  • Antibody and antibody fragment refers to any polyclonal, monoclonal, chimeric, human, 5 mammalian, single chains, dimeric and tetrameric antibody or antibody fragment.
  • Such biological carrier when conjugated (attached) to a functionalized chelate (metal complex of a chelant), serves to carry the attached metal ion to specific targeted tissues.
  • the term "antibody” refers to any polyclonal, monoclonal, chimeric antibody or heteroantibody.
  • the antibodies used in the conjugates of the present invention are monoclonal l o antibodies having high specificity for the desired cancer cells.
  • Antibodies used in the present invention may be directed against, for example, cancer, tumors, leukemias, autoimmune disorders involving cells of the immune system, normal cells that need to be ablated such as bone marrow and prostate tissue, virus infected cells including HIV, mycoplasma, differentiation and other cell membrane antigens, patogen surface antigens
  • Antibody fragments include Fab and F(ab') 2 fragments, single chain Fv fragments, and any portion of an antibody having specificity toward a desired epitope or epitopes.
  • the antibodies that may be used in the conjugates of the present invention can be prepared by techniques well known in the art. Highly specific monoclonal antibodies can be produced by hybridization techniques well known in the art,
  • conjugate means a biotargeting molecule covalently linked to either a bifunctional chelant which can sequester a metal ion, or some other payload such as, for example, a toxin or a growth factor that is to be carried to a biological
  • the term “half-life in the blood stream” is the time it takes for one- half of the material to completely clear the bloodstream.
  • the term “mammal” means animals that nourish their young with milk secreted by mammary glands, preferably humans. 30
  • “pharmaceutically acceptable salt” means any salt of a compound of the present invention that is sufficiently non-toxic to be useful in therapy of mammals.
  • salts which are formed by standard reactions, from both organic and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic, mucic, glutamic, d- camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic, bensenesulfonic, sorbic, picric, benzoic, cinnamic and other suitable acids.
  • the term "therapeutically effective amount” means an amount of the compound of the present invention that produces a therapeutic effect on the disease treated.
  • the therapeutically effective amount will vary depending on the compound, the mammal, and the method of its administration. A person of ordinary skill in the art can determine the therapeutically effective amount of the compound.
  • the compound may be administered per se or as a component of a pharmaceutically acceptable formulation.
  • the present invention may be practiced with the compound being provided in pharmaceutical formulation, for veterinary and/or for human medical use.
  • Such pharmaceutical formulations comprise the active agent (the compound) together with a physiologically acceptable carrier, excipient or vehicle.
  • the carrier(s) must be physiologically acceptable in the sense of being compatible with the other ingredient(s) in the formulation and not unsuitably deleterious to the recipient thereof.
  • the compound is provided in a therapeutically effective amount, as described above, and in a quantity appropriate to achieve the desired dose.
  • the formulations include those suitable for parenteral (including subcutaneous, intramuscular, intraperitoneal, and intravenous) administration. Formulations may be prepared by any methods well known in the art of pharmacy.
  • Such methods include the step of bringing the compound into association with a carrier, excipient or vehicle.
  • the formulations of this invention may further include one or more accessory ingredient(s), for example, diluents, buffers, surface active agents, thickeners, preservatives, and the like.
  • Injectable formulations of the present invention may be either in suspensions or solution form. In the preparation of suitable formulations it will be recognized that, in general, the water solubility of the salt is greater than the acid form. In solution form the compound of this invention (or when desired the separate components) is dissolved in a physiologically acceptable carrier.
  • Such carriers comprise a suitable solvent, preservatives such as free radical quenching agents, for example, ascorbic acid, benzyl alcohol or any other suitable molecule, if needed, and buffers.
  • suitable solvents include, for example, water, aqueous alcohols, glycols, and phosphonate or carbonate esters.
  • aqueous solutions contain no more than 50 percent of the organic solvent by volume.
  • injectable suspensions are compounds of the present invention that require a liquid suspending medium, with or without adjuvants, as a carrier.
  • the suspending medium can be, for example, aqueous polyvinylpyrrolidone, inert oils such as vegetable oils or highly refined mineral oils, polyols, or aqueous carboxymethylcellulose.
  • Suitable physiologically acceptable adjuvants may be chosen from among thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and the alginates.
  • thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and the alginates.
  • surfactants are also useful as suspending agents, for example, lecithin, alkylphenol, polyethyleneoxide adducts, naphthalenesulfonates, alkylbenzenesulfonates, and polyoxyethylene sorbitane esters.
  • the advantage to the chelates of this invention are the ability to use less radioisotope to accomplish the same amount of therapy and less damage to organ systems like the kidneys such due to less amount of the radioactivity passing through.
  • Ho- 166- DOTMP is used as a bone agent, as described in U.S. Patent No. 4,882,142, only about 15- 40%) of the radioactivity injected is taken up by the bone.
  • such bone uptake can be increased. Renal clearance also plays an important role when small proteins, peptides, or fragments are used to deliver isotopes to tumors.
  • the chelating agents that are used to label the targeting molecules are called bifunctional chelating agents.
  • the kidneys can be so efficient at removing the material from the bloodstream that high enough concentrations of the drug for long enough are not possible to give high uptake in tumors.
  • Another aspect of this invention is to provide bulky bifunctional chelating agents such that the renal clearance of the conjugate is decreased. This leads to higher retention in the blood, higher uptake in tumors, and less material cleared through the kidneys.
  • the outcome of this invention is a better therapeutic index by increasing the tumor uptake and decreasing the amount of activity that is processed through the kidneys.
  • the chelating agents for this application are based upon the tetraaza macrocychc backbone, (1,4,7,10-tetraazacyclododecane sometimes referred to as "Cyclen").
  • bifunctional derivatives such as ⁇ -(5-isothiocyanato-2- methoxyphenyl)-l,4,7,10-tetraazacyclododecane-l 5 4,7,10-tetraacetic acid are particularly preferred for two reasons: 1) the chelating portion of the bifunctional chelant is DOTA (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid) which forms stable chelates with the rare earth metals; and 2) the isothiocyante is readily functionalized with amine groups of lipophilic anchors.
  • a second approach is to utilize the mono-alkyl phosphonate ester ligating groups.
  • the tetraaza macrocychc ligand possesses ester functionality that can be varied to achieve the necessary lipophilicity.
  • the charge of the chelates used in the present invention can vary from —5 to +3. Examples of suitable chelates and their charges are as follows:
  • the bombesin peptide was provided by the University of Missouri.
  • the ⁇ -Amyloid (10-20) peptide was purchased from AnaSpec (San Jose, CA).
  • the apomyoglobin from horse skeletal muscle was obtained from Sigma-Aldrich (St. Louis, MO).
  • the 3,500 MWCO molecular porous membrane tubing was obtained from Spectrum Laboratories (Rancho Dominguez, CA).
  • Econo-Pac ® 10DG column was purchased from Bio-Rad Laboratories (Hercules,
  • Coomassie ® Plus-200 protein assay reagent was obtained from Pierce (Rockford, IL). SephadexTM C-25 cation exchange powder was purchased from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Lutetium-177 radionuclide was obtained from PerkinElmer Life Sciences, Inc (N.Billerica, MA) as 434.206 mCi/ml.
  • ESI/LC/MS spectra were obtained from a Waters Alliance 2690 ternary gradient liquid chromatograph coupled to a micromass QTOF2 SN#UC-175, quadrupole/time of flight MS/MS system via a Micromass Z-spray electrospray (ESI) interface operating in the positive ion (PI) mode.
  • ESI Micromass Z-spray electrospray
  • Chlates suitable for use with the present invention can be synthesized according to Scheme 1.
  • the synthesis and characterization of l-[(2-methoxy-5-nitrophenyl) acetate]-l,4,7,10- tetraazacyclododecane (1) is known to those of ordinary skill in the art. Hariprasad Glai, Gary L. Sieckman, Timothy J. Hoffman, Gary E. Kiefer, David T. Chin, Leonard R. Forte, Wynn A.
  • MeO-P chelating agent l-[(2-methoxy-5-nitrophenyl) acetateJ-1,4, 7,10- tetraazacyclododecane- 4, 7,10-(methylenephosphonic acid bis-methyl ester) (2).
  • l-[(2-methoxy-5-nitrophenyl) acetate] -1,4,7,10- tetraazacyclododecane (1) (1 gram, 2.53 mmol) and dry tetrahydrofuran (15 ml). After ⁇ 5 min stirring, paraformaldehyde (239 mg, 7.97 mmol) was added to this solution and stirring continued for 30 min.
  • the catalyst was removed by filtering and an equivalent volume of toluene added to the aqueous filtrate. To this two-phase system was added 2 ml thiophosgene followed by rapid stirring for 1.5 hours. The aqueous phase was then separated and freeze-dried to give the product as a white solid:
  • MeO-DOTA-PEG (19) was dissolved in 5 ml of a 1 M sodium acetate buffer (pH 6) in a 50 mL screw-cap Erlenmeyer flask. 3g of 0.01 M GdCl 3 was added to form complex. The flask was closed and incubated at 50 °C for 16 hours. After the solution was cooled to room temperature, the excess of GdCl was titrated with 0.01 M EDTA until a constant yellow color appeared using 0.05 mL 0.5 % xylenol orange indicator.
  • MeO-PCTA-NCS (1.24 ⁇ mol) were dissolved in water (0.85 ml, 0.88 ml, and 82 ⁇ l) and
  • MeO-P -bombesin, BuO-P-bombesin and MeO-PCTA-bombesin conjugates were adjusted to pH 7 using 0.5 N HCl for Lu complexation in subsequent animal experiments without any further purification.
  • NCS were dissolved in water (28 ⁇ l, 43 ⁇ l, 50 ⁇ l) and added to ⁇ -Amyloid (0.24 ml, 0.41
  • BuO-P-apomyoglobin conjugates were purified by gelfiltration chromatography using
  • the pH of the resultant complex solutions were adjusted to physiological (pH 7.4) with phosphate buffer. 177 Lu-labeling of MeO-DOTA- ⁇ -Amyloid, MeO-P- ⁇ -Amyloid, and BuO-P- ⁇ -Amyloid.
  • LuCl 3 (330000 counts/ ⁇ l) was added to 1 ml MeO-P-Apomyoglobin (0.73 mg/ml) and
  • BuO-P-Apomyoglobin (0.82 mg/ml) saline solutions respectively. These reaction mixtures were adjusted to pH 8 (4M NaOH) and incubated at room temperature for 75 min. The complex formations were evaluated by Sephadex C-25 cation exchange chromatography eluting with ammonium hydroxide (0.9 % NaCl 1 :4). Complexation efficiencies were determined by radioactive counting: 70 % and 93.7 % respectively. The pH of the resultant complex solutions were adjusted to physiological (pH 7.4) with phosphate buffer.
  • MeO-DOTA-Apomyoglobin MeO-DOTA-PEG, MeO-PCTA-N0 2 , and MeO-PCTA-Bombesin.
  • MeO-DOTA-apomyoglobin (0.57 mg/ml), MeO-DOTA-PEG (4.43 ⁇ mol), MeO-
  • MeO-PCTA-NCS were dissolved in water and complexed with 177 Lu + .
  • concentration of each chelate in the aqueous solution was 20 mM.
  • the resulting complex solutions were then purified by Sephadex C-25 cation exchange chromatography to remove any trace of free metal.
  • An equal volume of octanol was added to the four chelate aqueous solutions followed by rapid agitation.
  • the resulting emulsions were allowed to sit for 30 min to permit phase separation.
  • the organic and aqueous were then carefully separated and each one transferred to counting tubes for radiometric determination.
  • Normal BALB/cAnNHsd mice average weight
  • conjugates was injected into each animal via intraperitoneal route. Blood, livers and kidneys were excised from the sacrificed animals at 0.5 hour, 2 hours, 6 hours and 24 hours post injection. Three mice were used in each group. All organs were weighed and the radioactivity was counted. The percent injected dose per gram (%dose/g) and percent injected dose per organ (%dose) were then calculated.
  • MeO-PCTA-NCS The synthetic strategy for 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-l(15),l 1,13- triene-6-(2'-methoxy-5'-isothiocyanatophenyl)methy)- 3,6,9-tripotassium acetate (16) (MeO-PCTA-NCS) is outlined in Scheme 4.
  • PYCLEN 3,6,9,15- tetraazabicyclo-[9.3.1]pentadeca-l(15),l l,13-triene
  • Figure 12 shows the x-ray structure of
  • Step 2-4 in Scheme 4 show the conversion of aminomethanesulfonates to amino acids via nucleophilic substitution.
  • the aminomethanesulfonate was first converted to a bis-nitrile compound (11) with the addition of cyanide.
  • the crude product was estimated to be 90% organic purity (compound 11) with minor impurities arising from different levels of sodium formaldehyde bisulfite substitution.
  • Several purification methods were attempted to purify this intermediate utilizing selective extraction with pH adjusted aqueous rinses.
  • step 4 The hydrolysis reaction (step 4) has been studied by NMR under a variety of aqueous acid -conditions (4-12N HCl) and employing different reaction times (2hours, 5 hours, 9 hours and 24 hours). Following the hydrolysis by NMR shows that concentrated HCl is necessary for complete conversion to the carboxylic acid (14).
  • step 5 10%Pd/C was initially used as the hydrogenation catalyst and the hydrogenation reaction conducted in water at both low and high pH (pH 2 and pH 8) was monitored by HPLC and 1H NMR. Under these conditions, reduction of the pyri ' dine ring was observed along with the desired NO 2 -NH 2 conversion. Eventually, over-reduction was controlled using a different catalyst (PtO 2 ) under acidic condition (pH ⁇ 2). The nitro (14) subsequent conversion to isothiocynate 16 was conducted under basic condition (pH ⁇ 9) in high purity, c).
  • MeO-DOTA-PEG Polyethylene glycol (PEG) has been widely used in covalently formulating macromolecules or low molecular weight drugs for biomedical and biotechnical applications.
  • PEG polyethylene glycol
  • PEG-peptides conjugation PEG is usually designed with an electrophilic functional group which can react with the amino group in the peptides.
  • PEG-lipid conjugates are commonly synthesized with different phosphatidyl ethanolamines (PE) derivatives.
  • Solid-phase method has been used to attach PEG to oligonucleotides as well. Basically, the PEG unit was first attached to porous bead supports and this modified beads were used in the sequential assembly of the desired nucleotide. Examples of PEG-dextran conjugation have also been reported.
  • PEG conjugation with low molecular weight drugs The most common approach utilizes attachment ' of the carboxylic acid group of the drugs to the terminal PEG hydroxyl group.
  • Another method is to use amino-PEG (PEG-NH 2 ) or its 5 derivatives to couple drugs by forming amide, sec-amine, carbamate, or urea linkages.
  • Amino-PEG reacts with MeO-DOTA-NCS at molar ratio of 1 : 10 in o DMF and excess MeO-DOTA-NCS was removed by dialysis (3,500 MWCO) after the conjugation was complete.
  • the product was purified by freeze-drying and analyzed by quantitative titration and mass spectrometry.
  • the titration method utilizes excess Gd 3+ to form complex with MeO-DOTA-PEG and the remaining free Gd 3+ in solution was coordinated by later introduced EDTA.
  • Xylenol orange was used as the indicator for the5 endpoint where no free Gd exists in solution.
  • Aromatic isothiocynate derivatives have become a standard tool for covalent attachment protein/peptide conjugation procedures. Isothiocynate groups readily react5 under mild basic conditions with ⁇ -amino groups of lysine in proteins or peptides to form the stable thiourea linkage. The isothiocynates can also react with sulfhydryl, imidazolyl, tyrosyl and carboxyl groups of the proteins, however, these conjugates are very unstable. Therefore, only the amino group functionality was considered in our studies.
  • Bombesin used in this study contains 8 amino acid residues with a 5 -amino valeric acid residue incorporated as a spacer group for the
  • ⁇ -Amyloid was also selected for this study because of its similar size to bombesin ( ⁇ 1400 Da) and available lysine residue which can couple to the isothiocynates.
  • apomyoglobin was selected for direct comparison with a larger protein. Apomyoglobin( ⁇ 17 KDa) is nearly 15 fold larger than
  • bombesin or ⁇ -Amyloid contains 19 lysine residues which increases the chance of
  • MeO-P-Bombesin conjugation the excess MeO-P- NCS was hydrolyzed into MeO-P-NH2, which has higher polarity and shorter HPLC l o retention time than the isothiocynate.
  • the ⁇ -Amyloid has only one lysine residue in the sequence to conjugate with isothiocynates in bifunctional chelates.
  • the ⁇ -Amyloid has similar molecular size/weight to the bombesin. Therefore, the ⁇ -Amyloid conjugation with BFCs is similar to bombesin with respect to amine residues available for coupling.
  • 177 Lu 3+ is an important radioisotope for therapeutic that employs chelating agents such as DOTA.
  • 177 Lu 3+ is a ⁇ " (500 KeV) as well as a ⁇ (208 KeV) emitter and has half- life of 6.7 days. These properties make 177 Lu 3+ a suitable radionuclide for peptide/protein-
  • the radioactive distribution in the water and octanol is representative of the chelate distribution in the two phases.
  • the partitioning constants of the four chelates are listed in table 4. A higher value means the chelate is more water soluble. From table 4, the polarity of the chelates can be ranked in two series: MeO-DOTA-NCS > MeO-P-NCS > BuO-P-NCS and MeO-DOTA-NCS >5 MeO-PCTA-NCS.
  • the phosphonate ester side-chains have higher lipophilicities than the acetate acid side-chains and the butyl substitute is more lipophilic than the methyl substitute.
  • ⁇ Amyloid, 177 Lu-MeO-P- ⁇ Amyloid, 177 Lu-BuO-P- ⁇ Amyloid, 177 Lu-MeO-DOTA- 1 7 • 1 7 apomyoglobin, Lu-MeO-P-apomyoglobin, and Lu-BuO-P-apomyoglobin were studied at 0.5, 2, 6 and 24 hours post injection.
  • 1 and 3 hours post injection time points results were estimated from trend line formulas and incorporated with the experimental results for the 0.5, 2, 6 and 24 hours post injection time points. .
  • Figure la shows the blood retention study of 177 Lu-MeO-DOTA-NO 2 , 177 Lu-MeO- P-NO 2 , 177 Lu-BuO-P-NO 2 and the bombesin conjugations of these three bifunctional chelates.
  • the left group of columns represents 1 hour post injection results and the right represents 3 hours post injection results. All the unconjugated chelates and chelate- bombesin conjugates were eliminated from blood over time.
  • the blood retention of the unconjugated chelates and MeO-DOTA-bombesin have no significant difference while the phosphonate l A ester bombesin conjugates have significantly elevated retention in blood.
  • ⁇ -amyloid has similar molecular size (MW ⁇ 1400) as bombesin (MW ⁇ 1100).
  • Figure lb
  • the MeO-P- ⁇ Amyloid conjugate did not affect on
  • conjugate did show an increase in blood activity.
  • the observed differences in blood retention between these conjugates might be due to structural differences in tertiary
  • bombesin and ⁇ -amyloid have only one covalently bound chelate.
  • the chelate coupling amine group (from 5-aminovaleric acid residue) of bombesin is close to the C-terminus of the peptide sequence while the chelate coupling amine group (from 5-aminovaleric acid residue) of bombesin is close to the C-terminus of the peptide sequence while the chelate coupling amine group (from 5-aminovaleric acid residue) of bombesin is close to the C-terminus of the peptide sequence while the chelate coupling amine group (from 5-aminovaleric acid residue) of bombesin is close to the C-terminus of the peptide sequence while the chelate coupling amine group (from 5-aminovaleric acid residue) of bombesin is close to the C-terminus of the peptide sequence while the chelate coupling amine group (from 5-aminovaleric acid residue) of bombesin is close to the C-terminus of the
  • the BuO-P-apomyoglobin did not display any blood retention increasing while the MeO-P-apomyoglobin increased the blood retention significantly.
  • One explanation for this observation may be related to the fact that the apomyoglobin sequence has 19 conjugating positions.
  • the conjugation study revealed that the numbers of the chelate-coupled lysine residues in apomyoglobin are different depending upon the chelate. For MeO-DOTA-apomyoglobin and BuO-P-apomyoglobin conjugation, 2 lysine residues 5 were coupled with the bifunctional chelate. In contrast, MeO-P-apomyoglobin conjugation produced 7 lysine residues that were coupled with the chelate.
  • the apomyoglobin is a large peptide compared to the bifunctional chelates, when there are only two chelates conjugated to the peptide, the chelate lipophilicity probably has little effect on blood retention. Therefore, the BuO-P-apomyoglobin did not have a significant o influence on blood relative to MeO-DOTA-apomyoglobin. However, because of a higher substitution (7 chelates) level, the MeO-P-apomyoglobin conjugate could actually have a larger overall increase in the lipophilic character as observed and affect the blood retention more significantly. Therefore, the blood retention of MeO-P-apomyoglobin has noticeable increase.
  • Figure 2 shows the blood retention study of MeO-PCTA-NO 2 and MeO-PCTA- bombesin, using MeO-DOTA-NO 2 as the control. These chelates and conjugates efficiently excreted from blood and reached a very low level ( ⁇ 1 %) at 24 hours post injection (figure 2a). Similar to the phosphonate V% ester chelates, the MeO-PCTA- NO 2 chelate did not have obvious effect on the blood retention by itself. However, within 3 0 hours of post injection, the MeO-PCTA-bombesin conjugate increased blood retention more than 2 fold (figure 2b). The summary of bombesin conjugates is illustrated in figure 3.
  • PEG Polyethylene glycol
  • the pegylation of macromolecules provides the modified molecules ability to avoid quick recognition and clearance in vivo, increase plasma half-life, reduce immunogenicity and antigenicity, and increase solubility.
  • the pegylation of low molecular weight compounds is mainly used in aqueous tow-phase partitioning, PEG cofactor and or catalysts, and delivery control of biologically active substances. Besides these bioactive usages, other PEG functions have been studied. Deguchi and coworkers used PEG as a spacer between the peptide radiopharmaceutical and a MAb transport vector to keep the vector's function. In this research, PEG (MW ⁇ 5500) was employed as a carrier to conjugate with MeO-DOTA-NCS.

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Abstract

La présente invention a trait à une composition pharmaceutique présentant une rétention améliorée, la composition comportant : une molécule de ciblage biologique ; et un agent chélateurs conjugué à la molécule de ciblage biologique ; dans laquelle la composition présente une demi-vie entre 15 minutes et 24 heures dans la circulation sanguine. La présente invention a également trait à l'utilisation de chélateurs fonctionnalisés pour la modification de la pharmacocinétique de produits radiopharmaceutiques. En comparaison avec des chélateurs non fonctionnalisés, des chélateurs fonctionnalisés avec des groupes agglomérés et/ou lipophiles peuvent ralentir la clairance rénale et améliorer la rétention sanguine de complexes métalliques en contenant. Cette rétention sanguine améliorée entraîne une absorption accrue de métal dans un tissu cible.
PCT/US2004/037769 2003-11-14 2004-11-12 Chelateurs a retention sanguine amelioree WO2005049096A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
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WO2005120700A2 (fr) * 2004-06-14 2005-12-22 Novo Nordisk A/S Purification de peptides par chromatographie d'affinites pour les ions metalliques durs
US8926945B2 (en) 2005-10-07 2015-01-06 Guerbet Compounds comprising a biological target recognizing part, coupled to a signal part capable of complexing gallium
US8986650B2 (en) 2005-10-07 2015-03-24 Guerbet Complex folate-NOTA-Ga68

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005120700A2 (fr) * 2004-06-14 2005-12-22 Novo Nordisk A/S Purification de peptides par chromatographie d'affinites pour les ions metalliques durs
WO2005120700A3 (fr) * 2004-06-14 2006-07-20 Novo Nordisk As Purification de peptides par chromatographie d'affinites pour les ions metalliques durs
EP2399938A3 (fr) * 2004-06-14 2012-05-09 Novo Nordisk A/S Purification de peptide au moyen d'une chromatographie d'affinité ionique de métal dur
EP2412729A3 (fr) * 2004-06-14 2012-07-25 Novo Nordisk A/S Purification de peptide au moyen d'une chromatographie d'affinité ionique de métal dur
US8926945B2 (en) 2005-10-07 2015-01-06 Guerbet Compounds comprising a biological target recognizing part, coupled to a signal part capable of complexing gallium
US8986650B2 (en) 2005-10-07 2015-03-24 Guerbet Complex folate-NOTA-Ga68

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