WO2002098447A1 - Cytotoxic or radioactive conjugates able to bind to oxytocin receptors - Google Patents

Cytotoxic or radioactive conjugates able to bind to oxytocin receptors Download PDF

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WO2002098447A1
WO2002098447A1 PCT/EP2002/005687 EP0205687W WO02098447A1 WO 2002098447 A1 WO2002098447 A1 WO 2002098447A1 EP 0205687 W EP0205687 W EP 0205687W WO 02098447 A1 WO02098447 A1 WO 02098447A1
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conjugates
dota
agents
lvt
otr
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PCT/EP2002/005687
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French (fr)
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Giovanni Bussolati
Paola Cassoni
Bice Chini
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Giovanni Bussolati
Paola Cassoni
Bice Chini
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/095—Oxytocins; Vasopressins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/084—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being oxytocin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins

Definitions

  • the present invention refers to conjugates of oxytocin (OT) or OT- analogues with cytotoxic or radioactive agents, useful for the therapy or imaging of oxytocin-receptors (OTR) expressing tumors.
  • OT oxytocin
  • OTR oxytocin-receptors
  • the invention more particularly refers to conjugates of OT-analogues with chelating agents binding radioactive or paramagnetic metals or with cytotoxic agents such as taxol (paclitaxel).
  • OTR are expressed in several cell types in organs such as the breast, uterus, brain, vessels and prostatic gland.
  • OTR are expressed or over-expressed in tumor cells in several tumor types such as breast and endometrial carcinomas, neuroblastomas, glioblastomas, endometrial carcinomas, osteosar-comas, choriocarcinomas, Kaposi sarcomas, carcinomas of the prostate and of the lung (Endocrinology 142: 1377-1379, 2001).
  • Oxytocin analogues have been shown to inhibit growth of breast and endometrial carcinomas, neuroblastomas and glioblastomas, both in vitro and in vivo (Int. J. Cancer 66:817-820, 1996; Ibiden 72:340-344, 1997).
  • a radioactive or cytotoxic ligand specific for OTR would offer new strategies for the diagnosis and therapy of OTR-positive tumors.
  • a similar strategy proved successful in case of somatostatin receptors-expressing tumors, when the chelating agent DOTA was conjugated to a free amino group of somatostatin and of its analogues (Cancer Res. 153: 1-13, 2000; Clin. Cancer Res. 5: 1025-1033, 1999).
  • oxytocin analogues may be conjugated to suitable chelating agents or to cytotoxic agents such as taxol without loosing their affinity for OTR.
  • OT- analogues substituted in position 8 still have a reactive amino group in position 1 , which can interfere with the reaction.
  • blocking or removal of this amino group in position 1 such as in desamino-lysine-vasotocin (dLVT) does not affect the affinity for OTR of OT-analogues.
  • the invention therefore provides conjugates of OT-analogues having an amino acid residue in position 8 bearing a free reactive group to which cytotoxic agents or metal chelating compounds are bound.
  • lysine-vasotocin (LVT) and desamino- lysine-vasotocin (dLVT), described in Helv. Chirur. Acta 43: 182-185, 1960 and Justus Liebig Ann. Chem. 745:8-19, 1971 as well as the above peptides, where however the Glutamine (Glu) in position 4 can be substituted with Threonine (Thr) are particularly preferred.
  • Glu Glutamine
  • Thr Threonine
  • These compounds differ from oxytocin in having the amino acid in position 8 substituted with lysine.
  • Their affinity and specificity for OTR is similar to that of OT, in the range of 10 "9 M.
  • the OT-analogues can be bound to Paclitaxel derivatives for a targeted therapy of cancer, using an approach similar to that proposed by Savafy et al. J. Med. Chem. 42, 4919-24, 1999.
  • the conjugation of OT-analogues to Paclitaxel is obtained using polyethylene glycol (PEG) as a linker.
  • Metal-chelating agents such as DOTA, BOPTA, DOTMA, hydrazinonicotinamide (HYNIC) can be linked by amide bonds to the free amino group of lysine.
  • cytotoxic agents such as toxins, antibodies, lymphocyte-binding reagents, taxol can be bound.
  • the chelating agents e.g. DOTA
  • can bind radioactive substances such as 123 I, 125 I, 131 I, 75 Br, 76 Br, 74 Br, 77 Br e 82 Br, 99m Tc, 03 Pb, 67 Ga, 68 Ga, 72 As,
  • the radioactive substances can act to trace (imaging) and treat OTR- expressing tumors and organs, while the paramagnetic metals or halogens might be used for the magnetic resonance of OTR-expressing tumors or organs.
  • the conjugates ot the invention may optionally be labelled with a tracer such a Fluoride for the localization of tumors and organs with PET (Positron Emission Tomography).
  • the invention also relates to pharmaceutical and diagnostic kits containing effective quantities of the conjugates LVT, d LVT or similar OT- analogues.
  • the compounds of the invention will be administered, at a dosage related to the type of chelating agents, metal cytotoxic drugs, in order to achieve the therapy and diagnosis best for the patient.
  • DOTA was dissolved in anhydrous DMSO at 80°C and the solution allowed to cool under argon atmosphere.
  • DCC DOTA:NHS:DCC molar ratio
  • DOTA and LVT and DOTA and dLVT was carried out at a molar ratio of 50:1 by adding a suitable volume of the DOTA activated ester solution to the LVT or dLVT dissolved in phosphate buffer 0.1 M, pH 8.0. After reacting overnight, the conjugate was purified by means of a reversed phase column (Resource RPC 1ml, particle size 15 ⁇ m; Amersham Pharmacia Biotech, Uppsala Sweden) in a FPLC system (LCC-501 Plus controller, Pharmacia Biotech) coupled with an UV detector (LKB-UV-MII, Pharmacia Biotech) and a radiodetector (Flow scintillation analyzer, radiomatic 150 TR, Packard, Meriden, CT, USA).
  • Resource RPC 1ml, particle size 15 ⁇ m; Amersham Pharmacia Biotech, Uppsala Sweden a reversed phase column
  • FPLC system LCC-501 Plus controller, Pharmacia Biotech
  • UV detector LLB-UV-MI
  • a linear gradient method was applied using a solution of distilled water with 0.1% TFA (solvent A) and methanol (solvent B).
  • the eluents were delivered at a flow of 4 ml/min starting from 0% of solvent A to 100% of solvent B in 37 ml.
  • Two peaks corresponding to the LVT or dLVT conjugates with DOTA were evident in the UV profile.
  • the retention volume was 8.4 ml for the first compound (A) and 10.0 ml for the second (B), whereas unconjugated LVT and dLVT eluted at 7.0 ml in the same conditions.
  • An integrated fraction collector (Frac-100, Pharmacia Biotech) performed the recovery of each compound.
  • Each peak was subsequently analyzed by MALDI-TOF mass spectrometry, using a Reflex III instrument (Bruker, Germany).
  • Example 2 Radiolabeling of DOTA-LVT (peak A) and of DOTA-dLVT with [ ⁇ n In].
  • [ ul In]-DOTA-LVT, and [ ⁇ l In]-DOTA-dLVT 3.7 MBq of [ ⁇ ⁇ In]Cl 3 , diluted in acetate buffer (0.1 M, pH 5.5), were added to 0.07 ⁇ mol of fraction obtained from peak A. The solution was heated for 25' at 80°C and a labeling yield was checked by FPLC.
  • Example 3 Affinity studies.
  • Fig. 2 shows that dLVT and DOTA-dLVT showed high affinity for OTR, in the range of 10 "9 M and confirms that dLVT-DOTA displays an affinity for OTR much higher than that for other receptors such as V1A,V1B and V2. Table 1
  • Binding experiments were performed on intact human breast carcinoma (MCF7), glioblastoma (MOG-U-V-W) and colon carcinoma (HT29 and TS/A mouse mammary carcinoma cells, 20xl0 5 cells, suspended in 100 ⁇ l of culture medium were incubated for 30 min at 4°C in presence of 1 ⁇ M [ m In]-DOTA- LVT and [ u l In]-DOTA-dLVT. The cell suspension was then centrifuged for 5 min at 2000 g, the pellet was washed and re-suspended in fresh medium. Centrifugation and cell washing were repeated twice. The entity of the radiolabeling was evaluated measuring the radioactivity bound to cells (cpm/10 5 cells) by a Packard auto-gamma counter.
  • Binding specificity was determined by evaluating the radioactive displacement. Briefly, the cell suspension was incubated for 5 min in presence of LVT or OT 100 ⁇ M and 1 mM, or in presence of unrelated peptides (such as somatostatin 100 nM and 1 ⁇ M, and hexarelin 1 ⁇ M) followed by 20 min of incubation with 1 ⁇ M [ ⁇ n In]-DOTA-LVT. Cells were then centrifuged and washed twice as reported above. The activity of the two different agonists and of the unrelated peptides to compete with the radioligand was evaluated measuring the radioactivity bound to cells. All experiments were done in triplicate. Statistical analysis was carried out by ANOVA. Cut off for significance was 0.05.
  • Binding specificity was assayed by displacement with cold radio- ligands; in OTR+ cells, more than 90% of the specific [ ⁇ ⁇ In]-DOTA- LVT binding was displaced by 5 min preincubation with 100 ⁇ M and 1 mM non-radioactive OT or LVT (Fig. 3). A further confirmation of binding specificity was provided by the lack of radio-ligand displacement following incubation with two OT-unrelated peptides, somatostatin and hexarelin, used at 100 nM and 1 ⁇ M concentrations.
  • TS/A mammary carcinoma cells (lxl 0 6 , in 0.2 ml of medium) were injected subcutaneously.
  • the animals were injected intraperitoneally with a mixture of 1.1 MBq of [ l x ⁇ nj-DOTA-LVT and 74 kBq of [ FJ-DOTA-TOC.
  • the animals were divided into groups and sacrificed at 3 and 24 h post- injection, respectively. Tumor, blood, liver, kidney and brain were removed and weighed.
  • the radioactivity was measured in a gamma ray detector with a well counter geometry (Silena, Milano, Italy) together with standards of the injection mixture at two different time points in order to calculate the contribution of each isotope: immediately after the removal of the tissues and after two weeks, corresponding to 5 physical half-lives of [ l n In].
  • Activity was expressed as a percentage of injected doses per mg of tissue and ratios of uptake between tumor and brain vs. blood were calculated.
  • a statistical test (T-test) was performed to determine the significant difference between the groups. Cut off for significance was 0.05.
  • Tumour to blood ratios between the two peptides were remarkably different at both time points but especially at 24h (7.2 ⁇ 4.5 vs 2.7 ⁇ 1.4) demonstrating a receptor mediated accumulation in tumour.

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Abstract

Conjugates of oxytocin (OT) or OT-analogues with cytotoxic or radioactive agents, useful for the therapy or imaging of oxytocin-receptors (OTR) expressing tumors, more particularly conjugates of OT-analogues with chelating agents binding radioactive or paramagnetic metals or with cytotoxic agents such as taxol (paclitaxel).

Description

CYTOTOXIC OR RADIOACTIVE CONJUGATES ABLE TO BIND TO OXYTOCIN RECEPTORS
The present invention refers to conjugates of oxytocin (OT) or OT- analogues with cytotoxic or radioactive agents, useful for the therapy or imaging of oxytocin-receptors (OTR) expressing tumors.
The invention more particularly refers to conjugates of OT-analogues with chelating agents binding radioactive or paramagnetic metals or with cytotoxic agents such as taxol (paclitaxel).
OTR are expressed in several cell types in organs such as the breast, uterus, brain, vessels and prostatic gland.
Moreover, OTR are expressed or over-expressed in tumor cells in several tumor types such as breast and endometrial carcinomas, neuroblastomas, glioblastomas, endometrial carcinomas, osteosar-comas, choriocarcinomas, Kaposi sarcomas, carcinomas of the prostate and of the lung (Endocrinology 142: 1377-1379, 2001).
Oxytocin analogues have been shown to inhibit growth of breast and endometrial carcinomas, neuroblastomas and glioblastomas, both in vitro and in vivo (Int. J. Cancer 66:817-820, 1996; Ibiden 72:340-344, 1997). A radioactive or cytotoxic ligand specific for OTR would offer new strategies for the diagnosis and therapy of OTR-positive tumors. A similar strategy proved successful in case of somatostatin receptors-expressing tumors, when the chelating agent DOTA was conjugated to a free amino group of somatostatin and of its analogues (Cancer Res. 153: 1-13, 2000; Clin. Cancer Res. 5: 1025-1033, 1999).
We have now found that oxytocin analogues, particularly those having an amino acid bearing reactive groups in position 8, may be conjugated to suitable chelating agents or to cytotoxic agents such as taxol without loosing their affinity for OTR. However, OT- analogues substituted in position 8 still have a reactive amino group in position 1 , which can interfere with the reaction. We have found that blocking or removal of this amino group in position 1 such as in desamino-lysine-vasotocin (dLVT) does not affect the affinity for OTR of OT-analogues.
The invention therefore provides conjugates of OT-analogues having an amino acid residue in position 8 bearing a free reactive group to which cytotoxic agents or metal chelating compounds are bound.
Among the OT-analogues, lysine-vasotocin (LVT) and desamino- lysine-vasotocin (dLVT), described in Helv. Chirur. Acta 43: 182-185, 1960 and Justus Liebig Ann. Chem. 745:8-19, 1971 as well as the above peptides, where however the Glutamine (Glu) in position 4 can be substituted with Threonine (Thr) are particularly preferred. These compounds differ from oxytocin in having the amino acid in position 8 substituted with lysine. Their affinity and specificity for OTR is similar to that of OT, in the range of 10"9 M.
The OT-analogues can be bound to Paclitaxel derivatives for a targeted therapy of cancer, using an approach similar to that proposed by Savafy et al. J. Med. Chem. 42, 4919-24, 1999. The conjugation of OT-analogues to Paclitaxel is obtained using polyethylene glycol (PEG) as a linker.
Metal-chelating agents such as DOTA, BOPTA, DOTMA, hydrazinonicotinamide (HYNIC) can be linked by amide bonds to the free amino group of lysine. In a similar way, cytotoxic agents such as toxins, antibodies, lymphocyte-binding reagents, taxol can be bound. The chelating agents (e.g. DOTA) can bind radioactive substances such as 123I, 125I, 131I, 75Br, 76Br, 74Br, 77Br e 82Br, 99mTc, 03Pb, 67Ga, 68Ga, 72As,
JIn, J 1JIn, wYt, y/Ru, β πιRb, 0 Cu, 04Cu, J Fe, zmMn, 140La, 175Yb, 153Sm,
166 Ho, 149Pm, 177Lu, 142Pr, 159Gd, 212Bi, 47Sc, 149Pm, 67Cu, nιAg, 199Au, 188Re, 186Re, 161Tb and 51Cr or para-magnetic metals or halogens.
The radioactive substances can act to trace (imaging) and treat OTR- expressing tumors and organs, while the paramagnetic metals or halogens might be used for the magnetic resonance of OTR-expressing tumors or organs. The conjugates ot the invention may optionally be labelled with a tracer such a Fluoride for the localization of tumors and organs with PET (Positron Emission Tomography).
The invention also relates to pharmaceutical and diagnostic kits containing effective quantities of the conjugates LVT, d LVT or similar OT- analogues.
The compounds of the invention will be administered, at a dosage related to the type of chelating agents, metal cytotoxic drugs, in order to achieve the therapy and diagnosis best for the patient.
The processes for the preparation of the conjugates of the invention are known from the literature, and are similar to those described for somatostatin analogues.
The following examples further illustrate the invention. Example 1
DOTA was dissolved in anhydrous DMSO at 80°C and the solution allowed to cool under argon atmosphere. A solution of N-hydroxy-2,5- pyrrolidinedione (NHS) in DMSO was added dropwise to a stirred solution of
DOTA, followed by the dropwise addition of N,N'-dicyclohexylcarbodiimide
(DCC) in DMSO. The DOTA:NHS:DCC molar ratio was 1 : 1.4:0.8.
The mixture was allowed to react overnight under stirring and then filtered to separate the by-product dicyclohexylurea. The conjugation between
DOTA and LVT and DOTA and dLVT was carried out at a molar ratio of 50:1 by adding a suitable volume of the DOTA activated ester solution to the LVT or dLVT dissolved in phosphate buffer 0.1 M, pH 8.0. After reacting overnight, the conjugate was purified by means of a reversed phase column (Resource RPC 1ml, particle size 15 μm; Amersham Pharmacia Biotech, Uppsala Sweden) in a FPLC system (LCC-501 Plus controller, Pharmacia Biotech) coupled with an UV detector (LKB-UV-MII, Pharmacia Biotech) and a radiodetector (Flow scintillation analyzer, radiomatic 150 TR, Packard, Meriden, CT, USA). A linear gradient method was applied using a solution of distilled water with 0.1% TFA (solvent A) and methanol (solvent B). The eluents were delivered at a flow of 4 ml/min starting from 0% of solvent A to 100% of solvent B in 37 ml. Two peaks corresponding to the LVT or dLVT conjugates with DOTA were evident in the UV profile. The retention volume was 8.4 ml for the first compound (A) and 10.0 ml for the second (B), whereas unconjugated LVT and dLVT eluted at 7.0 ml in the same conditions. An integrated fraction collector (Frac-100, Pharmacia Biotech) performed the recovery of each compound. Each peak was subsequently analyzed by MALDI-TOF mass spectrometry, using a Reflex III instrument (Bruker, Germany).
Example 2 - Radiolabeling of DOTA-LVT (peak A) and of DOTA-dLVT with [ιnIn]. To prepare [ulIn]-DOTA-LVT, and [π lIn]-DOTA-dLVT, 3.7 MBq of [π ιIn]Cl3, diluted in acetate buffer (0.1 M, pH 5.5), were added to 0.07 μmol of fraction obtained from peak A. The solution was heated for 25' at 80°C and a labeling yield was checked by FPLC. Example 3 - Affinity studies.
To determine LVT, DOTA-LVT, dLVT and DOTA-dLVT affinity constants, heterologous competition experiments were performed on membranes prepared from monkey kidney COS7 cells transiently transfected with the human OTR cDNA (17). Briefly, electroporated cells were homogenized in a Dounce glass potter, washed twice and resuspended in binding buffer (50 mM Tris HC1, 5 mM MgCl, pH 7.4). 5-10 μg of membrane proteins were incubated with a fixed concentration of [^HJ-OT (1-2 nM) for 30 minutes at 30°C in the presence of increasing concentrations of unlabeled peptides. Non-specific binding was determined in the presence of 1 mM OT. Bound and free radioactivities were separated by filtration over Whatman GF/C filters pre-soaked in 10 mg/ml BSA. Binding isotherms were analyzed with the iterative curve-fitting program LIGAND.
As reported in Fig. 1 , unlabeled LVT inhibited [H]-OT binding with high affinity (Ki = 1.87 ± 0.418 nM; n=3); DOTA-LVT from peak A was also able to compete [HJ-OT binding with a calculated Ki of 238 + 52.36 nM; n=3. Displacement curves were parallel and their slopes were not significantly different from the unit. Fig. 2 shows that dLVT and DOTA-dLVT showed high affinity for OTR, in the range of 10"9 M and confirms that dLVT-DOTA displays an affinity for OTR much higher than that for other receptors such as V1A,V1B and V2. Table 1
Figure imgf000006_0001
Example 4 - Binding studies on OTR+ and OTR- tumor cells.
Cell lines were purchased from ATCC (Manassas, Virginia, USA). The (20), growing in Balb/c mice was a gift of Prof. Guido Forni, University of Torino, Italy. All the cells were grown as monolayers in RPMI medium (Gibco, Burlington) with 10% fetal calf serum (Gibco), in 25 cm T flasks in a 5% C02 humidified atmosphere, at 37°C. As proved in previous experiments (1, 3, 5), MCF7, MOG-U-V-W and TS/A cells are OTR+, while HT29 cells represent the OTR- control. Further, lack of expression of somatostatin receptor type 2 (SSTR2) in TS/A cells was tested using RT-PCR procedures similar to those used in our laboratory to study SSTR2 expression in human tumors (21).
Binding experiments were performed on intact human breast carcinoma (MCF7), glioblastoma (MOG-U-V-W) and colon carcinoma (HT29 and TS/A mouse mammary carcinoma cells, 20xl05 cells, suspended in 100 μl of culture medium were incubated for 30 min at 4°C in presence of 1 μM [mIn]-DOTA- LVT and [u lIn]-DOTA-dLVT. The cell suspension was then centrifuged for 5 min at 2000 g, the pellet was washed and re-suspended in fresh medium. Centrifugation and cell washing were repeated twice. The entity of the radiolabeling was evaluated measuring the radioactivity bound to cells (cpm/105 cells) by a Packard auto-gamma counter.
Binding specificity was determined by evaluating the radioactive displacement. Briefly, the cell suspension was incubated for 5 min in presence of LVT or OT 100 μM and 1 mM, or in presence of unrelated peptides (such as somatostatin 100 nM and 1 μM, and hexarelin 1 μM) followed by 20 min of incubation with 1 μM [ι nIn]-DOTA-LVT. Cells were then centrifuged and washed twice as reported above. The activity of the two different agonists and of the unrelated peptides to compete with the radioligand was evaluated measuring the radioactivity bound to cells. All experiments were done in triplicate. Statistical analysis was carried out by ANOVA. Cut off for significance was 0.05.
After 30 min. incubation with [πlIn]-DOTA-LVT, the presence of a specific radiolabeling was observed in all the OTR+ cell lines (MCF7, TS/A, MOG-U-V-W), and was negligible in HT29 (OTR-) cells. The amount of labeling, expressed as cpm/105cells, was 1711 ± 97 for MCF7 cells, 7252 ± 83 for TS/A cells and 1811 ± 101 for MOG-U-V-W cells, while only 34 + 6 for HT29 cells.
Binding specificity was assayed by displacement with cold radio- ligands; in OTR+ cells, more than 90% of the specific [π ιIn]-DOTA- LVT binding was displaced by 5 min preincubation with 100 μM and 1 mM non-radioactive OT or LVT (Fig. 3). A further confirmation of binding specificity was provided by the lack of radio-ligand displacement following incubation with two OT-unrelated peptides, somatostatin and hexarelin, used at 100 nM and 1 μM concentrations. Example 5 - In vivo studies.
To determine the entity and the specificity of receptor mediated uptake of [1 HIn]-DOTA-LVT in tumors in comparison to the non-specific compound [125I]-DOTA-TOC, the OTR+ and SSTR2- TS/A tumor growing in Balb/c mice was used as experimental model. A total of 8 Balb/c female mice, weighing 25 g were used. TS/A mammary carcinoma cells (lxl 06, in 0.2 ml of medium) were injected subcutaneously. Twenty days later, when the growing tumor reached a size of about 2 cm in diameter, the animals were injected intraperitoneally with a mixture of 1.1 MBq of [l x ^nj-DOTA-LVT and 74 kBq of [ FJ-DOTA-TOC. The animals were divided into groups and sacrificed at 3 and 24 h post- injection, respectively. Tumor, blood, liver, kidney and brain were removed and weighed. The radioactivity was measured in a gamma ray detector with a well counter geometry (Silena, Milano, Italy) together with standards of the injection mixture at two different time points in order to calculate the contribution of each isotope: immediately after the removal of the tissues and after two weeks, corresponding to 5 physical half-lives of [l nIn]. Activity was expressed as a percentage of injected doses per mg of tissue and ratios of uptake between tumor and brain vs. blood were calculated. A statistical test (T-test) was performed to determine the significant difference between the groups. Cut off for significance was 0.05.
The results of the biodistribution study in Balb/c mice bearing TS/A tumors showed that neither [nιIn]-DOTA-LVT nor [125I]-DOTA-TOC accumulated in the brain: 1.97xl0"5 ± 1.44xl0"5 and 2.14x 10"3+ 1.80xl0"3 %ID/mg respectively at 3 h; 1.71x 10"6 ± 1.09xl0"6 and 1.98xl0"4 ± 1.84xl0"4 respectively at 24h. Tumor uptake was 1.69xl0"4 ± 6.69xl0"5 vs. 8.11xl0"4 + 4.61xl0"4 respectively at 3h and 4.33xl0"5 ± 1.07xl0"5 vs. 1.16xl0"4 + 2.04xl0"4 at 24h. Liver uptake was also low at both time points: 4.20xl0"4 + 3.47xl0"4 vs. 2.11xl0-3 ± 1.3xl0"3 at 3h and 1.27xl0"4 ± 9.28xl0~5 vs. 3.72x10"4 + 2.51xl0"4 at 24h. As expected, kidney uptake was rather high although lower for [mϊn]-DOTA-LVT than for [1 5I]-DOTA-TOC especially at 24h (l . l lxlO"3 + 2.59xl0-4 vs. 1.49xl0'2 ± 3.51xl0-3). The tumor to blood ratios for both radio-labeled peptides are in shown in the following Table 2. Table 2 Tumour/blood Uptake ratios: in 8 Balb/c mice carrying TSA mammary tumor
3h 24h mIn-DOTA-LVT 125I-DOTATOC 1 HIn-DOTA-LVT 125I-DOTATOC
2.7 0.96 2.8 4.86
4.1 1.85 4.4 2.07
1.2 0.81 9.1 2.13
N.A. 0.49 12.6 1.88
mean±s.d. 2.7±1.4 1.02±0.58 7.2±4.5 2.7±1.4
Tumour to blood ratios between the two peptides were remarkably different at both time points but especially at 24h (7.2 ± 4.5 vs 2.7 ± 1.4) demonstrating a receptor mediated accumulation in tumour.

Claims

1. Conjugates of oxytocin analogues having an amino acid residue in position 8 bearing a free reactive group to which cytotoxic agents or metal chelating compounds are bound.
2. Conjugates according to claim 1, wherein the OT analogue is LVT (lysine-vasotocin) .
3. Conjugates according to claim 1 , wherein the OT analogue is blocked in position 1 or is de-aminated dLVT (deamino-lysine-vasotocin).
4. Conjugates according to any one of claims 1 to 3, wherein the cytoxic or anti-proliferative agents are antibodies, toxins, paclitaxel, cytoxic drugs, chelating agents of radio-active or para-magnetic metals, biotin.
5. Conjugates according to claim 4, wherein the cytotoxic agents are able to chelate metals or halogens or para-magnetic substances.
6. Conjugates according to claim 5, wherein the chelating agents are selected from DOTA, BOPTA, DOTMA, HYNIC.
7. Conjugates of claim 6 carrying radioactive substances selected from 1231, 125I, 131I, 75Br, 76Br, 74Br, 77Br e 82Br, 99mTc, 203Pb, 67Ga, 68Ga, 72As, n ιIn, U3In, 90Yt, 97Ru, 82mRb, 62Cu, 64Cu, 52Fe, 52mMn, 140La, 175Yb, 153Sm, 166Ho, I49Pm, 177Lu, 142Pr, 159Gd, 212Bi, 47Sc, 149Pm, 67Cu, Ag, 199Au, 2ssRe, 186Re, 161Tb and 51Cr.
8. Conjugates of claims 1-7 as tracers or radio-therapic agents for the localization or treatment of OTR-expressing tumors.
9. Pharmaceutical compositions or diagnostic kits containing the conjugates of claims 1-8 in admixture with suitable carriers.
PCT/EP2002/005687 2001-06-01 2002-05-24 Cytotoxic or radioactive conjugates able to bind to oxytocin receptors WO2002098447A1 (en)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BATHGATE ROSS ET AL: "Structure and expression of the bovine oxytocin receptor gene.", DNA AND CELL BIOLOGY, vol. 14, no. 12, 1995, pages 1037 - 1048, XP001093995, ISSN: 1044-5498 *
BUSSOLATI GIANNI ET AL: "111In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-Lys8-vasotocin: A new powerful radioligand for oxytocin receptor-expressing tumors.", CANCER RESEARCH, vol. 61, no. 11, 1 June 2001 (2001-06-01), pages 4393 - 4397, XP002209600, ISSN: 0008-5472 *
CASSONI PAOLA ET AL: "Activation of functional oxytocin receptors stimulates cell proliferation in human trophoblast and choriocarcinoma cell lines.", ENDOCRINOLOGY, vol. 142, no. 3, March 2001 (2001-03-01), pages 1130 - 1136, XP001098558, ISSN: 0013-7227 *
CHINOL M ET AL: "-DOTA-Lys8-vasotocin: A new promising peptide for oxytocin receptor expressing tumors.", JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, vol. 44, no. Supplement 1, May 2001 (2001-05-01), Fourteenth International Symposium on Radiopharmaceutical Chemistry;Interlaken, Switzerland; June 10-15, 2001, pages S679 - S681, XP001098513, ISSN: 0362-4803 *
SAFAVY A ET AL: "PACLITAXEL DERIVATIVES FOR TARGETED THERAPY OF CANCER: TOWARD THE DEVELOPMENT OF SMART TAXANES", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, vol. 42, 1999, pages 4919 - 4924, XP000919106, ISSN: 0022-2623 *

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