CZ261199A3 - Method of increasing accumulation of radiolabelled compounds in tissues - Google Patents

Abstract

Administration of a radiolabeled compound in an over lasting period of time »2 hours, better than 12 hours, greatly increases the maximum of radioactivity that accumulates in the target cells. Efficiency administration of the radiolabelled compound may be about five times higher than the bolus injection tone or short-term injection. This method increases the ratio of radioactivity between the tumor and the background by increasing the actual amount of radioligand accumulated in tumor cells. This method can be used in any way a radiolabeled compound whose uptake in the cells is limited by binding to cellular receptors or transport proteins. Where radiolabelled compounds are bound and In general, the ability of an unlabelled compound to compete with is radioligandemically reduced. The primary factor influencing retention time after intemalization is the physical half-life of the radioisotope, not its biological half-life.

Description

Technical field

The present invention relates to a method for increasing the accumulation and retention of radiolabeled compounds (radioligands) in tissues, thereby increasing their therapeutic and diagnostic value.

BACKGROUND OF THE INVENTION

Radiolabeled compounds are used for both tumor detection and tumor therapy. Many tumor cells have higher cell density for various circulating compounds than non-tumor cells; for example, endocrine tumors have a higher surface density of somatostatin receptors, and brain gliomas have a higher density of epidermal growth factor receptors. Therefore, radiolabeled compounds that bind to these cell receptors may preferentially bind to tumor cells. In addition, angiogenesis, the formation of new blood vessels from an already existing microvasculature, is an essential process in tumor growth. Primary tumors and metastases do not grow above 2 mm in diameter unless they have sufficient vascular supply. Angiogenic cells also have higher cell density for various circulating compounds than non-angiogenic vascular tissue; for example, the amount of somatostatin receptors and vascular endothelial growth factor is higher in angiogenic tissue. Therefore, tumors can also be detected using radiolabeled compounds that bind to angiogenic cells that are closely associated with tumor cells.

• ·

An ideal tumor imaging agent will maximize radioactivity in the target cells and minimize background signal, resulting in a well defined tumor focus image. For example, ^1u -DTPA-D-Phe-1-octreotide and ¹²³ Ivasoactive intestinal peptide, two radioligands for receptors, have been used to localize primary endocrine tumors as well as localize metastatic lesions in the liver. See A. Kurtaran et al., Vasoactive Intestinal Peptide and Somatostatin Receptor Scintigraphy for Differential Diagnosis of Hepatic Carcinoid Metastasis, The Journal of Nuclear Medicine, Vol. 38, pp. 880-881 (1997).

The ideal agent for radioligand therapy will be selectively accumulated in the target cells. The effect of radiotherapy is caused by the destruction of dividing cells, which is caused by radioactivity induced damage to cellular DNA. See W.D. Bloomer et al., Therapeutic Application of Iodine-125 Labeled Iododeoxyuridine in the Early Ascites Tumor Model, Current Topics in Radiation Research Quarterly, Volume 12, pages 513-525 (1977). In both therapeutic and diagnostic applications, the circulating radioligand is rapidly secreted by secretion systems, protecting normal organs and tissues. The radioligand may also be degraded by metabolic processes that increase clearance of the free radioisotope. See G.A. Wiseman et al., Therapy of

Neuroendocrine Tumors wit Radiolabelled MIBG and Somatostatin Analogues, Seminars in Nuclear Medicine, Volume XXV, No. 3, pp. 272-278 (1995).

In both therapeutic and diagnostic applications, the clinical goal is to maximize the amount of radiolabeled compound that is taken up by the tumor. The amount of radioligand that accumulates in target cells depends on many factors, such as: (1) concentration gradient

blood-target radioligand; (2) the number of cellular receptors, membrane or intracellular, and the affinity of these receptors for the radioligand; (3) at relative concentrations of labeled and unlabeled ligands competing for the receptor; (4) the cell receptor recycling rates; (5) on the capacity of the cells to store the radioligand; and (6) for degrading the radioligand within cells. See R.K. Rippley et al., Effects of Cellular Pharmacology on Drug Distribution in Tissues, Biophysical Journal, Volume 69, pp. 825-839 (1995).

Radiolabeled compounds are usually administered intravenously by bolus injection. In a few cases, radiolabeled compounds were administered as an infusion over 30 to 60 minutes, usually to reduce the side effects of the drug, not to increase efficacy. See, for example, H.P. Kalofonos et al., Antibody Guided Diagnosis and Therapy of Brain Gliomas using Radiolabeled Monoclonal Antibodies Against Epidermal Growth Factor Receptor and Placental Alkaline Phosphatase ,,

The Journal of Nuclear Medicine, Vol. 30, pp. 1636-45 (1989); Virgolini et al., Vasoactive Intestinal PeptideReceptor Imaging for Localization of Intestinal Adenocarcinomas and Endocrine Tumors, The New England Journal of Medicine, Vol. 331, pp. 1116-21 (1994); G.A. Wiseman et al.,

Therapy of Neuroendocrine Tumors wit Radiolabelled MIBG and Somatostatin Analogues, Seminars in Nuclear Medicine, Volume XXV, No. 3, pp. 272-278 (1995); S.W.J. Lamberts et al., Somatostatin-Receptor Imaging in the Localization of Endocrine Tumors, The New England Journal of Medicine, Vol. 323, pp. 1246-49 (1990); E.P. Krenning et al., Somatostatin-Receptor Scintigraphy with Indium-III-DTPA-D-Phe-1-Octreotidein Man: Metabolism, Dosimeters and Comparisons in iodine-123-Tyr-3Octreotide, The Journal of Nuclear Medicine, Volume 33, p.

652-58 (1992); E.P. Krenning et al.,

Localization of •

Endocrine-Related Tumors with Radioiodinated Analogue of Somatostatin, The Lancet, Vol. 1989, No. 1, pp. 242-244 (1989). There is one article that describes the duration of an infusion of two (2) hours. See J.A. Carrasquillo et al., Indium-111 T101 Monoclonal Antibody Is Superior to Iodine-131 T101 in Imaging of Cutaneous T-Cell Lymphoma, The Journal of Nuclear Medicine, Vol. 28, pp. 281-87 (1987).

The ability of cells to take up radiolabeled compounds in a short period of time is limited by the number of cell receptors or transport proteins for compounds on the cell membrane or within cells. When the radioligand is administered by bolus injection, the pharmacokinetic characteristics of the binding determine that uptake of the radioligand is proportional to the amount only at low radioligand concentrations. At higher concentrations, radioligand receptors are saturated. See H. Zhu et al., Potential and Limitations of Radioimmunodetection and

Radioimmunotherapy with Monoclonal Antibodies, The Journal of Nuclear Medicine, Vol. 38, No. 5, pp. 731-41 (1997); and R.M. Kessler et al., High Affinity Dopamine D 2 Receptor

Radioligands. 1, Regional Rat Brain Distribution of Iodinated Benzamides, The Journal of Nuclear Medicine, Vol. 32, pp. 1593 - 1600 (1991). Such saturated receptors are unable to bind another radioligand until the receptor releases the radioligand or the receptor-radioligand complex is transported to another part of the cell and the receptor is recycled to re-bind the new radioligand molecule. Since the circulating unbound radioligand is rapidly eliminated, the radioligand may no longer be present in the circulation by the time the receptor is capable of receiving another radioligand molecule. Therefore, the accumulation of radioligand depends on the availability of free radioligand and on the recycling time of cellular receptors and transport proteins.

• 9

Recycling of cell receptors depends on the fate of the ligand receptor complex. Many - if not more? - peptide compounds (including peptides and protein hormones) that bind to surface receptors are internalized as a ligand-receptor complex by endocytosis, ie invagination into the plasma membrane. Examples of peptides that are internalized as part of a ligand-receptor complex include nerve growth factor, fibroblast growth factor, epidermal growth factor, platelet growth factor, cholecystokinin, vascular endothelial growth factor, vasoactive intestinal peptide, gastrin releasing peptide, leukemia inhibitory factor, somatostatin , oxytocin, bombesin, calcitonin, arginine vasopressin, angiotensin II, atrial natriuretic peptide, insulin, glucagon, prolactin, growth hormone, gonadotropin, thyrotropin triggering hormone, triggering hormone for growth hormone, triggering hormone for gonadotropin, triggering hormone, triggering hormone , interferons, transferrin, substance P, neuromedin, neurotensin, neuropeptide Y and various opiates. This internalization takes minutes or even hours. See G. Morel, Internalization and Nuclear Localization of Peptide Hormones, Biochemical Pharmacology, Vol. 47 (1), pp. 63-76 (1994); D. Nouel et al., Differential Internalization of Somatostatin in COS-7 Cells Transfected with SSTi and SST ₂ Receptor Subtypes: A Confocal Microscopic Study Using Novel Fluorescent

Somatostatine Derivatives, Endocrinology, Volume 138, pp. 296-306 (1997); L.H. Wang et al., Ligand Binding,

Guanine Nucleotides of Bombesin Receptor Subtypes: A Comparative Study, Biochimica et Biophysica Acta, Vol. 1175, 232-232 (1993). Monoclonal antibodies have also been reported to be internalized into cells; See O.W. Press et al., Comparative Metabolism and Retention of Iodine-125, Yttrium-90 and Indium

111 Radioimmunoconjugates by Cancer Cells, Cancer Research, Vol. 56, pp. 2123-29, (1996).

After internalization, many peptides move to the nucleus where they bind to DNA. Peptides that have been shown to accumulate in the nuclei of target cells include insulin, growth hormone, prolactin, nerve growth factor, somatostatin, epidermal growth factor, fibroblast growth factor, platelet growth factor, and interferons. Nuclear binding sites have been described for gonadotropin trigger hormone, gonadotropin, growth hormone, angiotensin II, prolactin, transferrin, insulin, various interleukins, glucagon, various opiates and growth factors (including epidermal growth factor, nerve growth factor, platelet growth factor and fibroblast) factor). Specific nuclear effects have been demonstrated for insulin and epidermal growth factor. See G. Morel, Internalization and Nuclear Localization of Peptide Hormones, Biochemical Pharmacology, Vol. 47 (1), pp. 63-76 (1994); and P.M. Laduron From Receptor Internalization to Nuclear Translocation - New Targets for Long-Term

Pharmacology, Biochemical Pharmacology, Volume 47, pp. 3-13 (1994).

Steroid hormones are known to diffuse across the plasma membrane and either bind to intracellular receptors and move to the nucleus, or directly bind to receptors in the nucleus. See W.V. Welshons et al., Nuclear Localization of Unoccupied Oestrogen Receptors, Nature, vol. 307, pp. 747-49, (1984). Classes of steroid hormones known to bind to intracellular receptors include progestins (eg progesterone), androgens (eg testosterone), glucocorticoids (eg hydrocortisone), mineralocorticoids (eg aldosterone) and estrogens ·· AA • · A • AA (eg estradiol). See D.J. Sutherland et al., Hormones and Cancer, The Basic Science of Oncology, 2, Ed. (I.F. Tannock and R. P. Hill, Ed.), Chapter 13, pp. 207-231, (1992). Breast and prostate cancer cells are known to have an increased number of steroid hormone receptors.

One method that has been used to increase the tumor-background ratio for a radioligand in therapy or detection is to reduce the uptake of radioactivity by the surrounding tissues while changing the rate of degradation or excretion. As background radiation decreases, the tumor-background ratio increases; however, the amount of radioligand accumulated by the tumor cells remains the same. Thus, the actual therapeutic dose (intra-cell dose) does not change, even though the image of the tumor against the background is more contrasted.

Methods that have been used to increase radioligand uptake by tumor cells and thus to increase the therapeutic or diagnostic dose include the following: use of a more targeted tumor cell radioligand, use of a higher tissue diffusion rate radioligand, change of radioligand elimination rate, use of longer radioligand half-life, use of a radioisotope with a longer physical half-life, and use of higher doses of radioligand. The radioligand was administered either as a single bolus dose or as a short infusion over up to 2 hours. Models have been developed attempting to identify parameters that can be optimized for more efficient uptake. These models allow for the common assumption that radiolabeled compounds are administered in a single bolus dose. See Rippley et al. (1995); S.E. Strand et al., Pharmacokinetic Modeling, Medical Physics, Volume 20 (2), Pt. 2, pp. 515-27 (1993); and H. Zhu et al., Potential and Limitations of Radioimmunodetection and Radioimmunotherapy with Monoclonal Antibodies, The Journal of Nuclear Medicine, Vol.

• BBB • BBB

38, No. 5, pp. 731-41 (1997). There is a need in the art for methods for increasing radioligand accumulation in target cells without increasing damage to normal cells.

Radiolabeled somatostatin peptide analogues were studied for their efficacy in tumor detection and therapy. See E.A. Woltering et al., The Role of Radiolabelled Somatostatin Analogs in the Management of Cancer Patients, Principles and Practice of Oncology, Volume 9, pp. 1-15 (1995); U.S. Pat. U.S. Patent No. 5,590,656; and U.S. Pat. Endogenously produced somatostatin, a tetradecapeptide, inhibits the release of several pituitary and intestinal factors that regulate cell proliferation, cell motility or cell secretion, including secretion of growth hormone, adrenocorticotropic hormone, prolactin, thyroid stimulating hormone, insulin, insulin gastric inhibitory peptide (GIP), vasoactive intestinal peptide (VIP), secretin, cholecystokinin, bombesin, gastrin trigger peptide (GRP), adrenocorticotropic hormone (ACTH), thyrotropic hormone trigger hormone (TRH), cholecystokinin (CCK), pancreatic polypeptide (PP), various cytokines (e.g. interleukins, interferons), various growth factors (e.g. epidermal growth factor, nerve growth factor), and various vasoactive amines (e.g. serotonin).

Because somatostatin has a short half-life (1-2 minutes), many somatostatin analogs have been produced, by eliminating amino acids, substituting the natural L-amino acids with the corresponding D-isomers of the amino acids, addition of alcohol to the carboxyl terminus of the molecule, or using different combinations of these approaches. See U.S. Pat. Examples of somatostatin analogs include octreotide acetate, lantreotide, vapreotide.

9 (RC-160) and pentetreotide, all of which have a longer half-life. Multi-tyrosinated somatostatin analogs were produced and were shown to bind to cell receptors for somatostatin. See U.S. Pat. No. 5597894.

Receptors for somatostatin are found in all parts of cells, including the cell membrane, Golgi apparatus, endoplasmic reticulum, vesicles and nucleus. Somatostatin and its analogs are internalized by endocytosis of the ligand receptor function. See LJ Hofland et al., Internalization of the Radioiodinated Somatostatin Analog [ ¹²⁵ I-Tyr ³ ] Octreofcideby Mouse and Human Pituitary Tumor Cells: Increase by Unlabeled Octreotide, Endocrinology, Volume 136, pp. 3698 - 3706 (1995); Wiseman et al., (1995).

High densities of somatostatin receptors, particularly somatostatin receptor subtype 2 (SST-2), have been found on cells from many tumors, including endocrine tumors, melanomas, breast cancer, Merkel cell tumors, lymphomas, small cell lung cancer, gastrointestinal tumors, astrocytomas , gliomas, meningeomas, carcinoids, pancreatic B cell tumors, kidney tumors, neuroblastomas, and pheochromocytomas. See EA Woltering et al., The Role of Radiolabelled Somatostatin Analogs in Management of Cancer Patients, Principles and Practice of Oncology, Vol. 9, pp. 1-15 (1995); and EA Woltering et al., Somatostatin Analogs: Angiogenesis Inhibitor with Novel Mechanisms of Action, Investigational New Drugs, Volume 15, pp. 77-86 (1997). Radiolabelled somatostatin analogue ^{1: L1} In-pentetreotide, known to bind to SST-2 receptors on the cell membrane, has been shown to bind to pituitary tumors, pancreatic endocrine tumors, carcinoids, paragangliomas, pheochromocytomas, medullary thyroid carcinomas, small cell lung carcinomas, neuroblastomas, meningiomas, breast cancers, renal cancers, gliomas, astrocytomas, melanomas and lymphomas. ⁿⁱ -pentetreotide has also been used to treat metastatic glucagon and carcinoid. See Wiseman et al., 1995; Krenning et al., Radiotherapy with a radiolabelled somatostatin analogue, [beta] -DTPA-D-Phe-1] octreotide. A Time History, Annals of the New York Academy of Sciences, Volume 733, pp. 469-506 (1996); and M. Fjalling et al., Systemic Radionuclide Therapy Using Indium-111-DTPA-DPhe-1-Octreotide in Midgut Carcinoid Syndrome, The Journal of Nuclear Medicine, Vol. 37, pp. 1519-21 (1996).

Radiolabeled somatostatin or somatostatin analogs were used for tumor detection and / or therapy and were given either by bolus injection or short-term infusion (within 2 hours).

See S.W.J. Lamberts et al., Somatostatin-Receptor Imaging in the Localization of Endocrine Tumors, The New England Journal of Medicine, Vol. 323, pp. 1246-49 (1990); E.P. Krenning et al., Somatostatin-Receptor Scintigraphy with Indium-111DTPA-D-Phe-1-Octreotides in Man: Metabolism, Dosimetry and Comparison in iodin-123-Tyr-3-OctreoticE, The Journal of Nuclear Medicine, Volume 33, pp. 652-58 (1992); E.P.

Krenning et al., Localization of Endocrine-Related Tumors with Radioiodinated Analogue of Somatostatin, The Lancet, Vol. 1989, No. 1, pp. 242-244 (1989); WAP Breeman et al., Studies on Radiolabelled Somatostatin Analogues in Rats and Patients, The Quarterly Journal of Nuclear Medicine, Vol. 40, pp. 209-220 (1996); and EP Krenning et al., Somatostatin Receptor Scintigraphy with [ ¹¹¹ In-DTPA-D-Phe ¹ ] - and [ ¹²³ I-Tyr ³ ] -octreotide: The Rotterdam Experience With More Than 1000 Patients, European Journal of Nuclear Medicine, Vol. 20, pp. 716-31 (1993).

···· ·

Radiolabeled somatostatin analogues that have been used for tumor detection or therapy include ⁱⁿ Inpentetreotide ((In In-DTPA-D-Phe ¹ ) -octreotide), (In In-DOTA ^ D-Phe ¹ Tyr ³ ) -octreotide) , ( ⁹⁰ Y-D0TA ° -D-Phe ¹ -Tyr ³ ) -octreotide), (® ^S Y-DOTA ° D-Phe ^x -Tyr ³ ) -octreotide), ( ¹¹¹ In-DTPA-D-Phe ¹ ) -RC-160, ^99m Tc-RC-160, ^99m Tc-octreotide, ¹⁸⁸ Re-RC-160, ¹²³ I-Tyr ^3- octreotide, ¹²⁵ I-Tyr ³ octreotide, ¹²⁵ I-lantreotide, ⁹⁰ Y-DOTA-lantreotide , and ¹³¹ I-WOC-3.

See Woltering et al., 1995; M.L. Thakur et al., Radiolabelled Somatostatin Analogs in Prostate Cancer, Nuclear Medicine and Biology, Volume 24, pp. 105-113, (1997); M. de Jong et al.,

Yttrium-90 and Indium-111 labeling, Receptor Binding and Biodistribution of [DOAT °, D-Phe ¹ , Tyr ³ ] octreotide, and Promising Somatostatin Analogue for Radionuclide Therapy, European Journal of Nuclear Medicine, Volume 24, pp. 368-371 (1997); WAP Breeman et al., A New Radiolabelled Somatostatine Analogue [ ¹¹¹ In-DTPA-D-Phe ¹ ] RC-160: Preparation, Biological Activity, Receptor Scintigraphy in Rats and Comparison with [^ ”in-DTPA-D-Phe ^ octreotide , European Journal of Nuclear Medicine, Vol. 21, No. 4, pp. 323-355 (1994); WAP

Breeman et al., Studies on Radiolabelled Somatostatin Analogues in Rats and Patients, The Quarterly Journal of Nuclear Medicine, Vol. 40, pp. 209-220 (1996); M. Fjalling et al., Systemic Radionuclide Therapy Using Indium-III-DTPA-DPhe-1-Octreotide in Midgut Carcinoid Syndrome, The Journal of Nuclear Medicine, Vol. 37, pp. 1519-21 (1996); H. Kolan et al., Sandostatin ^R Labeled with ^99m Tc: In Vitro Stability, In Vivo Validity and Comparison With ^{11: L} In-DTPA-Octreotide,

Peptide Research, Vol. 9, No. 3, pp. 144-150 (1996); Virgolini et al., 'MAURITIUS ¹ ': Biodistribution, Safety and Tumor Dose in Patients Evaluated for Somatostatin Mediated Radiotherapy, Paper Submitted to the Journal of Nuclear Medicine, (1997); and US Patent No. 5597894.

inhibit C: Cp ::?

O'Reillv.

Somatostatin analogues have also been shown to angiogenesis in tumors. The primary tumor initiates neovascularization by simulating angiogenesis. See M.S.

Angiostatin: An Endogenous Inhibitor of Angiogenesis and of Tumor Growth, in I. Goldberg et al., (Ed.), Regulation of Angiogenesis, pp. 273-294 (1997). The growth of solid tumors is dependent on neovascularization. This angiogenic tissue has been shown to be rich in somatostatin 2 receptor subtype (SST-2) and is inhibited by somatostatin analogues known to bind to SST-2 receptors such as octreotide acetate, RC-160 and lantreotide. See E.A. Woltering et al., The Role of Radiolabelled Somatostatin Analogs in the Management of Cancer Patients, Principles and Practice of Oncology, Volume 9, pp. 1-15 (1995); E.A. Woltering et al., Somatostatin Analogs: Angiogenesis Inhibitor with Novel Mechanisms of Action, Investigational New Drugs, Volume 15, pp. 77-86 (1997); P.C. Patel et al., Postreceptor Signal Transduction Mechanism Involved in Octreotide-Induced Inhibition of Angiogenesis, Surgery, Vol. 116, pp. 1148 52 (1994); R. Barrie et al., Inhibition of Angiogenesis by Somatostatin and Somatostatin-like Compounds Is Structurally Dependent, Journal of Surgical Research, Vol. 55, p. 446 450 (1993); and E.A. Woltering et al., Somatostatin Analogues Inhibit Angiogenesis in the Chick Chorioallantoic Membrane, Journal of Surgical Research, Vol. 50, pp. 245-251 (1991).

Angiogenic vessels have SST-2 receptors at a higher density than those of normal tissues. See J.C. Watson et al., Up-Regulation of Somatostatin Receptor Subtype 2 (SST-2) mRNA Occurs During The Transformation of Human Endothelium to Angiogenic Phenotype, published at the 12th International Symposium on Regulators Peptides, Copenhagen, Denmark, September 1996; and J.C. Watson et al., SST-2 Gene Expression Appears During Human Angiogenesis, Peptides Regulators, Volume 64, p. 206 ··· ··············· 4 Abstract (1996). Radiolabeled somatostatin analogues that bind. SST-2 receptors on tumor vessels were used for radiodetection and radiotherapy. Tumor size decreases because vascular growth is inhibited by the radiolabeled compound. See J.C. Reubi et al., High Density

Somatostatin Receptors in Veins Surrounding Human Cancer Tissue: Role in Tumor-Host Interaction ?. International Journal of Cancer, Volume 56, pp. 681-88 (1994). Pathological vascular growth is also contemplated in several other diseases, including premature retinopathy, diabetic retinopathy, glaucoma, tumor growth, rheumatoid arthritis, and inflammation. See, Barrie et al., 1993. Indeed, ⁱⁿ -pentetreotide has been used to locate areas of joints affected by rheumatoid arthritis. See PM Vanhagen et al., Somatostatin Receptor Imaging: The Presence of Somatostatin Receptors in Rheumatoid Arthritis, Arthritis and Rheumatism, Vol. 37, No. 10, pp. 1521-27 (1994).

Angiogenic cells have also been shown to express the kdr gene for the vascular endothelial growth factor (VEGF) receptor, while resting vascular cells do not express the gene for this receptor. Both angiogenic and resting cells express the flt-1 VEGF receptor gene. See J.C. Watson et al., Initiation of kdr Gene Transcription is Associated with Conversion of Human Vascular Endothelium to an Angiogenic Phenotype, Surgical Forum, Volume 47, pp. 462-64, (1996).

Non-radiolabeled somatostatin analogues have been used to inhibit growth hormone secretion by infusing from an implantable pump to prevent intermittent growth hormone release between injections and to avoid the inconvenience of frequent subcutaneous injections. See G. Hildebrandt et al., Results of Continuous Long Term Intravenous Application of • Φ • φ • φ

• φφφ • Φ ΦΦ · · · φ

J · · φ • φ φφφ

1 ·· ΦΦ »· Φ I ¹ φ φ« ··· φφφ

ΦΦ

ΦΦ

Octreotide via an Implantable Pump System in Acromegaly

Resistant to Operative and X-ray Therapy, Acta

Neurosurgery, Vol. 117, pp. 160-65 (1992). Administration of the hormone by infusion allows a constant concentration of the hormone in the blood to be achieved. Hormone accumulation within the cells was not increased.

Infusion was also designed to capture a radiolabeled pyrimidine analog for incorporation into DNA during DNA synthesis when the cell is in the growth phase. Pyrimidine does not bind to cellular receptors but instead is used as a building block for the synthesis of new DNA. Because radiolabeled pyrimidine is rapidly dehalogenated in the circulation, infusion administration has been proposed to increase uptake in solid tumors (e.g. breast cancer) known to have a slower growth rate and therefore a lower percentage of cells in the growth phase at any time than hematological tumors (e.g., leukemia). It was assumed that the infusion would maintain the concentration of the radiolabeled compound in the circulation so that cells dividing at different cycles would have access to the radioligand and be able to incorporate it into DNA during DNA synthesis. No corresponding experimental data is given. No reference is made to radiolabeled compounds that bind to cell receptors. See Bloomer et al., 1977.

U.S. Patent No. 5,590,656 describes the use of bolus injection of radiolabeled somatostatin to detect and distinguish neoplastic tissue.

No. 5597894 discloses the use of multi-tyrosinated somatostatin analogs administered by bdlus injection or short infusion (up to 60 minutes) for the diagnosis and treatment of tumors with surface receptors specific for peptides.

♦ ···· · «·

• · ·

International Application (PCT) No. WO 91/01144 describes the use of labeled polypeptide derivatives administered by a single bolus injection or short-term infusion (up to 60 minutes) for in vivo imaging of target tissues or for therapy.

Radioactive detection and radiotherapy are increasingly important in identifying and killing unwanted tumor cells.

The efficacy of the radioligand depends on the concentration at which it accumulates in the target cells. Although methods have been developed to increase the tumor-background ratio, only a few methods actually increase the radioligand concentration in tumor cells or closely related angiogenic cells. Therefore, there is a need for a method for increasing the accumulation and retention of radioligand in target cells without increasing destruction of normal cells.

SUMMARY OF THE INVENTION

We have found that administration of a radiolabeled compound over an infusion lasting more than 2 hours, preferably more than 12 hours, greatly increases the maximum of radioactivity that accumulates in the target cells. The accumulation of radiolabelled compound in target cells may be approximately five times higher than that of bolus injection or short-term infusion. This method increases the ratio of radioactivity between tumor and background by increasing the amount of radioligand accumulated in the tumor cells. This method can be used with any radiolabeled compound whose uptake in cells is limited by binding to cellular receptors or transport proteins. Once the radiolabeled compound is internalized, the half-life has only a minor role at the time the compound is present in the body. The primary factor affecting the presence in the body after internalization is the physical half-life of the radioisotope.

Description of the figures in the attached drawings

Giant. 1 depicts time-dependent uptake of N-pentetreotide in one patient for various routes of administration.

Giant. 2 shows the data of FIG. 1 in a logarithmic graph.

Giant. 3 show e binding and internalization ¹²⁵ I-WOC-4a. Giant. 4 show e binding and internalization ¹³¹ I-WOC-4a. Giant. 5 show e binding and internalization ¹²⁵ I-WOC-3b. Giant. 6 show e binding and internalization ^{11: L} -pentetreotide.

Giant. 7 shows the rate of disappearance of radioactivity from cells exposed to ¹²³ I-WOC-4a.

Giant. 8 shows the effect of a competitor on internalized ⁿⁱ -pentetreotide.

Giant. 9A shows the degree of internalization and transfer of ¹²⁵ I-WOC-4a at 1 hour.

Giant. 9B depicts the degree of internalization and transfer of ¹²⁵ I-WOC-4a at 4 hours.

Giant. 9C shows the degree of internalization and transfer of ¹²⁵ I-WOC-4a at 24 hours.

Giant. 10A shows the degree of internalization and transfer of ¹²⁵ isomatostatin per hour.

Giant. 10B shows the degree of internalization and transfer of ¹²⁵ Isomatostatin at 4 hours.

Giant. 10C shows the degree of internalization and transfer of ¹²⁵ Isomatostatin at 24 hours.

• · · ·

Giant. 11A shows the degree of internalization and transfer ^it Inpentetreotidu in 1 hour.

Giant. 11B depicts the degree of internalization and transfer ^{of 1} µp of Inpentetreotide at 4 hours.

Giant. 11C depicts the degree of internalization and transfer of ⁿⁱ Inpentetreotide at 24 hours.

Giant. 12 shows the rate of ^{1: L1} binding of ¹ -pentetreotide to DNA from IMR-32 cells.

Giant. 13 depicts the effect of protease on ^{βη-pentetreotide} binding to DNA from IMR-32 and SKNSH cells.

Giant. 14 depicts the effect of various concentrations of ¹²⁵ I-WOC-4a on the viability of IMR-32 cells.

Giant. 15 depicts the long-term effect of ¹²⁵ I-WOC-4a on the viability of IMR-32 cells.

Giant. 16A shows the rate of binding of ¹²⁵ I-17-3 estradiol to the T47-D cell line.

Giant. 16B shows the binding rate of ¹²⁵ I-17-β-estradiol to the MCF-7 cell line.

Giant. 16C shows the rate of binding of ¹²⁵ I-17-β-estradiol to the ZR-75-1 cell line.

Giant. 17 depicts the binding and internalization of ¹²⁵ I-JIC-2D in human angiogenic vascular endothelial cells.

Giant. 18 shows the effect of compound emitting Auger electrons, ^U1 LN-pentetreotide, on initiation of human angiogenic vascular tissue.

Giant. 19 shows the effect of compound emitting Auger electrons, ^U1 LN-pentetreotide, on vessel area of human angiogenic vascular tissue.

Giant. 20 shows the effect of compound emitting Auger electrons, ^LU In-pentetreotide, on growth rate of human angiogenic vascular tissue.

• * • · · · · · · · · · · ·

We have shown that an infusion lasting longer than 2 hours, preferably longer than approximately 12 hours, preferably longer than 24 hours, significantly increases the measured radioactivity within the target cells. Infusion increases accumulated radioactivity in tumor or angiogenic cells for almost all receptor-bound peptides and steroids. The mechanism of action is believed to be as follows: Binding to a cellular receptor (whether membrane, cytoplasmic or nuclear) or to a transport protein that is required for peptides and steroids to affect cell processes occurs in the finite time. When excess radiolabeled compound is administered (more than the receptor or transport protein binding capacity at any time), cell uptake by the cell is limited by the number of available receptor or transport protein molecules. The remaining free compound is subject to degradation and excretion. By the time the receptor is released to bind another molecule, the circulating radiolabeled compound is partially or fully eliminated. However, if the rate of administration of the radiolabeled compound is adjusted according to the rate of receptor binding and subsequent transport of the radiolabeled compound, the circulating free radioligand is accessible to the binding to recycled receptors. Thus, an effective dose of the drug, i.e. its intracellular accumulation, is optimized.

This theory is consistent with the observation that compounds that are internalized (e.g., peptides, hormones, cytokines, growth factors, and steroids) are resistant to competition or have a lower degree of competition than compounds that only bind to cell surfaces. Experiments that monitored the rate of internalization or radioligand transfer showed all short binding periods (0 to 4 hours). We have demonstrated in vitro for radiolabeled somatostatin analogues for a period of at least 120

9 ·

9 9 9 hours, there is a progressive transfer of membrane and cytoplasmic receptors to the nucleus. Since radiolabeled compounds bind not only to nuclear receptors but also specifically to DNA, an effective radiotherapeutic technique can utilize radiolabeled compounds labeled with elements emitting low energy particles, such as Augerl / electrons, which have a small radius of emitted radioactivity.

These electrons must be incorporated into the nucleus to cause DNA damage. Auger electron emitting elements have minimal effect on surrounding cells.

To optimize cellular accumulation of the radiolabeled compound, the infusion rate is adjusted to correspond to the rate of uptake in cells and transfer, preferably to the nucleus and preferably to DNA. Adjusting the rate of binding, internalization and transfer results in more efficient accumulation in cells than accumulation when administered as a bolus dose or short infusion. The new long-term infusion technique allows for repeated recycle of receptors, resulting in increased internalization, transfer and ultimately storage in the cytoplasm, organelles, and nucleus DNA. This increased accumulation of radioactivity allows optimal detection and optimal in situ radiotherapy.

More efficient uptake of the radiolabeled compound by the cells allows for more efficient use of the compound, i.e., more compound will bind to target cells and normal cells will be exposed to less toxic compound.

We have shown that after internalization of a compound in a cell it is less available for competition or degradation. Therefore, the biological half-life of the compound becomes less significant and the physical half-life of the radioisotope becomes critical. Many peptides, growth factors and hormones have a very short half-life. When radiolabeled peptides, growth factors or hormones are infused and transferred to cells where they are no longer subject to active competition with unlabeled hormones and are protected from degradation and secretion, the physical half-life of the isotope becomes more important than the amount of radioactivity accumulated in the cells. half-life.

The uptake of the radiolabeled compound for radiodiagnostics or radiotherapy can be monitored by scintigraphic techniques well known in the nuclear medicine art, for example by scintigraphic camera. The regions of interest can be displayed in the form of computer images to evaluate the uptake of the radiolabeled compound. Radioactivity and its persistence in tumor or angiogenic cells can be calculated. Infusion therapy allows the administration of higher effective doses that persist in tumor or angiogenic cells for extended periods of time.

The new method increases the tumor-background ratio by increasing the amount of radioactivity accumulated in the target cells, not just by reducing the background signal.

The term "target cell" refers to tumor cells or angiogenic cells that contain higher concentrations of membrane or internal receptors or transport proteins that bind to receptor-dependent peptides or steroids than concentrations of the same receptor or transport protein in normal cells.

The term radiolabeled compound or radioligand refers to a receptor-dependent peptide or steroid that forms a complex with a radioisotope and is useful as a pharmaceutical, eg, a radiopharmaceutical, for in vivo detection of target tissues or for in vivo therapy. The term radiolabeled compound also includes a compound with a chelating agent coupled to a receptor-dependent peptide for radioisotope binding.

The term receptor-dependent describes a compound whose effect on cells depends on binding to a cellular receptor, including membrane and intracellular receptors. The term receptor-dependent peptides refers to natural peptides or proteins isolated from natural cells or from fermented cells, for example obtained by genetic processing, or synthetic peptides, as well as derivatives and analogs thereof. Examples of receptor-dependent peptides are, but are not limited to, antibodies, growth factors, peptide and protein hormones, interferons, cytokines, and analogs and derivatives thereof. Specific examples of receptor-dependent peptides are, without limitation, nerve growth factor, fibroblast growth factor, epidermal growth factor, platelet growth factor, cholecystokinin, vascular endothelial growth factor, vasoactive intestinal peptide, gastrin releasing peptide, leukemia inhibitory factor, somatostatin, oxytocin, bombesin, calcitonin, arginine vasopressin, angiotensin II, atrial natriuretic peptide, insulin, glucagon, prolactin, growth hormone, gonadotropin, thyrotropin triggering hormone, trigger hormone for growth hormone, trigger hormone for gonadotropin, progestin hormone, trigger hormone interferons, transferrin, substance P, neuromedin, neurotensin, neuropeptide Y, opiates and their derivatives and analogs.

The terms derivatives and analogues refer to certain peptides or proteins in which one or more amino acid units have been omitted or replaced by one or more different amino acid units, or in which one or more functional groups have been replaced by one or more amino acid units, or several different functional groups, or in which one or more groups have been replaced by one or more different isosteric groups. In general, the term encompasses all derivatives of a receptor-dependent peptide having qualitatively similar effects to the unmodified peptide. For example, they may have higher or lower potency than the native peptide, may bind to another receptor subtype, or may have a longer half-life.

The term also includes agonists and antagonists of the native peptide that bind to the same receptor. Radiolabeled peptides may be obtained commercially (for example, from New England Nuclear or from ICN Pharmaceuticals, Inc.) or may be prepared by techniques known in the art (see, for example, U.S. Patent No. 5,597,894 and PCT / WO 91/01144).

The term receptor-dependent steroid includes natural steroids or synthesized steroids as well as derivatives and analogs thereof. Examples of receptor-dependent steroids that can be used are steroid hormones such as estrogen, progesterone, testosterone, glucocorticosteroids, mineralocorticoids and analogs thereof.

Radiolabeled steroids may be prepared by techniques known in the art (see, for example, CS Dence et al., Carbon-11-Labeled Estrogens and Potential Imaging Agents for Breast Tumors, Nuclear Medicine and Biology, Vol. 23, pp. 491-496 ( 1996) and RB Hochberg Iodine-125-Labeled Estradiol: A Gamma-Emitting Analog of Estradiol That Binds to the Estrogen Receptor, Science, Vol. 205, pp. 1138-1140 (1979), or may be purchased from commercial suppliers.

• · »· ···

Suitable radioisotopes include radioisotopes that emit alpha, beta, and gamma radiation, preferably gamma radiation, which are more readily viewable by the present art. Examples are radioisotopes derived from gallium, indium, technetium, yttrium, ytterbium, rhenium, platinum, thallium, and astatine, such as ^S7 Ga, ^l In, ^{99mTc, 90} Y, ⁸⁶ Y, ¹⁶⁹ Y, ¹⁸⁸ Re, ^{195 meters} Pt ²⁰¹ Ti and ²¹¹ At. Radioisotopes suitable for therapeutic treatment include Augerovs | electrons, for example ¹²⁵ I, ¹²³ I, ¹²⁴ I, ¹²⁹ I, ¹³¹ I, ⁿⁱ In, ⁷⁷ Br, and other radiolabeled halogens. The choice of a suitable radioisotope depends on many factors including the type of radiation emitted, the energy emitted by the radiation, the range of the emitted radiation and the physical half-life of the isotope. Preferred radioactive isotopes are those having a half-life equal to or longer than the receptor-dependent half-life of the compound. Preferably, the radioactive isotope has a half-life of between 1 hour and 60 days, more preferably between 5 hours and 60 days, and most preferably between 12 hours and 60 days. ¹²⁵ I has an advantage over other emitters that emit high-energy gamma rays (ie ^Lu In and ¹³¹ I) that require hospitalization and isolation after administration to the patient. ¹²⁵ I allows treatment without hospitalization because a limited amount of radiation is leaking from the body.

The term radiolabeled somatostatin includes, for example, ^1:11 In-DTPA-somatostatin, ⁹⁰ Y-DOTA-somatostatin, and ¹²⁵ Isomatostatin.

The term radiolabelled somatostatin analogues include, for example ^U1 LN-pentetreotide ^{((111In-DTPA-D-Phe 1)} -octreotid), ^(Iu In-DOTA ° D-Phe ¹ -Tyr ³⁾ -octreotid) (Y- ⁹⁰ DOTA-D-Phe ¹ -Tyr ³ ) octreotide), ( ⁸⁶ Y-DOTA-D-Phe ¹ -Tyr ³ ) -octreotide), ( ⁿⁱ In-DTPA-DPhe ¹ ) -RC-160, ^99m Tc- RC-160, ^99m Tc-CTTA-RC-160, ¹²³ I-RC-160, ¹²⁵ IR-160, ¹³¹ I-RC-160, ^99m Tc-octreotide, ¹⁸⁸ Re-RC-160, ¹²³ I-Tyr ³ octreotide , ¹²⁵ I-Tyr ^3- octreotide, ¹³¹ I-Tyr ^3- octreotide, ¹²⁵ I24

9 «· · 99 99«

9 9 9 lanthreotide, ¹²³ I-lantreotide, ¹³¹ I-lantreotide, ⁹⁰ Y-DOTAlantreotide, ^8S Y-DOTA-lanthreotide, ⁿⁱ In-DTPA-lanthreotide, ^lu InDOTA-lanthreotide, ^ul In-DPTA-somatostatin, ⁹⁰ Y-DOTA -somatostatin, ^{86 I} -DOTA-somatostatin, ¹²⁵ I-somatostatin, ¹³¹ I-WOC-3, ¹²⁵ I-WOC-3b, ¹³¹ I-WOC-4a, ¹²⁵ I-WOC-4a, ¹²⁵ I-JIC-2D, ¹²³ I-JIC-2D and ¹³¹ I-JIC-2D.

The term cell receptor includes membrane receptors, intracellular receptors, and binding sites within organelles, including endosomes, ribosomes, lysosomes, mitochondria, nucleus, vesicles, endoplasmic reticulum, sarcoplasmic reticulum, and Golgi.

The infusion may be administered by any conventional route, including intravenous, oral, intraperitoneal, intratumoral, subcutaneous, intraarterial, intramuscular, or by use of a sustained release formulation. The term infusion includes the use of repeated bolus doses administered at short intervals, whereby the physical and / or biological characteristics of the administered compound cause such repeated bolus doses at short intervals to be physiologically equivalent to continuous infusion. The radiolabeled compound may be administered by infusion in free form or together with one or more pharmaceutically acceptable carriers or diluents.

The term retention time refers to the accumulation of a radiolabeled compound by a target cell per dose of radiolabeled compound administered at a retention time. The accumulated radiolabeled compound is measured as the area under the curve in the radioactivity graph in target cells versus time.

The amount of radiolabeled compound that is given in detection ^ is the amount that is effective to allow imaging φφφφφφφφφφφφφφφφφφφφφφ φφφφφφφφφφφ

ΦΦ ΦΙ φ φ φφφ φ <φ

ΦΦ Φ <

nuclear medicine techniques, such as single photon emission computed tomography (SPECT) or positron emission tomography (PET), or allows detection by an ionisation chamber or gamma probe or a semiconductor detector. The amount is determined by the biological and physical half-life of the radiolabeled compound, the type and location of the tumor, and the characteristics of the patient, including the size, weight and volume of the tumor.

The amount of radiolabeled compound to be administered in radiotherapy is determined by the particular disease being treated, the radiolabeled compound used, and the patient's characteristics, including size, weight, receptor density on the target cells, and the severity of the disease. The efficacy of therapy can be assessed using techniques well known in the art, including radiodetection and monitoring as described above.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

A 36-year-old woman with metastatic small cell lung cancer received a high dose of ⁱⁿ -pentetreotide in ten individual sessions, with the first two doses given before the start of tissue accumulation monitoring. During each of the next eight doses, the tumor on the left side of the neck was monitored for radioligand uptake using an ionization chamber, the Victoreen 450P ionization chamber, which measured the external dose rate of gamma radiation. The first seven doses were administered by bolus injection. The last three doses were infused with different radioligand activities.

Tttt tttt tttt tt ttt ttt ttt ttt ttt

• · • tttt · <

Tt tt

Each of the following five months, a bolus injection of 180 mCi ⁱⁿ -pentetreotide (OctreoScan ^R , Mallinckrodt Medical, Inc, St. Louis, MO) was administered. The external gamma radiation dose rate from radioligand accumulation in the neck region of the neck was measured immediately after and 24 hours after injection. At month 5, the patient's radioligand uptake was measured over a longer period -

-2, 4 and 8 days after injection. Earned 1 and also shown in FIG data are shown in the whiteboard as the bottom curve is marked Bolusová dose. Table 1 : Bolus injections Moon Amount Time after Accumulated Ratio filed filed inj ekci (h) external dose (mR / h / mC bolus radioligand (mR / h) benefits (mCi) 1 179, 9 24 80 0.444691 2 160, 6 24 28 0.174346 3 182.6 24 33.20 0.181818 4 169, 5 24 27.2 0,160472 5 178.8 24 34.4 0.192394 5 72 19, 85 5 120 11.46 5 216 3, 82

Four months after the last bolus dose, the first dose of infusion (infusion 1) was administered. A dose equivalent to the previous bolus dose (175.9 mCi) was administered, but the radioligand was infused over 72 hours at a constant rate of 2.5 mCi / h. These data are shown in Table 2. The data is also shown as the curve indicated by Infuse 1 in Figure 1.

· · * * * * * * * * * * * *

Table 2: Infusion dose Duration Amount Time since Accumulated Maximal infusion (h) filed initiation external dose ratio radioligands infusion (h) (mR / h) (mR / h / mCi) (mCi) * 24 53.52 0 2,0 1,027729 24 55 24 52, 62 $ 4,5 54, 8 0.659505 48 70 24 50, 75 49 69, 7 0.446169 72 70 137 32.9 161 25 185 20 May 214 15, 4 72 156, 89 * Modified for radioactive disintegration 0 one a month later it was total infusion dose . 2 increased to 389.2 mCi. Infusion was 189, 8 mCi of the radioligands administered

during the first 24 hours, 108.6 mCi during the second 24 hours and 90.8 mCi during the third 24 hours. For each 24-hour period, the infusion rate was constant. These data are shown in Table 3 and are also shown as the curve labeled Infuse 2 in Figure 1.

• · ··· · ···

Table 3: Infusion dose 2 Duration Amount Time since Accumulated Maximal infuse (h) administered launch external dose ratio radioligand infusion (h) (mR / h) (mR / h / mCi) (mCi) * 20 May 189.76 0 2, 0 1,106654 16 180 17 210 20 May 181 22nd 108.62 22nd 171 0.670287 23 180 42 200 24 90, 84 44 200 0,565236 47 183 65 220 68 210 144 81 161 66 66 389.22 * Adapted for radioactive disintegration 0 one a month later she was infusion administered third dose (infuse 3). The 301 mCi dose was infused during the first 24 hours. IN next two 24 hours periods were filed 25 a 21 mCi. Data are shown in the table 4 and also on FIG j sou

marked as infusion 3.

• · • ·

29 - · · · · · · · · · · · · · · Table 4: Infusion dose 3 Duration Amount Time since Accumulated Maximal infuse (h) administered initiation external dose ratio radioligand infusion (h) (mR / h) (mR / h / mCi) (mCi) * 17 301.07 0 0, 68 1,561 2 36 3 90 4 144 8 260 9 270 13 400 16 470 17 460 19 Dec 410 24 29, 93 20 May 410 22nd 380 23 375 25 360 26 350 29 345 30 340 31 335 40 310 24 20, 48 46 280 47 275 67 230 90 179 164 70 65 346.49 -

* Adapted for radioactive decay • · ·

···················· · 9

Based on the results of the final bolus dose and the first two infusion doses, it is clear that equivalent doses administered in different ways accumulated at very different rates. At a bolus injection of 178.8 mCi, the maximum external gamma dose dose rate occurred soon after injection and decreased to approximately 34 mR / h 24 hours post injection, which corresponds to a 24 hour 0.19 mR / h measured external dose rate for each mCi administered. For infusion 1, with an infusion rate of 2.5 mCi per hour for 3 days, an increase was observed both in the area under the accumulation curve (total amount of radioactivity absorbed by the tumor) and in the maximum dose rate of external radiation (mR / h on contact surface). ). After infusion of a third of the radioligand, the measured dose rate was approximately 55 mR / h and this rate was maintained or increased for the next three days. The highest mR / mCi ratio of 1.03 was observed after the first 24 hours of infusion and was almost five times higher than the highest rate achieved with a bolus injection. The slope of the accumulation curve was maximal within the first 24 hours and then flattened over the next 48 to 72 hours, indicating that the system reached the limit with minimal subsequent uptake. After 24 hours, the cellular feedback mechanism slowed uptake.

When a larger dose was infused during the first 24 hours, as is the case with infusions 2 and 3, when 189.9 and 301 mCi were administered, respectively, and the infusion rate was reduced for the following 24-hour periods, greater area under the curve and higher maximum activity (220 and 470 mR / h). these data show the importance of administering a higher dose during the first 24 hours before the cellular feedback mechanism slows uptake.

• · • ♦ · ··· ·· <

• <·· ··

The differences between the four curves are not intuitive. Data show that the radioligand was protected from degradation removal, competition or secretion after incorporation into cells, and that cellular transport mechanisms continued to take up radioligand despite higher concentrations within the cell until the feedback mechanism slowed uptake at 16-24 hours. .

When plotting data in a semi-logarithmic scale, as shown in Figure 2, and evaluating the total area under the curves, two conclusions are evident:

(1) Initial peptide removal was much more significant for bolus administration and left less peptide available for incorporation into cells. The incorporation of radioligand by the cells showed an exponential decay in which the half-life approached the physical half-life of the radioligand (67.3 h). The slope of the decay curve was relatively stable, regardless of the dose sequence.

(2) In normalization to the administered dose, the area under the curve or retention time increased by simply adjusting the time and dose administered up to 5-fold. For bolus injection, the area under the curve in Figure 1 is 4.030 mR, which corresponds to the accumulated 23 mR per mCi administered. Comparable areas for infusion are 10.398 mR, 37.370 mR and 41.993 mR. These data correspond to the ratios of accumulated radioligand per administered mCi of 66 mr / mCi, 96 mR / mCi and 121 mR / mCi, respectively. Thus, the infusion ratios were 3, 4 and 5 times higher than the bolus ratios. There was more radioligand on infusion that could be incorporated into tumor cells.

• · · · ·

• · • · ···

At infusion 3, the same patient was followed by a scintigraphic gamma camera. Photographs showed that not only increased uptake of the radiolabeled compound, but also that multiple tumor foci could be identified. Further, radioactivity remained in the tumor region for a longer period of time than with bolus administration. In this patient, tumor foci could be identified by gamma scintigraphy even 13 days after the end of the last infusion.

Example 2

In vitro experiments were performed with radiolabeled somatostatin analogs and human cell lines to determine the uptake of radiolabeled peptide in different cell types over time.

Cell lines:

The cell lines used were: (1) IMR-32 (ATCC No. CCL-127), a human neuroblastoma cell line with very high expression of somatostatin receptors (SST), in particular SST-2; (2) SKNSH (ATCC No. HTB-11), human neuroblastoma cells metastasized to bone marrow, lacking somatostatin receptors, as determined by either the binding assay or the reverse transcriptase polymerase chain reaction (RT-PCR); and SK-R2, a cell line derived from SKNSH cells that has been transfected with genewi for the SST-2 receptor. IMR-32 and SK-R2 cells express somatostatin type 2 (SST-2) receptors. SKNSH cells do not express somatostatin receptors and serve as a negative control. Cell lines were obtained from ATCC (IMR-32 and SKNSH) or from Ohio State University (Columbus, Ohio) (SK-R2). All cells were cultured in 5% CO ₂ at 37 ° C in the medium defined by the cell line supplier.

Peptide Binding Assay and Internalization

Cells were harvested, counted in a hemocytometer, and resuspended in binding buffer (Minimum Essential Medium (MEM), 10 mM HEPES, 0.01% BSA). The standard assay utilized 500,000 cells in 1 ml binding buffer. The radioactive ligand alone (500,000 cpm) or in combination with at least a 1000-fold molar excess (10 ^-6 M) of the non-radioactive compound (to determine specific binding) was added to a final volume of 1 ml. At the end of the experiment, the incubation medium was removed, the cells were washed twice with Hank's balanced salt solution (HBSS), and the radioactivity in the cells was determined using a gamma camera. This level of radioactivity represents the total binding, which includes both the membrane bound fraction and the internalized fraction. Specific binding was determined by calculating the difference between the measured radioactivity without unlabeled competitor minus radioactivity with unlabeled competitor.

To distinguish between the membrane bound and intracellular fractions, the cells were further incubated for 10 minutes at 4 ° C with acidified HBSS (pH = 4-5), rinsed in HBSS and radioactivity again determined by gamma camera. Since the acid preferably elutes the radioactive ligand from the external cell surface, the remaining radioactivity belongs to the internalized fraction. The membrane binding is then calculated as the difference between the total binding and the internalized fraction.

Ligand binding and internalization versus time

The times for ligand binding and internalization were analyzed using the standard assay described above. All radioactive ligands and competitors were somatostatin analogues that preferentially bind to SST-2 cell receptors. In the first series of experiments, IMR-32 cells that were exposed to radioligand for various periods of time (up to 72 hours) were used. The incubation medium was replaced at least once every 48 hours. The radioligand was ¹²⁵ I-WOC-4a, ¹³¹ I-WOC-4a, ¹²⁵ I-3b and ^W0C-ni In-pentetreotide. The unlabeled competitor was octreotide acetate. WOC-4a and WOC-gb are multityrosinated somatostatin analogs with preferential binding to SST-2, which can be labeled with high specific activity (8800 Ci / mmol) while still retaining high biological activity and receptor binding affinity (Kd = 1) nM). WOC-3b and WOC-4a have the same structures and method of synthesis as described in US Patent No. 5597894.

Specific binding and internalization data for the radiolabeled compounds ¹²⁵ I-WOC-4a, ¹³¹ I-WOC-4a, ¹²⁵ I-WOC-3b and ⁱⁿ Inpentetreotide are shown in Figures 3, 4, 5 and 6, respectively. These data show that the binding of somatostatin analogs to their cell receptors increases with time with maximum binding between 48-72 hours. Significant fractions of total radioactivity (40-75%) were incorporated into the cells.

Example 3: Determination of the rate of radioactivity loss

Cell lines and assay techniques were the same as in Example 2. IMR-32 cells were incubated for 48 hours with ¹²⁵ I-WOC-4a (1 cpm / cell) and washed three times in HBSS. Basic radioactivity of the cells was determined. Cells were cultured in MEM with 10% fetal bovine serum (FBS) and the medium was changed daily. The radioactivity of the removed medium was measured and the percentage of radioactivity lost from cells over time was calculated. These results are shown in Figure 7, which shows that the retention time of ¹²⁵ I-WOC-4a is approximately 11 days. This result is very surprising because the described half-life of somatostatin is measured in minutes and the half-lives of all known somatostatin analogues are given in hours (octreotide acetate - 90-120). minutes; lantreotide - 60-90 minutes).

This experiment has shown that once the radiolabeled compound is internalized into a cell, its retention time and thus its ability to irradiate the cell is significantly increased. The internalized peptide is protected from degradation and the physical half-life of the isotope is then decisive for the radiation dose. Since the measured dose of radioactivity includes any losses due to cell death and consequent loss of membrane integrity, the actual retention time of the radioligand within intact cells may be slightly higher than the measured value of 11 days.

The results of Examples 2 and 3 provide three significant observations that support the use of this method for radiodetection and radiotherapy: (a) a significant portion of the receptors and its associated radioligand were sequestered within cells; (b) the progressive receptor-mediated internalization has been carried out over a longer period of time (at least 48 hours); and (c) internalization significantly prolonged the retention time.

Example 4: Binding to cells with and without somatostatin receptors

To determine whether the radioligand actually binds to the SST-2 receptor, experiments were performed with the IMR-32, SK.R2 and SKNSH cell lines. In-pentetreotide (1 cpm / cell) was used as a radioligand and octreotide acetate as a competitor. The test was performed in the same manner as in Example 2. The data are shown in Table 5.

··· ··· ···

Table 5: Binding of ^{U1-pentetreotide} LN cells with and without receptors for somatostatin

Total Intracellular Membránová IMR-32 Total binding (cpm ± SD) 4.780 ± 928 2.408 ± 511 2.373 ± 417 Specific Binding (CPM) K d = 4x10 ^-9 M 3, 908 1,536 2,372 SKR-2 Total binding (cpm ± SD) 36, 261 ± 2.085 2, 607 ± 672 33.655 ± 2.728 Specific Binding (CPM) K d = 1x10 ^-9 M 35,475 1,792 33,683 SKNSH Total binding (cpm ± SD) 861 ± 30 775 ± 107 87 ± 114 Specific binding (CPM) 0 0 0

As shown in Table 5, no specific binding was detected for SKNSH cells lacking somatostatin receptors. However, significant levels of specific binding to the cell membrane and within cells were observed in IMR-32 cells. Specific membrane binding was also observed for SK-R2 cells (genetically engineered cells producing excessive amounts of SST-2 membrane receptor). In the SK-R2 cell line, 95% of the binding was coupled to the plasma membrane and the ligand internalization was relatively weak. Scatchard analysis showed φφφ · φ

Dissociation constant (which measures ligand-receptor binding affinity) 1 x 10 ^-9 M to 4 x 10 ^-9 M for SK-R2, respectively. IMR-32 cells, a value falling within the range predicted for these receptor-ligand interactions.

This experiment has shown that (a) the binding of somatostatin analogs to their receptors and subsequent internalization is specific for cells expressing somatostatin receptors; and (b) internalization requires additional cellular components (presumably signal transduction elements that are not present in genetically engineered cells) and is not a function of the membrane receptor itself.

Example 5: Competition Study

To determine the significance of competition after internalization, IMR-32 cells were incubated with radioligand for a period of time sufficient to effect internalization, and then incubated with unlabeled radioligand. IMR-32 cells were incubated overnight with ^lu Inpentetreotidem for filling the cells with radioactive ligand as described in Example 2. One set of culture tubes were then rinsed with acid and incubated for 2 hours with excess unlabeled oktreotidacetatu (competitor). The amount of radioactivity was then compared to the radioactivity in tubes not exposed to the competitor.

As shown in Fig. 8, the radioactivity trapped in the cells was similar for both cases, indicating that there is no competition with the unlabeled compound after internalization of the radioligand. These observations are of great importance for the use of somatostatin analogs in radiotherapy: the intracellular radioactive ligand cannot be displaced by an unlabeled competitor and therefore remains captured in the cells.

Example 6: Core Transfer Study

To determine intracellular shifts of labeled somatostatin analogs, IMR-32 cells were incubated in T-150 culture flasks containing Minimum Essential Medium with L-glutamine and non-essential amino acids for various times with ¹²⁵ I-WOC-4a, ¹²⁵ I-somatostatin, and ^µL In- pentetreotide. The cells were washed twice in medium at 4 ° C and resuspended in cold isotonic sucrose solution. Labeled cells were pushed three times with a balloon homogenizer to disrupt cell membranes, leaving about 95% of the cell nuclei intact as described by Balch et al., Arch. Biochem. Biophys., Vol. 240, p. 413 (1985). The cell homogenate was mixed with Percoll to give a final Percoll concentration of 0.292% and centrifuged at 20400 rpm on a Beckman 40.2 centrifuge for 45 minutes to obtain a high density mass. Density determination beads (Pharmacia) are used for density determination in tubes. The contents of the tube are fractionated in an ISCO Density Gradient Fractionator. The final contents of the tubes are assayed for radiolabeled somatostatin or analogues thereof, for DNA showing the presence of nuclei, proteins, 5'-nucleotidase plasma membrane marker and N-acetylglucosaminidase lysosome marker. It is believed that the radioactivity measured in fractions rich in certain cellular components is indeed associated with such components; for example, the radioactivity measured in the plasma membrane rich fraction is due to the association of the radiolabeled compound with the plasma membrane.

Data are shown in Figures 9A-C, 10A-C, and 11A-C. The density distribution is represented by triangles in the figures. Nuclear fractions were detected in a density gradient represented by tubes 12-15 and membrane fractions by tubes 0-6. Giant. 00 00 0 0 0 * 0 0 000 000 shows that the early cellular distribution of ¹²⁵ I-WOC-4a is limited in essentially on the plasma membrane (tubes 1-5). Giant. 9A shows that very weak nuclear activity is observed at hour 1. At 4 o'clock, Fig. 9B, membrane binding is still indicated, but the activity of the nuclear fraction begins to slowly increase. At 24 hours, Fig. 9C, almost all activity was observed in the nuclear fraction, indicating that ¹²⁵ I-WOC-4a was transferred to the nucleus. Thus, these data show that the binding of the radiolabeled compound to membrane receptors, the internalization of these receptors into cells and the transfer of radioligand to the nucleus lasted several hours. Radioactive decay in the nucleus, which ionizes DNA, leads to cell damage. Giant. 11A (1 hour) and 11B (4am) and 11C (24 hr) indicate that the time required for transmission and internalization ^U1 LN-pentetreotide was similar to ¹²⁵ I-WOC-4a.

However, Figure 10 shows that the transfer time of native ¹²⁵ Isomatostatin was different. Already at 1 hour, Fig. 10A, both membrane binding and weak core binding were present. At 4 hours, Fig. 10B, plasma membrane binding peaked and the nuclear fraction decreased. At 24 hours, Fig. 10C, this very short-lived radiolabeled peptide (half-life of 1-2 minutes) was almost completely excluded from cultured cells and virtually no plasma membrane or core binding was observed.

The internalization time of the radiolabeled compound is a function of the cell receptor type, the particular compound and the particular isotope.

Example 7: Binding of somatostatin to nuclear DNA

To determine that somatostatin or its analogs bind to genetic material, DNA was extracted from cells that were •

incubated with radioligand, 1 ^H -pentetreotide, and was φ

• • φ · φ analyzed for radioactivity.

Three T75 flask containing IMR-32 and SKNSH cells were incubated overnight with ^lu In-pentetreotide as in Example 2 were used for DNA extraction. Cells were removed from the culture surface using a sterile rubber loop. The suspended cells were washed three times in cold Dulbecco's phosphate buffered saline and were processed by the DNA extraction procedure according to the manufacturer's instructions (GenomicPrep ^R cells and Tissue DNA Isolation Kit, Pharmacia Biotech, Piscataway, NJ). The cells were incubated in a Cell Lysis Solution kit for DNA isolation at 37 ° C for 10 minutes. 3 μΐ of RNAse solution was added to the cell lysate and mixed thoroughly and incubated at 37 ° C for 1 hour. The samples were cooled to ambient temperature and 200 μΐ of protein precipitation solution was added. After mixing, the contents of each tube were centrifuged at 14,000 xg for 1 minute and the DNA pellet washed with 70% ethanol. After drying, the DNA was hydrated in a DNA hydration solution and the DNA concentration was determined by spectrometry at 260 nm. 100 µg of each sample was transferred to radioactivity counting tubes and the amount of radioactivity in each sample was measured on a Beckman 5500 gamma camera. Data as shown in Table 6 was obtained as counts per minute (cpm). Differences between radioactivity measured in cells incubated with radioligand only and cells incubated with both radioligand and unlabeled ligand were statistically analyzed using ANOVA. The accumulation rate of ^1u Inpentetreotide in the DNA of IMR-32 cells is shown in Figure 12.

β Λί > Φ Ρ β β β ρ Λ φ υ Λ β υ τ5 Ρ • Η β Ρ > ο ή φ Ρ β β -Ρ tn Φ ο β +1 C4 | β Η τ) ω • 1—1 (Ό -Ρ ο 0 φ Λ β φ + J β φ Ρ Ρ β υ φ Ή Λ 1 β > β • Η H Ρ <—4 Ή ς-β Ν Φ Ο řX υ β 04 / β 1 ο Ρ and ω β ω ο Μ β * ζ; Ρ Ο

β f —I β

- Λ β

ω β

r-Η

O,

C β β fcn Ή i — I O • H Ό β β β

'> 1 β

-P o

Ή β

φ β

II w

(Λ

Β) β

β fcn

Η

Ο

Ή

Ό β

β

Ο β

• η

Ν φ

β> ι

Ή

Ό

Ν

X (X «—ι

Ϊ> 1 β

φ>

Ο β

β

Ρ

Μ> ι γ — I> 1 Λ

Β β β tn β ι — ι '> Ί β

Υ> υ β

β

Ν

Φ β

Next, DNA from each well was split in half and 1 mg of DNA from each well was treated with proteinase K (Lite Technologies, Gaithersburg, MD) for 1 hour at 37 ° C. The enzyme treated DNA was then reprecipitated with ethanol and sodium acetate, washed with cold ethanol, and redissolved in the DNA hydration solution. The concentration was determined by a spectrophotometer at 260 nm. 100 gg of protease-treated DNA and the same amount of untreated DNA were transferred to radioactivity counting tubes, and the amount of radioactivity in each sample was measured on a Beckman 5500 gamma camera. minute (cpm).

Table 7: Binding of ^X11 ln-pentetreotide to cellular DNA ⁽¹⁾

Try IMR-32 cells SKNSH cells Processed Unprocessed- Processed Unprocessed- proteasou no (cpm) proteasou no (cpm) (cpm) (cpm) 1 10276 11512 122 316 2 9564 10122 412 319 3 12648 14566 208 220 Diameter ± SD 10829 ± 1614 12066 ± 2273 247 ± 149 285 ± 56 ⁽¹⁾ Details are given in impulses for minute (cpm) for 100 gg samples DNA for each cell type in triplicate experiments. How is shown in Tables 6 and 7, considerable amount of ^lu In- pentetreotide was associated with IMR-32 cellular DNA cells while substantially no In-pentetreotide ^it has not been established on DNA SKNSH cells. How it was written supra in Example 4, SKNSH cells do not

receptors for somatostatin type 2 and do not internalize radiolabeled somatostatin analogs.

• · • ·

• · · · ·························

Surprisingly, it was found that ¹¹¹ In-pentetreotide bound to the DNA of IMR-32 cells was resistant to nonspecific proteinase K. Overall, only 1013% of the total cpm bound was removed by protease processing, indicating that the radioligand-DNA linkage was not mediated by non-specific protein binding, such as histones.

Kinetics of radioligand binding to DNA were comparable to those found for intracellular shifts in IMR-32.

Therefore, the rate of intracellular transfer of radioligands is about the same as that of its binding to DNA.

Example 8: Cytotoxicity ¹²⁵ I-WOC-4a

WOC-4a was synthesized by the solid phase synthesis technique as described in Example 2 and Patent No. 5597894. IMR-32 Human Neuroblastoma Cells (ATCC No. CCL-127, American Type Culture Collection, Rockville, MD) of which known to exert SST-2 receptors, and PANC-1 human pancreatic epithelial carcinoma cells (ATCC No. CRL-1469) that are SST-2 negative were cultured in culture at 37 ° C in humidified air with 5% CO ₂ . IMR-32 cells were cultured in Eagle's Minimum Essential Medium (MEM) with Earl salts (Gibco, Grand Island, NY) supplemented with non-essential amino acids, 15% fetal bovine serum (Gibco, Grand Island, NY), and antibiotic-antifungal agents. reagent (Gibco, Grand Island, NY). PANC-1 cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY), supplemented with 10% fetal calf serum and an antibiotic antifungal agent. Both cell lines were passaged once a week using 0.25% trypsin and 1 mM EDTA.

• ·

For short-term cytotoxicity experiments, cells were harvested, washed and plated in 96-well cell culture plates (Costar, Cambridge, MA) at a density of 5 x 10 ³ cells / well. In experiments investigating short-term dose-dependent toxicity, cells were incubated in binding buffer (MEM, 10 mM HEPES, 0.01% BSA). ¹²⁵ I-WOC-4a was added to appropriate wells at concentrations of 0.1-100 cpm / well. After exposure, the radioactive medium was replaced with culture medium and the cells were cultured for an additional five days. Cell viability was analyzed using an enzymatic cell viability assay using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) as described by RF Hussain et al., A New Approach for Measurement of Cytotoxicity Using Colorimetric Assay, Journal of Immunological Methods, Volume 160, p. 89 (1993). The colorimetric results were then analyzed using a microplate reader (Dynatech, Dynatech Labs., Chantilly, VA). The results were expressed in optical density (OD).

In long term exposure experiments, 25 million IMR-32 cells were placed in seven T75 vessels (Corning, Cambridge, MA). The cells were incubated for 48 hours at 37 ° C in a humidified 5% CO ₂ /95% air atmosphere and were exposed to one of the following samples: control medium (binding buffer), 1 cpm / cell ¹²⁵ I-WOC-4a; 1 cpm / cell ¹²⁵ I-WOC4a with 10 ^-6 M octreotide acetate (Novartis Pharmaceuticals, East Hanover, NJ); 1 cpm / cell ¹²⁵ L alone; 1 cpm / ¹²⁵ I cell with 10 ^-6 octreotide acetate; and 10 ^-6 M octreotide acetate alone. Then, the cells were harvested, washed three times with fresh culture medium and cryopreserved for 4 weeks at -85 ° C in 1 ml of 90% culture medium and 10% DMSO (Sigma, St. Louis, MO). After cryopreservation, cells were thawed rapidly and cell viability was tested by trypan blue exclusion (n = • · n n n n). ·

* .......... ·· 99

3 / group). The remaining cells were replated in a 96-well plate (n = 21 / group) (Costar, Cambridge, MA) and cultured under standard conditions for 24 hours.

The viability of these cells was then confirmed in the MTT assay.

After short-term drug exposure, the effect of each dose of ¹²⁵ I-WOC-4a on the viability of IMR-32 and PANC-1 cells was determined. Mean cell viability (± SD) was calculated and dose effect was analyzed by variance analysis (ANOVA). Student ttest was used to compare one drug concentration between two cell types. At long-term exposure, the mean cell viability (± SD) was calculated for each group and compared to control group values using ANOVA (p <0.05).

When IMR-32 cells were incubated for 48 hours with ¹²⁵ IWOC-4a at doses from 0.1 to 100 cpm / cell, a dose-dependent decrease in viability was observed as found in the MTT assay. ¹²⁵ I-WOC-4a induced a 33% decrease in viability at 100 cpm / cell (Fig. 14). These differences were statistically significant by ANOVA (p <0.05). However, when the SST-2 negative cell line (PANC-1) was exposed to the same concentrations of ¹²⁵ I-WOC-4a, no statistically significant difference in cell viability was observed between the control and test groups (n = 21 per group) as shown in FIG.

14. These results indicate that ¹²⁵ I-WOC-4a induces SST-2 dependent cytotoxicity at short-term exposure (48 hours).

To evaluate the effect of long-term (week 4) ¹²⁵ IWOC-4a exposure on cells expressing SST-2, IMR-32 cells were exposed to 1 cpm / ¹²⁵ I-WOC-4a cell; 1 CPM / cell ¹²⁵ I-WOC-4a with 10 ^"6 M oktreotidacetatu; 10 ⁶ M octreotide acetate alone; ¹²⁵ I alone; or ¹²⁵ I, 10 ^"6 oktreotidacetatem. Viability

cells for each group were compared to control values after a four-week, cryopreserved exposure. These data are shown in Figure 15. ¹²⁵ I-WOC-4a induces statistically significant toxicity; however, no toxicity is observed after exposure to similar doses of ¹²⁵ L alone or ¹²⁵ L with octreotide acetate. In addition, the cytotoxicity of ¹²⁵ I-WOC-4a is not inhibited by the addition of a 10,000-fold excess of octreotide acetate, meaning that intracellular incorporation of small amounts of ¹²⁵ IWOC-4a may be cytotoxic to SST-2 positive cells. This cryopreservation technique allows long-term exposure to radioligands, but prevents cell proliferation that could mask the cytotoxic effects of radioligands.

These studies have shown that ¹²⁵ I-WOC-4a induces cytotoxicity in neuroblastoma cells that express SST-2 receptors. Thus, ¹²⁵ I-WOC-4a will be an effective therapeutic agent for SST-2 containing tumors.

Example 9: Cell uptake of steroid hormones

To monitor the time course of steroid hormone uptake, three cell lines were incubated with radiolabeled estrogen, using a technique similar to that of Example 2. Three breast cancer cell lines were used: MCF7 (ATCC No. HTB-22), T47D (ATCC No. HTB- 133) and ZR-75-1 (ATCC No. CRL-1500). The estrogen used was 17-3-estradiol, the major secreted estrogen in humans. The radiolabeled compound was ¹²⁵ I-17-β-estradiol, which was purchased from New England Nuclear, or was synthesized as follows:

ml Sephadex G-10 column was equilibrated with 0.1 M PBS over 1 hour. A solution containing 12.5 Hg of conjugate estradiol (BL-43), 25 μΐ of methanol, 50 μΐ of 0.05 M phosphate buffer, pH 7.5, 3 mCi Na ^{125 was} prepared! and 25 μΐ of a 10 mg mixture • ·

chloramine-T and 10 ml of 0.05 M phosphate buffer. After mixing for 40 seconds, 25 μΐ of sodium sulfite 20 mg / 10 ml of 0.05 M phosphate buffer was added and mixing was continued for a further 40 seconds. The mixture was then added to a Sephadex column and eluted first with 0.1 M PBS. Finally, radiolabeled estradiol was eluted using 0.1 M PBS and 1,4-dioxane (70:30).

A standard binding assay procedure as described in Example 2 was used, except that radiolabeled estradiol was used as a ligand with a 1000-fold excess of nonradioactive estradiol as a competitor. Unlike peptides that must first bind to the plasma membrane, steroids only bind to intracellular receptors. Therefore, no radioactivity on the membrane can be detected. The data from this experiment is shown in Table 8.

Table 8: Specific binding of ¹²⁵ I-17-β-estradiol versus time

Cellular 1 hour 4 hours 24 hours 48 hours line (cpm) (cpm) (cpm) (cpm) T-47D 401 506 532 455 ZR-75-1 4102 4865 3370 3057 MCF-7 1605 2253 4109 731

The data show specific binding for these three cell lines at different densities. This binding and radioactivity was maintained for at least 24 hours. These data are similar to peptide hormone uptake. Progressive accumulation of radiolabeled steroid in three cell lines is demonstrated in Figures 16A, 16B and 16C. The rate of accumulation depends on the cell line. Therefore, infusion therapy may lead to greater accumulation rate and greater retention of radiolabeled steroids.

··················

Example 10: Preliminary Results of a Pilot Clinical Trial with ^{1: L1} Inpentetreotide Infused at 180 mCi Once Monthly

A pilot clinical study was conducted for 6 months in ten human patients with progressive metastatic indolent or symptomatic neuroendocrine tumors expressing somatostatin receptors. Ten patients were given from one to six doses ^l In-pentetreotide. The first monthly dose was a bolus injection of 180 mCi. The second monthly dose was an infusion of 180 mCi over 72 hours. The third monthly dose was an infusion of 180 mCi within 24 hours. Other monthly doses were all infusions of 180 mCi within 24 hours. Clinical benefit (as assessed by pain reduction, weight gain, reduced weakness, etc.) was seen in 6 out of 10 patients with neuroendocrine tumors (carcinoids / pancreatic B cell tumors). Partial radiographic response (more than 50% reduction in vertical tumor diameters) occurred in 2 patients and significant tumor necrosis occurred in 7 out of 10 patients with neuroendocrine tumors. Treatment-related toxicity included 2x Grade III thrombopenia, 1x Grade II leucopenia, 1x Grade 1 leucopenia, and 2x Grade 1 anemia according to the NCI (National Cancer Institute) scale. This experiment demonstrated that ⁿⁱ Inpentetreotide at a dose of 180 mCi once a month was a potent and well tolerated antitumor agent in some individuals with tumors expressing somatostatin receptors.

Example 11: Study Plan Overview

Future clinical trials will begin with a non-randomized study comparing bolus and infusion of 180 mCi ⁿⁱ Inpentetreotide. Up to 30 participants will be selected from approximately 100 patients with metastatic neuroendocrine tumors expressing receptors for somatostatin, as defined by a positive diagnostic 6 mCi ⁿⁱ -pentetreotide scan (Octreoscan ^R ). Most of these patients have received octreotide and still show signs of slow progression by CT scans, biochemical markers and / or clinical signs (status performance, weight loss, increasing weakness, etc.).

Study population and selection criteria

Subjects will have completed standard therapy and still show signs of disease progression. Most subjects with endocrine malignant tumors will have at least one year of octreotide biotherapy. interferon. Patients with non-endocrine malignancies will receive standard chemotherapy and radiotherapy. A subset of patients will receive external radiotherapy to the bone and / or cytotoxic chemotherapy.

After dosimetric N-pentetreotide scanning and CT scans, subjects who have demonstrated pathological radioisotope uptake in areas corresponding to metastatic sites are selected for study. Following administration of bolus and infusion doses, subjects who respond to therapy will be suitable to continue dosing once a month until either disease progression or regression occurs, which means cessation of the therapy.

Method of administration ^In the In-pentetreotide will be given either as a rapid intravenous (IV) bolus or as an iv infuse'během 24 or 72 hours. Each entity will serve as its own control. IV administration will be initiated and the radioactive dose will be administered either within a few seconds or within 24 hours or 72 hours. During therapy, patients will be given iv hydration (100 cm ³ / h) with normal saline.

Some individuals will then enter the classic phase 1 study to monitor dose escalation. Three patients will be monitored at the following ⁱⁿ -pentetreotide doses: 180, 360, 540,

720, 900, 1080, 1260 and 1440 mCi. These doses are expected to be given as a continuous iv infusion over 24 hours.

Objectives

The main aim of the first study will determine the optimal rate of administration ^U1 LN-pentetreotide at 180 mCi. Blood counts and serum biochemical values will be analyzed before administration and weekly for 3 weeks after each dose to determine any toxic effects. Scintigraphic examination will be performed daily for 3 days and on days 7, 14, and 21 post-dose to determine uptake and excretion rates. Plasma samples will be collected and stored prior to therapy and monthly for 6 months for tumor marker testing. Additional plasma and urine samples will be collected daily to determine the clearance of the radioligand. CT scans of the chest, abdomen and pelvis (to determine the radiographic degree of response) will be taken before the first and the third dose. Radioactivity uptake ratios (tumor to background), therapeutic ratios (tumor to kidney), excretion rates and dose of radioactivity (areas under the mRoentgen / h vs time) will be calculated.

The main objective of the second study will be to determine the maximum tolerated dose of ^1u -pentetreotide. In groups of three patients will be evaluated following doses ^U1 LN-pentetreotide: 180, 360, 540, 720, 900, 1080, 1260 and 1440 mCi. The same φφφ ·· · provedena will be performed

φ φ

φ ΦΦ φ

φ blood tests, biochemical parameters, scintigraphic examinations and radiodiagnostic examinations as described above. If at least 2 NCI grade IV toxicities occur, treatment will be discontinued. The number of series that can be administered will then be determined as the cut-off dose.

Patients will be examined before each dose and once a month for 3 months after therapy. Their quality of life will be assessed by clinical benefit, including reduced pain, weight gain and / or reduced weakness. Clinical responses will be identified as present, unchanged and absent. Biochemical responses will be measured by plasma chromogranin A concentration and / or urinary 5-HIAA levels at 24 hours or by other elevated tumor markers. Partial response is defined as a 50% or greater reduction in tumor marker. Radiodiagnostic responses will be determined by comparing perpendicular tumor diameters, using the following WHO criteria: (a) Complete response: complete disappearance of the disease; (b) Partial response: 50% or greater reduction; (c) Stable disease: less than 50% reduction and less than 20% magnification; and (d) Progressive disease: greater than 20% magnification.

Analysis of results

The sample size for the Phase 1 pilot study is determined by the objective of the study as above. In preliminary studies, if the investigational drug is 20% or more effective, one treatment success should be observed in the first 14 patients treated, with a 95% confidence interval. If treatment success is not observed in the first 14 patients, the drug is not suitable for further research. Therefore, the sample size for this Phase 1 study should be 14 assessable patients. Another 15 individuals will be

9

9

9 studied after interval analysis of the first 14 subjects. This second group will help to determine toxicity and efficacy. Indolent neuroendocrine tumors are expected to predominate in the first 15 cases, as this therapy will have the greatest potential benefit.

To determine whether bolus or infusion administration of ^1u -pentetreotide is optimal, areas below 3 curves (mRo X-ray / hr vs time) will be compared and half-lives calculated. The optimal method will be one that results in the largest area under the curve, with standard statistical tests for comparison between subjects and for one subject, where this comparison depends on the distribution of variance and whether parametric measurements are given. NCSS or Number Crunching Statistical Software is used.

Example 12: Accumulation of radiolabeled somatostatin analogues in angiogenic cells

To demonstrate that radiolabeled somatostatin analogues accumulate in angiogenic blood vessels, human neuroblastoma SKNSH cells lacking somatostatin receptors were implanted in nude mice. Tumors were allowed to grow to a size of about 1 cm in diameter, which took about 3 to 4 weeks. Mice were then injected with 50 μθϊ ¹²⁵ I-WOC-4a. The radioligand was allowed to act for 1 week. On day 7, a photograph was taken on a photosensitive film and a standard X-ray film. The resulting photographs were placed on top of each other to locate the radioligand. The radioligand was located in the tumor. Since tumor cells do not have receptors for somatostatin analogs and cannot bind radioligand, the radioligand detected in the tumor must be bound to angiogenic vessels.

φ φ · φ φ φ

Human placental vascular endothelial cells were used to test the binding of somatostatin analogs to cells.

The radioligand, a somatostatin analog, ¹²⁵ I-JIC-2D, was produced by solid phase synthesis as described in US Patent No. 5597894. This peptide had the following amino acid sequence: D-Lys-D-Tyr-D-Lys-D -Tyr-D-Lys-C [Cys-Phe-D-Trp-Lys-Thr-Cys] -Thr-NH ₂ . Cells were harvested by culturing human placental vein disks in fibrin clots for 14 days. Using this technique, the disc fraction has an angiogenic response. These discs and their angiogenic shoots were obtained mechanically and were dissociated by vortexing. The venous discs were then removed and endothelial cells collected. Cells were harvested, counted in a hemocytometer, and resuspended in binding buffer (Minimum Essential Medium (MEM), 10 mM HEPES, 0.01% BSA). The standard assay utilized 500,000 cells in 1 ml binding buffer. The radioactive ligand alone (500,000 cpm) or in combination with at least a 1000-fold molar excess (10 ^-6 M) of the non-radioactive compound (to determine specific binding) was added to a final volume of 1 ml. At the end of the experiment, the incubation medium was removed, the cells were washed twice with Hank's balanced salt solution (HBSS), and the radioactivity in the cells was determined using a gamma camera. This level of radioactivity represents the total binding, which includes both the membrane bound fraction and the internalized fraction. Specific binding was determined by calculating the difference between the measured radioactivity without unlabeled competitor minus radioactivity with unlabeled competitor.

To distinguish between the membrane bound and intracellular fractions, the cells were further incubated for 10 minutes at 4 ° C with acidified HBSS (pH = 4-5), rinsed in HBSS, and the radioactivity again determined by gamma camera. Because acid

0

000

000 000 • 00 0 ♦ * 0 00 00 preferably elutes the radioactive ligand from the external cell surface, remaining radioactivity belongs to the internalized fraction.

As shown in Figure 17, a radiolabeled somatostatin analog binds to angiogenic cells and most of the radioligand is internalized into the cells.

Example 13: Inhibition of angiogenic cell growth by radiolabeled somatostatin analogue

To demonstrate that radiolabeled somatostatin analogs inhibit the growth of angiogenic blood vessels, the Human Placental Vein Angiogenesis Model as described by J.C. Watson et al., Up-regulation of Somatostatin Receptor Subtype 2 (SST-2) mRNA Occurs During the Transformation of Human Endothelium to the Angiogenic Phenotype, published at the 12th International Symposium on Regulators Peptides, Copenhagen, Denmark, September 1996; and J.C. Watson et al., SST-2 Gene Expr <3sion Appears During Human Angiogenesis, Peptides Regulators, Vol. 64, p. 206 (Abstract) (1996); and J. C. Watson et al., Breast Cancer Increases Initiation of Angiogenesis Without Accelerating Neovessel Growth Rate, Surgery, Vol. 122, pp. 508-14 (1997). Human placental veins were cut into 2 mm diameter rings and inserted into 0.3% fibrinogen gel. This gel was overlaid with tissue culture medium containing 1020% fetal calf serum (FCS). Under normal conditions, endothelial processes begin to spread out of the rings within 6 days.

These venous discs were placed in the wells of three separate culture plates. One culture plate - control - was left without any treatment. Another plate was treated with 50 gCi / ml radiolabeled analogue

somatostatin, ¹¹¹ In-pentetreotide; and the third plate was treated with an equivalent amount of ⁱⁿ -Cl. The discs were then incubated for 14 days. After 14 days, the culture plates were examined for the number of wells in which angiogenic growth was initiated. The percentage of initiation is shown in Figure 18. The% of initiation observed in both treated plates was significantly lower than for the control plate. The lowest% initiation was observed for the ¹¹¹ In-pentetreotide-treated culture plate. The molar concentrations of radiolabeled somatostatin analog added to the culture plates were in the order of femtomols (10 ¹⁵ ). This concentration is 100x or more times the concentration of the unlabeled somatostatin analogue known to inhibit angiogenesis; ie a concentration of 10 ^-5 M to 10 ⁸ M. Therefore, any effect was due to the presence of the radioactive compound and not to the presence of the somatostatin analogue. The difference between ^it and the In-Cl ^lu In-pentetreotide is due to the effect of Auger emission on angiogenic cell growth that is caused by effect of Auger electrons to DNA. Only a radiolabeled compound bound to a somatostatin analog can be incorporated into DNA. Thus, this higher reduction is due to this incorporation and the effect of Auger electron emission on DNA.

The average area of the projections around the disks is measured using a computer image analyzer. As shown in FIG. 19, the vessel area (mm ² ) was significantly smaller for both treated plates. Again, the ⁱⁿ -pentetreotide-treated plate showed the least growth area.

To measure the growth rate of angiogenic vascular tissue, a measurement of the length of the processes was performed every two days during the 14-day incubation period. The growth rate, as measured in mm / day for all three culture plates, is shown in Figure 20. Again, it is ·· «· · ···

The growth rate for the two treated plates was significantly lower than for the control plate. However, there is no apparent difference in growth rate between the two treated samples.

Thus, it has been shown that the radiolabeled somatostatin analogue inhibits angiogenic vascular growth even at concentrations much lower than necessary to inhibit angiogenic growth by the unlabeled analogue. This suggests that gamma emission causes some inhibition and Auger electron emission causes further inhibition. The most potent inhibition is due to a radiolabeled analogue, which has both the effects of both gamma emission and Auger electron emission.

Other somatostatin analogs were tested using the same vascular disks of the human placental vein. These discs were placed in wells in two separate culture plates. One culture plate was left untreated until maturity. The second culture plate was subjected to various treatments: (1) radiolabeled somatostatin analogue ( ¹²⁵ I-WOC-4a, ¹³¹ I-WOC-4a, ¹¹¹ In-DPTA-W0C-4a, ^11x InDPTA-JIC-2D, ¹²⁵ I- JIC-2D, or dual labeled ^{ni in-125} I-DPTA- WOC4a or ⁱⁿ DPTA- ^in-125 I-JIC-2D); (2) cold, unlabeled somatostatin analogue (WOC-4a or JIC-2D); (3) by radioisotope alone ( ¹²⁵ L or ¹³¹ L); (4) a combination of a radiolabeled somatostatin analogue and its corresponding unlabeled analogue (e.g. ¹²⁵ I-WOC-4a and WOC-4a); or (5) a combination of a cold, unlabeled somatostatin analog and an unbound radioisotope (e.g. ¹²⁵ I and WO-4a). The discs were incubated for 3 days with the analog at a dose of 10 to 10,000 counts per well. After three days fresh medium was added and the discs were monitored to maturity. For treated and untreated wells, the percentage of wells that initiated angiogenesis was calculated. Results for various treatments were analyzed using ANOVA.

• · • ·

Alternatively, to increase cell destruction by radioactivity, venous disks were cryopreserved and stored in liquid nitrogen after three days of exposure to the radioligand. Freezing inhibits cell division but does not affect radioactivity. Another set of plates was prepared with identically treated venous discs but without a source of radioactivity. After three days of treatment, these venous discs were harvested, washed and cryopreserved in tissue culture medium containing 10% dimethylsulfoxide (DMSO). These venous disks were cryopreserved in a controlled freezer freezer and stored in liquid nitrogen for 2 months, or for a period that is six times the physical half-life of the radioisotope used. After this time, the venous discs were thawed and replated in wells containing fibrinogen gel and cultured as described above. After two weeks of growth, treated and untreated wells were compared.

These experiments have shown that radiolabeled somatostatin analogs selectively inhibit the initiation or induction of an angiogenic response in humans in a manner similar to the method of inhibiting the growth of tumor cells containing somatostatin receptors. Therefore, when radiolabelled somatostatin analogues are used in radiodiagnostics and angiogenic vascular therapy, infusion will be more effective than bolus injection.

Example 14: Increasing PDGF Receptor Expression in Human Angiogenic Cells

Using the in vitro model of human placental vein angiogenesis described above, we replaced the previously used discs with discs taken from the human aortic vascular endothelium and vein cava inferior to test for the presence of

Φ · · · · · φ · φ · φ · · · · · · · · · · · · · · · · · · Platelet-derived growth factor (PDGF) and its receptors. A similar angiogenic response was observed after 14 days of incubation of 2 mm discs in a 0.3% fibrin clot supplemented with fetal bovine serum. RNA was extracted from the cava inferior and aorta obtained from organ donors and tissue-treated angiogenic explants cultured as above. Reverse transcription polymerase chain reaction (RT-PCR) was then performed on each sample with PDGF A chain, B chain and receptor specific primers using 500 ng of RNA per reaction. Primers were made according to the published sequence. RT-PCR products were analyzed by gel electrophoresis, which showed bands at the expected positions for these products in angiogenic explants from both the aorta and cava inferior, but not in control natural vessels. These results were repeated using vascular samples from three patients. This suggests that the A and B chains of PDGF may play an important role in the regulation of angiogenesis. Furthermore, it also suggests that radiotherapy directed at this growth factor or at its receptor may be effective in reducing the development of angiogenesis associated with tumor growth.

It will be understood by those skilled in the art that techniques similar to those described in the above examples can be used to determine the efficacy of bolus versus infusion of other radiolabeled peptides and steroids for radiotherapy and radiodiagnostics of tumors.

All publications cited in this application are hereby incorporated by reference. However, in the event of conflicting statements, this application should be reviewed.

S =!

Claims (53)

PATENT CLAIMS Use of a receptor-dependent radiolabeled compound for the manufacture of a medicament for use in therapy for selectively accumulating said radiolabeled compound in target cells in a patient, wherein the target cells are selected from the group consisting of tumor cells and angiogenic cells and wherein the target cells express receptors for a radiolabeled compound, said administration comprising administering a dose of the radiolabeled compound to an infused patient for more than two hours, wherein the resulting retention time of the radiolabeled compound in the target cells is at least one and a half times the retention time of the radiolabeled compound in the target cells. total doses of the same radiolabeled compound to a patient. The use of claim 1, wherein the infusion rate is approximately equal to the rate of uptake and transfer of the radiolabeled compound to the nucleus in the target cells. Use according to claim 1 or 2, wherein the infusion time is between about 4 hours and about 30 days. Use according to any one of the preceding claims, wherein the infusion time is between about 24 hours and about 5 days. Use according to any one of the preceding claims, wherein said target cells are tumor cells. Use according to any one of the preceding claims, wherein said radiolabeled compound comprises a receptor-dependent peptide bound to a radioisotope. Use according to claim 6, wherein said receptor-dependent peptide is selected from the group consisting of nerve growth factor, fibroblast growth factor, epidermal growth factor, platelet growth factor, cholecystokinin, vasoactive intestinal peptide, gastrin releasing peptide, leukemia inhibitory factor, somatostatin , oxytocin, bombesin, calcitonin, arginine vasopressin, angiotensin II, atrial natriuretic peptide, insulin, glucagon, prolactin, growth hormone, gonadotropin, thyrotropin triggering hormone, triggering hormone for growth hormone, triggering hormone for gonadotropin, triggering hormone, triggering hormone , interferons, transferrin, substance P, neuromedin, neurotensin, neuropeptide Y, opiates and derivatives and analogues thereof. Use according to claims 6 or 7, wherein said receptor-dependent peptide is somatostatin. Use according to claims 6 or 7, wherein said receptor-dependent peptide is a somatostatin analogue. The use of claim 9, wherein said somatostatin analog is selected from the group consisting of octreotide, pentetreotide, vapreotide, lantreotide, WOC-3, WOC-3b, WOC-4a and JIC-2D. Use according to claim 9 somatostatin pentetreotide. or 10, where Yippee mentioned analogue Use according to claim 9 or 10, where Yippee mentioned analogue somatostatin WOC-4a. Use according to claim 9 or 10, where Yippee mentioned analogue somatostatin WOC-3b. The use of any one of claims 6 to 13, wherein said radioisotope has a half-life of between about 1 hour and about 60 days. • 99 The use of any one of claims 6 to 14, wherein said radioisotope has a half-life of between about 2 hours and about 60 days. The use of any one of claims 6 to 15, wherein said radioisotope has a half-life of between about 12 hours and about 60 days. Use according to any one of claims 6 to 16, wherein said radioisotope is selected from the group consisting of ⁶⁷ Ga, ^1u In, ^99m Tc, 90 _in 86 _in 169 125 _T 124 _T 123-r 129 _T 131Y, Y ^° , ^Yoy , ¹⁸⁸ Re, ^1Z0 I, ^1zq I, ^1ZJ I, ^izy I, ^iJ1 I and ^{Z /} Br. Use according to any one of claims 6 to 17, wherein said radioisotope is ⁱⁿ In. Use according to any one of claims 6 to 17, wherein said radioisotope is ¹²⁵ I. Use according to any one of claims 6 to 17, wherein said radioisotope is ¹³¹ I. 21. Use according to claim 6, wherein said radiolabeled compound is selected from the group consisting of ln ^{U1-pentetreotide ((111In-DTPA-D-Phe 1)} -octreotid) ⁽¹¹¹ In-DOTA ° D-Phe ¹ -Tyr ³ ) octreotide), ( ⁹⁰ Y-DOTA-D-Phe ¹ -Tyr ³ ) -octreotide), ( ⁸⁶ Y-DOTA-D-Phe ^1- Tyr ³ ) -octreotide), ( ¹¹¹ In-DTPA-D- Phe ¹⁾ -RC-160, ^99m Tc-RC-L60, ^{99 m} TcCPTA-RC-160, ^RC-123 I-160 ^I-125 RC-160, ¹³¹ I-RC-160, ⁹⁹ⁿ Tcoctreotide, ¹⁸⁸ Re-RC-160, ¹²³ I-Tyr ^3- octreotide, ¹²⁵ I-Tyr-octreotide, 131 I-Tyr-octreotide, 125131I-lantreotid, ¹²³ Ilantreotid, ⁱ³ⁱ Ilantreotid, lantreotid, Y-DOTA-lantreotid, ⁸⁶ Y-DOTA-lantreotid, ^LII DTPAIn-In-DOTA-lantreotid, ¹¹¹ In-DPTA-somatostatin, ⁹⁰ Y125, DOTA-somatostatin, ⁸⁶ Y-DOTA-somatostatin, ^i2i> I-somatostatin, 131 I-WOC-3 125 I-WOC-3b, ¹³¹ I-WOC-4a, ¹²⁵ I-WOC-4a, ¹²⁵ I-JIC-2D 123 IJIC-2D a 131 I-JIC-2D. 90, 111 • · 6222. The use according to any one of the preceding claims, wherein said radiolabeled compound is accumulated in tumor cells selected from the group consisting of endocrine tumors, melanomas, breast cancer, Merkel cell tumors, lymphomas, small cell lung cancer, gastrointestinal tumors, astrocytomas, gliomas , meningiomas, carcinoids, pancreatic B cell tumors, kidney tumors, neuroblastoma, and pheochromocytomas. The use of any one of claims 1 to 18, wherein said radiolabeled compound is ¹¹¹ In-pentetreotide. The use of any one of claims 1 to 17, wherein said radiolabeled compound is ¹²⁵ I-WOC-4a. Use according to any one of claims 1 to 17, wherein said radiolabeled compound is ¹³¹ I-WOC-4a. The use of any one of claims 1 to 17, wherein said radiolabeled compound is ¹²⁵ I-WOC-3b. The use of any one of claims 1 to 17, wherein said radiolabeled compound is ¹²⁵ I-somatostatin. Use according to any one of the preceding claims, wherein said radiolabeled compound is accumulated in tumor cells selected from the group consisting of pituitary tumors, endocrine tumors of the pancreas, carcinoids, paragangliomas, pheochromocytomas, medullary thyroid carcinomas, small cell lung carcinomas, neuroblastomas meningiomas, lymphomas, glucagonons, breast cancers, renal cancers, gliomas, astrocytomas, and melanomas. The use of any one of claims 23 to 28, wherein said radiolabeled compound is accumulated in neuroblastoma cells. bz The use of any one of claims 23 to 28, wherein said radiolabeled compound is accumulated in small cell lung cancer cells. Use according to any one of claims 23 to 28, wherein said radiolabeled compound is accumulated in endocrine tumor cells. The use of any one of claims 1 to 5, wherein said radiolabeled compound comprises a receptor-dependent steroid coupled to a radioisotope. The use of claim 32, wherein said receptor-dependent steroid is selected from the group consisting of estrogen, testosterone, progesterone, glucocorticosteroids, mineralocorticoids and derivatives and analogs thereof. The use of claim 33, wherein said receptor-dependent steroid is estradiol. The use of any one of claims 32 to 34, wherein said radioisotope has a half-life of between about 1 hour and about 60 days. The use of any one of claims 32 to 35, wherein said radioisotope has a half-life of between about 5 hours and about 60 days. The use of any one of claims 32 to 36, wherein said radioisotope has a half-life of between about 12 hours and about 60 days. Use according to any one of claims 32 to 37, wherein said radioisotope is selected from the group consisting of ⁶⁷ Ga, ^at Tn, ^99m Tc, ⁹⁰ Y, ⁸⁶ Y, ¹⁶⁹ Yb, ¹⁸⁸ Re, ¹²⁵ I, ¹²⁴ I, ¹²³ I , ¹²⁹ I, ¹³¹ I and ⁷⁷ Br. · · · · »» »» »» »» » L U ·· ········ Π · 14 4 1 ··· · ··· · · 44 · · 4 4 4 11 Use according to any one of claims 32 to 38, wherein said radioisotope is ¹²⁵ I. The use of any one of claims 32 to 39, wherein said radiolabeled compound is ¹²⁵ I-17-β-estradiol. The use of any one of claims 32 to 40, wherein said radiolabeled compound is accumulated in breast cancer cells. The use of any one of claims 1 to 4, wherein said target cells are angiogenic cells. The use of claim 42, wherein said radiolabeled compound comprises a radioisotope bound to a group consisting of somatostatin, somatostatin analogs, vascular endothelial growth factor, and platelet-derived growth factor. The use of claim 43, wherein said somatostatin analog is selected from the group consisting of octreotide, pentetreotide, vapreotide, lantreotide, WOC-3, WOC-3b, WOC-4a and JIC-2D. The use of any one of claims 43 or 44, wherein said somatostatin analog is pentetreotide. The use of any one of claims 43 or 44, wherein said somatostatin analog is WOC-4a. The use of any one of claims 43 or 44, wherein said somatostatin analog is WOC-3b. The use of any one of claims 43 or 44, wherein said somatostatin analog is JIC-2D. The use of any one of claims 43 to 48, wherein said radioisotope has a half-life of between about 1 hour and about 60 days. The use of any one of claims 43 to 49, wherein said radioisotope has a half-life of between about 5 hours and about 60 days. Use according to any one of claims 43 to 50, wherein said radioisotope has a half-life of between about 12 hours and about 60 days. Use according to any one of claims 43 to 52, wherein said radioisotope is as defined in any one of claims 17 to 20. Use according to any one of claims 43 to 52, wherein said radiolabeled compound is the same as defined in any one of claims 21, 23, 24, 25, 26 and 27. Use according to any one of claims 43 to 52, wherein said radiolabeled compound is ¹²⁵ I-JIC-2D. Use according to any one of the preceding claims, further comprising the step of imaging radioactivity in the region of said target cells by scintigraphic examination. The use of any preceding claim, further comprising the step of repeating the process of claim 1 until the target cell number in the patient is significantly reduced. 57. A method for selectively accumulating a receptor-dependent radiolabeled compound in target cells in a patient, wherein the target cells are selected from the group consisting of tumor cells and angiogenic cells and wherein the target cells express receptors for the radiolabeled compound, 0 · Characterized in that it comprises administering a dose of the radiolabeled compound to the patient in an infusion lasting more than two hours, wherein the resulting retention time in the radiolabeled compound is: the target cells is at least one and a half times the retention time of the radiolabeled compound in the target cells by bolus administration of the same total dose of the same radiolabeled compound to the patient.