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WO1990013256A1 - Procede de localisation et de traitement de tumeurs a l'aide de nouveaux complexes - Google Patents

Procede de localisation et de traitement de tumeurs a l'aide de nouveaux complexes Download PDF

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Publication number
WO1990013256A1
WO1990013256A1 PCT/US1990/002458 US9002458W WO9013256A1 WO 1990013256 A1 WO1990013256 A1 WO 1990013256A1 US 9002458 W US9002458 W US 9002458W WO 9013256 A1 WO9013256 A1 WO 9013256A1
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Prior art keywords
polylysine
tumor
agent
complex
imaging
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PCT/US1990/002458
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English (en)
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Steven Kornguth
Patrick Turski
H. Ian Robins
Robert J. Nickles
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Wisconsin Alumni Research Foundation
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Publication of WO1990013256A1 publication Critical patent/WO1990013256A1/fr

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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
    • A61K51/08—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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • 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
    • A61K49/14—Peptides, e.g. proteins
    • A61K49/146—Peptides, e.g. proteins the peptide being a polyamino acid, e.g. poly-lysine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • the present invention relates to methods for the location and treatment of tumors and complexes for use in such methods.
  • the localization of tumors such as astrocytomas in the brain ⁇ n vivo and the determination of the margin between normal tissue and tumor can be useful for surgical, radiotherapeutic and chemotherapeutic approaches to the tumor.
  • gliomas generally do not metastasize, they do recur locally after surgical resection and carry a grave prognosis (1).
  • the grave prognosis results in part from the inability to delineate clearly the boundary between tumor and normal brain tissue, and from the restricted permeability of the blood brain barrier to imaging and therapeutic agents.
  • the successful delivery of magnetic resonance contrast agents or of radionuclides for positron or gamma imaging might contribute to the more precise localization of tumor margins.
  • Monoclonal antibodies prepared against the tumor have been proposed for use in the past as effective carrier molecules for the delivery of contrast and radionuclide agents (2,3).
  • the use of such monoclonal antibodies is accompanied by disadvantages.
  • Antibodies are very large molecules that also can carry cross-reactive antigenic determinants that could cause problems.
  • the monoclonal antibodies seldom bind more than 70% of cells, even in clonogenic tumors.
  • polypeptides which selectively binds to tumor cells as compared to normal brain cells, have been considered for use as carrier agents for therapeutic agents.
  • the objects of the present invention include disclosing novel methods for the localization and the treatment of tumors.
  • the objects also include the disclosure of novel complexes which can be used in such methods and a kit containing a complex.
  • a safe and effective amount of a novel complex of a polylysine, a linking molecule and an imaging agent or a chemothera ⁇ Commissionic agent having a high net positive charge is injected into the arterial system of an animal and the complex is carried to and selectively binds to tumors having a greater- net negative charge than non-tumor cells.
  • the method is especially useful for the imaging of polyanionic charged tumors such as Wilms tumors, brain tumors, small cell carcinoma of the lung and melanomas.
  • novel complexes of the present invention comprise polylysine, a linking molecule and an imaging agent or a chemotherapeutic agent.
  • the linking molecule is bound to less than all of the lysyls of the polylysine so that the complexes have a high net positive charge and will bind selectively to tumors having a higher net negative charge than non-tumor cells.
  • the ratio of lysyl residues to linking agent will range from about 5 to 1 to about 20 to 1.
  • polylysine in the complexes are: 1) the chemical homogeneity of the synthetic polymer, 2) the ease of preparation of polylysines having different molecular sizes, 3) the ease of modification of the polymer-linking agent complex with tri- or tetravalent radionuclides, such as gadolinium and zirconium, 4) the ease of labelling polylysine with iodide radionuclides using the Bolton Hunter reagent.
  • the ability to select polylysine polymers of low molecular weight can facilitate the delivery of the imaging or therapeutic agent through the areas where blood-brain barrier is intact without recourse to permeabilization with mannitol (2).
  • polylysines may be modified with multiple nuclides provides an approach to determine dosage and concentration of polymer that is required for successful imaging in a patient population and for simultaneous imaging and therapy (e.g. with Gd and 90 Y.)
  • the polylysine is not immunogenic as compared to immunoglobulins; therefore, the novel polylysine containing complexes are selective for any tumors having greater net negative charges than non-tumor cells and may be used for multiple imaging or multiple therapeutic procedures.
  • the primary advantage of immunoglobulins, such as antibodies resides in the specificity of these proteins for particular tumors and in the relative lack of toxicity as compared with polylysine which exhibits toxicity at concentrations above 1.0 mg per 100 gm body weight (8,10,11).
  • the unexpected sensitivity of the methods of the present invention permits the use of complexes containing polylysine concentrations that are not toxic.
  • the polylysine is coupled covalently to a linking molecule, diethylene triamine pentaacetic acid dianhydride (DTPA), with a ratio of lysine residue/DTPA in the range of 5-20 lysyls per DTPA. This ratio assures that the complexes will have an adequate net positive charge to bind to tumors having a greater net negative charge than non-tumor cells.
  • DTPA diethylene triamine pentaacetic acid dianhydride
  • the preferred degree of polymerization of the polylysine chain length is in the range of 50-100.
  • the polylysine-DTPA complex is preferrably separated from low molecular weight reagents by gel filtration chromatography on Sephadex G-25.
  • the imaging and chemotherapeutic agents such as metal ions that are paramagnetic (e.g. gadolinium, manganese), positron emitters (e.g. 89 Zr), gamma emitters (e.g. I53 Gd) or beta emitters (e.g. 90 Y), are then added to the polylysine-DTPA complex by citrate exchange and the low molecular weight materials present are separated from the polylysine-DTPA-metal ion complex by gel filtration.
  • a paramagnetic component in the complex permits MR imaging
  • a positron emitter permits PET imaging and the presence of a beta emitter in the complex provides a radiotherapeutic agent.
  • a selected complex is injected into the arterial system in the area of the suspected tumor so that the concentrations of polylysine are less than 100 ⁇ g per 100 gms body weight.
  • the method is an imaging method
  • the MRI or PET imaging is then done in a conventional manner after a suitable time delay (24 to 96 hours) to permit maximum contrast between tumor and surrounding tissue.
  • Novel complexes of polylysine-DTPA and the metallic ions ⁇ s 3 Qd and 89 Zr have been found to bind to C6 astro- cytoma and U87 MG glioblastoma cells six to eight times more readily than they bind to endothelial cells from the brain or the aorta and to provide superior imaging.
  • Many tumors have a greater net negative charge than non-tumor cells.
  • Polylysine in the complexes contributes a positive charge that increases their tendency to bind to tumor cells. Therefore, the high net positive charge on the polylysine-DTPA-metal ion complexes, prepared as described, results in selective binding of the complex to the tumor cells.
  • the ingredients for preparing the novel complexes of the present invention may be provided in kit form for the convenience of users.
  • the novel complexes of the present invention may also be supplied with other ingredients for use in test kits for the i__ vitro analysis of tumor cells, tumor cell fragments or tumor specific proteins in spinal fluid or plasma.
  • test kits for the i__ vitro analysis of tumor cells, tumor cell fragments or tumor specific proteins in spinal fluid or plasma are described.
  • Fluorescein-labelled polylysine hydrobromide (DP 88, weight average by light scattering), unmodified polylysine (DP 299 by light scattering, DP 267 by viscosity) and DTPA dianhydride were purchased from Sigma (St. Louis, MO).
  • the ⁇ s3Qd and the i25i Bolton-Hunter reagents (12) were purchased from DuPont-New England Nuclear (Boston, MA); the 89 Zr was generated from an yttrium target by the reaction 89 Y(P,n)8-Zr in the 11 MeV proton beam of the University of Wisconsin, Medical Physics cyclotron (CTI, Inc.).
  • the carrier free 89 Zr was purified by the method of Scadden and Ballou (13).
  • the 89 Zr was coupled to the polylysyl DTPA on the day it was generated from the yttrium. At 24 hours after preparation, 1 pico ole of carrier free 89 Zr contains 300 ⁇ Ci.
  • the polylysine hydrobromide was dissolved in bicarbonate buffer (0.1 mol/1, pH 9.0); then, DTPA, dissolved in anhydrous dimethyl sulfoxide (DMSO), was added immediately to the polylysine.
  • DMSO dimethyl sulfoxide
  • the polylysine-DTPA-Gd chelates were then passed through G-25 Sephadex gel filtration columns, which were preequilibrated with 0.15 mol/1 of sodium chloride, to separate the free gadolinium from the polylysine-DTPA-Gd complex.
  • the complexes containing zirconium and other metal ions can be prepared in a similar manner.
  • the C6 astrocytoma cells were cultured in Ham's F-10 medium, supplemented with 2.5% fetal calf serum (Hyclone Lab, Logan UT) , 15% horse serum (Gibco), 100 ⁇ g streptomycin and 100 unites penicillin per ml, and 1.2 gm bicarbonate buffer per liter.
  • the animals were catheterized through the femoral artery to the ascending aorta. Then 500 ul of saline solution containing 100 ug of 1251 polylysine (Bolton Hunter), or 100 ug of ⁇ s3Gd-and i5 7 Gd-labelled polylysine or 100 ug of 89 Zr labelled polylysine was injected into the aorta. Each rat received 0.23 Ci 89 Zr. The animals were permitted to recover for three additional days to permit the background radiation level to fall. On the 11th day after tumor implantation the animals were anestheized with chloral hydrate and imaged by the Signa MRI using the GE extremity coil (17 cm diameter).
  • the PET images were generated by a CTI Inc Model 933/04-12. It provides a 4 ring, 7 slice positron tomograph with 5 mm full width, half maximum (FWHM) spatial resolution (transverse) and 6 mm FWHM resolution axially.
  • the brains and kidneys of each animal were removed after imaging.
  • the liver, lungs, spleen, thyroid, testes, bone, heart and pancreas of animal three (a tumor recipient) were removed. All three nuclides were measured in the tissue samples by analysis with a Ge(Li)- type counter (15% efficiency germanium gamma spectrometer).
  • Tissue histology was performed on all brain samples to validate the location of the implanted C6 cells.
  • Frozen sections (10 urn) of formalin fixed brains were cut, stained with thionine and covered with DePex embedding material (Gurr Microscopy Ltd) and a cover slip. The sections were examined in a Leitz-DADS microscope and photographed.
  • Poly-L-lysine hydrobromide (DP88) was modified cova- lently with either the chelator, DTPA or with izsi-Bolton Hunter reagent. The polylysine-DTPA was then reacted either with the positron emitter 89 Zr, or paramagnetic stable gadolinium and the gamma emitter !5 3 Gd.
  • the 89 Zr was produced in the 11 meV cyclotron by the reaction 89 Y(p,n) 89 Zr and purified by fractional solubilization techniques utilizing acid and organic solvents.
  • the polylysine-DTPA-nuclide and the polylysine-iodide complexes were separated from the unbound nuclide or metal ion by gel chromatography.
  • Wistar Furth rats were implanted intracranially with C6 astrocytoma and 8 days later they were injected, through a catheter placed in the aorta, with the polylysine nuclide complexes. On the eleventh day after tumor implantation the rats were imaged by magnetic resonance imaging (MRI), and by positron emission tomography (PET).
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • the organs were removed from the rats and the amount of each nuclide was determined by Ge(Li) counting. Frozen sections of the brains were prepared and stained with thionine to validate the tumor growth and the margin between tumor and normal brain.
  • the signal intensity (SI) of the Tl weighted MR images revealed enhancement by the polylysine-DTPA-Gd; the central region of the tumor had a low SI with a high SI at the periphery in all cases.
  • Ge(Li) counting revealed a 3-8 fold higher level of 89 Zr in the tumor containing hemisphere than in the non-tumor hemisphere in 4 of 5 rats surviving 11 days with the implanted tumor.
  • the PET revealed the whole body distribution of the polylysine-DTPA- 89 Zr; the major organs labelled were the tumor, kidney, spleen, thymus, heart, bone, testes and liver and the radioactivity recorded on a counts per second per gram normalized basis. Cytological studies of the thionine stained sections revealed good correlation with the tumor morphology as demonstrated by MR imaging. These observations suggest that polylysine-DTPA-Gd and polylysine-DTPA- 89 Zr complexes may have utility in detecting the margin between astrocytoma and normal brain by MRI and possibly by PET. Polylysine-DTPA-beta emitting metal nuclide complexes may have utility in radiotherapy of such tumors in situ.
  • the magnetic resonance images of the brains of the rats injected with the C6 astrocytoma will be described first.
  • the MR images obtained pre- and post-injection of the modified polylysine will be shown.
  • the MR images obtained from tumor free rats that were injected with the modified polylysines will also be shown.
  • the positron emission tomographs obtained from the rats will be dis- cussed.
  • the images of the other 3 tumor bearing rats were similar. These images were obtained prior to injection of the gadolinium, zirconium and iodide labeled polylysines (DP88). It may be observed that some degree of asymmetry is detectable at this stage (animal 2 and animal 3) and one animal had a low signal intensity (SI) on Tl weighted images (TWI) in the region of the implanted tumor (animal 6). The control animals injected with cell free agarose, by contrast, revealed no unusual features at the same plane of section (animal 8). The images were taken at three mm thickness.
  • the central zone of the C6 astrocytoma in the rat brain is frequently necrotic, an observation consistent with the low SI images obtained.
  • the tumor region is clearly resolved in the 3 mm thick sections when the extremity coil is used.
  • the tumor histology section below indicates that there was no evidence of hemorrhage in the tumor area or surrounding brain.
  • PET of the Tumor Containing and Control Rats The positron emission tomographs of the rats were obtained in 7 planes, from the dorsal to the ventral surface of the rats. Four rats were imaged simul ⁇ taneously and ring sources were used to correct for position in the apparatus. Each set of animals was imaged for 10 hours or longer to obtain the data for image reconstruction. These images reveal that the majority of the 89 Zr-DTPA-polylysine was localized in the kidneys and a second major area of positron source was the snout. It was of interest in this regard that the animals, all of whom received 300 ug of polylysine, had blood in the urine and 2 evidenced nose bleeds.
  • the brain was separated into the right and left hemisphere for determination of the counts per second of each nuclide in the organ.
  • the tumor cells in all cases were implanted in the left frontal region of the brain.
  • Table I reports the counts per sec per gram tissue normalized to the whole rat body for each animal.
  • the nuclide distribution was determined with a Ge ⁇ Li) counter; the 89 Zr was measured from the 909 keV peak, the i53Qd from the 105 keV peak and the 1251 f rom the 27keV peak.
  • An aliquot of the injected polylysine nuclide material was used for calculating the organ distribution of the radionuclides.
  • the polylysyl-DTPA-Gd complex contained more metal ion (cold Gd was added) than the Zr complex, it is possible that the excess metal in the Gd-DTPA complex affected nuclide distribution.
  • the iodide label was equivalent in both hemispheres. This is consistent with the recognized loss of iodide from iodide labeled proteins in the presence of serum and other tissue fluids.
  • the Zr is the label of choice from these observations and the iodide is least preferred of the three nuclides.
  • the distribution of the nuclides in other organs of the body is illustrated in Table II using rat 3 as an example.
  • This table reveals that the organs with highest Zr contents are the kidney, spleen, heart, thymus, bone and testes.
  • the high nuclide content of the spleen, heart, thymus and testes is anticipated since the poly ⁇ lysine was injected directly into the aorta.
  • Positron emission tomographs of the rats reflect the Ge(Li) counts as anticipated and the PET may be used to follow tempo ⁇ rally the polylysine-DTPA- 8 Zr organ distribution.
  • the ratio of the Zr to the Gd dif- fers in several organs indicating that Zr uptake is high in bone and low in liver whereas Gd is high in liver.
  • the tumor proper contains small round cells and larger round cells with pale nuclei and condensed chromatin.
  • the histology confirms that the C6 tumors grew in the adult Wistar Fur h rats, that the cell type and structure is consistent with the properties of the C6 tumor line, and that the MRI images obtained in vivo correlate with the histological appearance of the tissue. There was no evidence of hemorrhagic changes in the tumor or surrounding brain even though necrotic central zone could be seen in some tumors. From the foregoing it is clear that polylysine derivatives, containing DTPA chelated to paramagnetic ions, such as Gd, enhances the MRI of intracerebral tumors.
  • These data represent the first successful use of a tumor selective carrier vehicle, polylysine, to deliver paramagnetic Gd and positron emitting 89 Zr in vivo to a syngenic model rat glioma for the purposes of neuroradiological imaging.
  • This delivery system enhanced the relaxation of water in the area of the C6 astrocytoma.
  • the proof of principle that polyly ⁇ sine-DTPA- 89 Zr may be used for the PET imaging of intra- cranial tumors has also been demonstrated.
  • the advantage of the polylysine-DTPA-Gd complex compared with DTPA-Gd alone resides in the selective binding of the polylysine to the tumor cell surface. Because the polylysine is bound by the tumor, the imaging may be performed after the blood level of the contrast material (i.e. polylysine) has fallen to very low levels. The signal to noise ratio is thus enhanced by the use of polylysine.
  • the polylysine can also be used to deliver several nuclides or chemotherapeutic agents simultaneously, because of the abundance of epsilon amino groups on the polymer. This permits an analysis of drug delivery or of radiation dosage effects by a comparison of the PET image with the MRI image.
  • Polylysine does exhibit toxic properties at concentrations exceeding 1.5 mg per 100 gm body weight in the rat (10,11). However the picomole concentrations of 89 Zr, Gd and 90 Y required for PET imaging, for MRI or for radiotherapy is several magnitudes below toxic concentrations. Polylysine has been used a complexing agent for poly I:poly C in the chemotherapy of tumors (18), and for the delivery of methotrexate to ovarian cells (19). The toxicity problem is therefore amenable to solution.
  • the C6 astrocytoma is a good model for human gliomas because the tumor produces S-100 (20), glutathione S transferase (21) and glial maturation factor (22).
  • the successful imaging of this tumor in vivo has direct applications to the imaging of human brain gliomas in vivo.
  • the polylysine-DTPA-nuclide complex will have greater utility than in the rat.
  • the placement of the catheter into the carotid artery in the area of the tumor is readily achieved as is discernment by PET of the tumor mass.
  • the polylysine polymers preferred for use in the complexes of the present invention are those lysine polypeptide or homopolymers having a molecular weight of about 5,000 to about 20,000 dalton. They can be made by the process described in U. S. Patent No. 3,215,684. Any polylysine which covalently bonds to the chelating agent and possesses an adequate net positive charge to be attracted to and bind to tumor cells and a favorable toxicity ratio can be used.
  • the preferred chelating agent for use in the present invention is DTPA which is also known as pentetic acid and diethylenetriamine pentaacetic acid.
  • the purpose of the chelating agent is to covalently bind to the polylysine and the metallic ions which are imaging or therapeutic agents.
  • DTPA can be prepared as described in U. S. Patent No. 2,384,816.
  • Other chelating agents that might be used include ethylene diamine tetraacetic acid and DOTA.
  • metallic agents that can be used as MR imaging agents in the novel complexes of the present invention are paramagnetic ions such as gadolinium, manganese, and cobalt.
  • Representative of the metallic ions which can be used as PET imaging agents are 89 Zr, and 152 Mn or s 5 Co.
  • Representative of the ions that can be used as ⁇ - camera imaging agents is X11 ln.
  • Representatives of metallic ions that can be used as therapeutic agents in the complexes of the present invention are 90 Y and 211 A+ (astatine).
  • Two other technologies that have clinical utility in tumor studies are possible because of the binding of polylysine containing complexes or probes to tumor cells: a) Spinal fluid samples may be centrifuged at low speed, lOOOg for 10 minutes, to recover any cell or cell fragments present. Spinal fluid normally does not contain cells but may contain cells or cell fragments in the case of central nervous system tumors. The resultant pellet is resuspended in bicarbonate buffer containing saline and the radiolabelled polylysine is added to the suspension.
  • the cell suspension is recentrifuged at the same force indicated above, the pellet recovered and washed three times with the buffer. The pellet is then counted to determine the number of cells per volume of spinal fluid, b)
  • Spinal fluid samples or samples of blood plasma can be incubated with polylysine probes (polylysine alone, polylysine-DTPA-metal ions, polylysine-fluorescein [or other fluorescent probe]).
  • the mixture is centrifuged at low speed, ⁇ 2000g, for 10 minutes.
  • the pellets are resuspended in bicarbonate buffer and applied to pure nitrocellulose membranes.
  • the polylysine probes bind avidly to the nitrocellu ⁇ lose membrane, even when the polylysine is complexed with other materials.
  • the tumor cell-polylysine complexes also will adhere to the membrane.
  • the cells on the membrane may then be incubated with immunoglobulins that are specific for particular tumor cell types. These immunoglobulins are available commercially or can be prepared. Then traditional western blot procedures can be employed to yield spot tests which identify the tumor cell fragments adherent to the nitrocellulose membrane.
  • the initial anti-tumor immunoglobulin may be of varied origin, i.e. from the patient, mice, sheep, goat etc.
  • a second immunoglobulin i.e.
  • anti-human, anti-mouse, anti-sheep, anti-goat IgG and IgM) coupled to a reporter molecule such as peroxidase or phosphatase is incubated with the nitrocellulose membrane.
  • the me - branes are washed after each step.
  • the membranes are incubated with an appropriate substrate which yields a new signal (e.g. color, electric output).
  • the complexes of the present invention when used in vivo as diagnostic agents or therapeutic agents are preferably combined with conventional diagnostic or pharmaceutical diluents, such as Sterile Water for Injection U.S.P., lactose, salts and the like and packaged as sterile preparations.
  • diagnostic or pharmaceutical diluents such as Sterile Water for Injection U.S.P., lactose, salts and the like and packaged as sterile preparations.
  • the preparations will normally contain a safe and effective amount of the metal ions, which are either known imaging or diagnostic agents, for their intended use.

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Abstract

L'invention concerne de nouveaux procédés de localisation sélective, d'imagerie et/ou traitement de tumeurs qui ont une charge négative nette, à l'aide de nouveaux complexes ayant une charge positive nette. Les complexes contiennent une polylysine, un agent de liaison lié à moins de tous les groupes lysile de la polylysine, et un agent d'imagerie ou un agent chimiothérapeutique qui est également lié à l'agent de liaison.
PCT/US1990/002458 1989-05-04 1990-05-02 Procede de localisation et de traitement de tumeurs a l'aide de nouveaux complexes WO1990013256A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5364613A (en) * 1989-04-07 1994-11-15 Sieving Paul F Polychelants containing macrocyclic chelant moieties
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US6958148B1 (en) 1998-01-20 2005-10-25 Pericor Science, Inc. Linkage of agents to body tissue using microparticles and transglutaminase
WO2009074274A1 (fr) * 2007-12-10 2009-06-18 Bayer Schering Pharma Aktiengesellschaft Nanoparticules polymères solides fonctionnalisées contenant des épothilones
WO2011009539A1 (fr) * 2009-07-20 2011-01-27 Merck Patent Gmbh CONJUGUÉS DE ε-POLYLYSINE ET LEUR UTILISATION
WO2012143508A1 (fr) * 2011-04-20 2012-10-26 Spheritech Ltd Particules d'epsilon-polylysine réticulée
WO2013138696A1 (fr) * 2012-03-16 2013-09-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Complexes de zirconium-89, procédés de marquage de cellules, cellules marquées, kits et leurs procédés d'utilisation
GB2562004A (en) * 2012-04-20 2018-10-31 Spheritech Ltd Cross-linked poly-e-lysine particles
US10736976B2 (en) 2016-12-01 2020-08-11 Regeneron Pharmaceuticals, Inc. Radiolabeled anti-PD-L1 antibodies for immuno-PET imaging

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Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5364613A (en) * 1989-04-07 1994-11-15 Sieving Paul F Polychelants containing macrocyclic chelant moieties
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US6958148B1 (en) 1998-01-20 2005-10-25 Pericor Science, Inc. Linkage of agents to body tissue using microparticles and transglutaminase
WO2009074274A1 (fr) * 2007-12-10 2009-06-18 Bayer Schering Pharma Aktiengesellschaft Nanoparticules polymères solides fonctionnalisées contenant des épothilones
WO2011009539A1 (fr) * 2009-07-20 2011-01-27 Merck Patent Gmbh CONJUGUÉS DE ε-POLYLYSINE ET LEUR UTILISATION
US20120122788A1 (en) * 2009-07-20 2012-05-17 Merck Patent Gesellschaft Mit Beschrankter Haftung E-polylysine conjugates and the use thereof
CN102470181A (zh) * 2009-07-20 2012-05-23 默克专利有限公司 ε-聚赖氨酸缀合物及其用途
AU2010275778B2 (en) * 2009-07-20 2016-05-05 Merck Patent Gmbh Epsilon-polylysine conjugates and use thereof
JP2012533579A (ja) * 2009-07-20 2012-12-27 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング ε−ポリリシン接合体およびその使用
EA022137B1 (ru) * 2009-07-20 2015-11-30 Мерк Патент Гмбх КОНЪЮГАТЫ ε-ПОЛИЛИЗИНА И ИХ ПРИМЕНЕНИЕ
US9078929B2 (en) 2009-07-20 2015-07-14 Merck Patent Gmbh ε-Polylysine conjugates and the use thereof
CN102470181B (zh) * 2009-07-20 2014-12-03 默克专利有限公司 ε-聚赖氨酸缀合物及其用途
JP2014513737A (ja) * 2011-04-20 2014-06-05 スフィリテック・リミテッド 架橋ポリ−ε−リシン粒子
US10266652B2 (en) 2011-04-20 2019-04-23 Spheritech Ltd. Cross-linked poly-E-lysine non-particulate support
GB2504641A (en) * 2011-04-20 2014-02-05 Spheritech Ltd Cross-linked poly-e-lysine particles
GB2504439A (en) * 2011-04-20 2014-01-29 Spheritech Ltd Cross-linked poly-e-lysine non-particulate support
GB2504439B (en) * 2011-04-20 2020-01-01 Spheritech Ltd Cross-linked poly-e-lysine non-particulate support
WO2013041250A1 (fr) * 2011-04-20 2013-03-28 Spheritech Ltd Support non particulaire en poly-e-lysine réticulée
WO2012143508A1 (fr) * 2011-04-20 2012-10-26 Spheritech Ltd Particules d'epsilon-polylysine réticulée
US9938378B2 (en) 2011-04-20 2018-04-10 Spheritech Ltd Cross-linked poly-E-lysine non-particulate support
GB2504641B (en) * 2011-04-20 2018-09-05 Spheritech Ltd Cross-linked poly-e-lysine particles
KR101976151B1 (ko) 2011-04-20 2019-05-07 스페리테크 리미티드 가교된 폴리-ｅ-라이신 비-미립자 지지체
KR20140038417A (ko) * 2011-04-20 2014-03-28 스페리테크 리미티드 가교된 폴리-ｅ-라이신 비-미립자 지지체
WO2013138696A1 (fr) * 2012-03-16 2013-09-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Complexes de zirconium-89, procédés de marquage de cellules, cellules marquées, kits et leurs procédés d'utilisation
GB2562004A (en) * 2012-04-20 2018-10-31 Spheritech Ltd Cross-linked poly-e-lysine particles
GB2562004B (en) * 2012-04-20 2019-10-23 Spheritech Ltd Cross-linked poly-e-lysine particles
US10736976B2 (en) 2016-12-01 2020-08-11 Regeneron Pharmaceuticals, Inc. Radiolabeled anti-PD-L1 antibodies for immuno-PET imaging
US12053534B2 (en) 2016-12-01 2024-08-06 Regeneron Pharmaceuticals, Inc. Radiolabeled anti-PD-L1 antibodies for immuno-PET imaging

Also Published As

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EP0471790A1 (fr) 1992-02-26
EP0471790A4 (en) 1992-07-08
CA2032169A1 (fr) 1990-11-05
AU628988B2 (en) 1992-09-24
FR2646608A1 (fr) 1990-11-09
AU5679490A (en) 1990-11-29

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