Conjugates containing gastrin-releasing peptide receptor antagonists or salts thereof and uses thereof

Abstract

The present invention relates to a conjugate having the formula: CLA, or a pharmaceutically acceptable salt thereof, wherein C is a chelator, L is a linker covalently attached to the chelator, and A is a gastrin-releasing peptide receptor antagonist covalently attached to the linker, wherein the chelator has formula (I) where the dotted line represents the covalent bond to the linker; the linker has the formula: -β-Ala-β-Ala-; and the gastrin-releasing peptide receptor antagonist has the amino acid sequence: -DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2. The present invention also relates to uses of the conjugate or salt thereof. TIFF2023542218000025.tif72105

Description

[Technical Field]

[0001] The present invention is in the field of radiopharmaceuticals.

[0002] More specifically, the present invention relates to conjugates or pharmaceutically acceptable salts thereof comprising a gastrin-releasing peptide receptor (GRPR) antagonist, which may be used to prepare radiopharmaceuticals or may be used as radiopharmaceuticals once labeled with a radionuclide.

[0003] The present invention also relates to compositions, radiopharmaceuticals, and kits of parts comprising the conjugates or salts thereof.

[0004] The present invention further relates to the use of the unlabeled conjugate or a salt thereof and the kit of parts for preparing a radiopharmaceutical.

[0005] The present invention further relates to radiopharmaceuticals for use in the in vivo imaging or treatment of cancers that overexpress GRPR, more particularly prostate, breast, and lung cancers. [Background technology]

[0006] Prostate cancer is the most common cancer among men, excluding skin cancer, and the second leading cause of cancer death in men in the United States.

[0007] Several treatment options are currently offered to prostate cancer patients, including active surveillance, surgery, external beam radiation therapy, cryotherapy, hormone therapy, high intensity focused ultrasound, and chemotherapy, depending on the type of cancer cells and the stage of progression of the cancer, the patient's age, and general health.

[0008] Nevertheless, there is a great need for improved treatments.

[0009] One promising approach to improved treatment for prostate cancer is the use of targeted radiopharmaceuticals, i.e., drugs labeled with radionuclides that can be targeted to cancer cells, thereby delivering toxic levels of radiation to the cancer cells while sparing normal, healthy tissue.

[0010] Typically, radiopharmaceuticals designed for prostate cancer are conjugates comprising a vector molecule, optionally linked via a linker (or spacer), to a chelator that has high affinity for prostate cancer cells and that retains the radionuclide by chelation.

[0011] GRPR, also known as bombesin (BBN) receptor subtype II, has been shown to be overexpressed in several human tumors, including not only prostate tumors but also breast and lung tumors. GRPR overexpression is observed in 63% to 100% of primary prostate cancers and in over 50% of lymphatic and bone metastases. GRPR density has been reported to be 26-fold higher in prostate cancer than in benign prostatic hyperplasia.

[0012] Therefore, various conjugates have been proposed to target GRPR-positive tumors, especially prostate cancer.

[0013] Recent reports have shown that GRPR antagonists have superior properties compared to conjugated GRPR agonists, with higher tumor uptake and less accumulation in physiological GRPR-positive non-target tissues. Furthermore, GRPR agonists have been shown to induce adverse effects in patients that are mediated by their physiological activity.

[0014] Therefore, particular attention has been focused on developing conjugates containing GRPR antagonists rather than GRPR agonists as vector molecules.

[0015] Examples of such conjugates are disclosed, for example, in European Patent Application No. 2252628. Summary of the Invention [Problem to be solved by the invention]

[0016] However, contrary to what the teachings of this reference may suggest, the pharmacokinetic and tumor-targeting properties of GRPR antagonist-based conjugates cumulatively depend on the choice of chelator, linker, and GRPR antagonist, and therefore the design of GRPR antagonist-based conjugates that can be readily used as targeted radiopharmaceuticals to target GRPR-positive tumors remains a major challenge. [Means for solving the problem]

[0017] The present invention precisely demonstrates that conjugates, as well as pharmaceutically acceptable salts thereof, have unexpectedly high and sustained uptake in GRPR-positive tumors, such as prostate tumors, while at the same time exhibiting low uptake in non-target organs and rapid clearance. The conjugate satisfies the formula: CLA, where C is a chelator, L is a linker covalently attached to the chelator, and A is a GRPR antagonist covalently attached to the linker; the chelating agent corresponds to the chelating agent known as DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) and has the formula:

[0018] TIFF0007824281000001.tif72108 characterized in that it has Where the dotted line represents a covalent bond to the linker; - the linker has the formula: -β-Ala-β-Ala-; - the GRPR antagonist is a peptide known as JMV594, which has the amino acid sequence: -DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (SEQ ID NO: 1). In other words, the conjugate has the formula:

[0019] TIFF0007824281000002.tif39157 is equivalent to

[0020] In the foregoing and in the following: - β-Ala refers to beta-alanine, also known as 3-aminopropanoic acid; - DPhe, Gln, Trp, Ala, Val, Gly, His and Leu refer to the α-amino acids phenylalanine, glutamine, tryptophan, alanine, valine, glycine, histidine and leucine, respectively, phenylalanine being in the D-form, while glutamine, tryptophan, alanine, valine, histidine and leucine are in the L-form; whereas - Sta is the formula:

[0021] TIFF0007824281000003.tif5082 refers to the gamma-amino acid statins Also known as (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid.

[0022] Furthermore, the term "pharmaceutically acceptable salt" refers to salts that have toxicity profiles within a range that makes them useful in pharmaceutical applications.

[0023] Suitable pharmaceutically acceptable salts may in particular be addition salts of free acids or free bases.

[0024] Acid addition salts may be prepared from inorganic or organic acids. Suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid, while suitable organic acids may be selected from aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, and the like. Examples of suitable hydroxybenzoic acids include lactic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic (pamoic) acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, beta-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid.

[0025] Base addition salts are, for example, metallic salts including alkali metal, alkaline earth metal, and transition metal salts, such as calcium, magnesium, potassium, sodium, and zinc salts, or organic salts made with basic amines, such as N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine.

[0026] For use as a radiopharmaceutical, the conjugate or salt thereof further comprises a radionuclide chelated by a chelating agent.

[0027] The present invention also relates to compositions comprising the conjugate or a salt thereof in unlabeled form (i.e., without any radionuclide) in a pharmaceutically acceptable vehicle, such as saline, metal-free water, ascorbic acid, ethanol, polysorbate 80 (i.e., polyoxyethylene (20) sorbitan monooleate, sold under the trademark Tween™ 80), a buffer solution, such as ammonium acetate buffer, or a mixture thereof, wherein the ascorbic acid and ethanol advantageously act as antioxidants, while the polysorbate 80 advantageously reduces viscosity.

[0028] The present invention further relates to a ready-for-use radiopharmaceutical comprising the conjugate or a salt thereof in radiolabeled form (i.e., comprising a radionuclide chelated by a chelating agent) in a pharmaceutically acceptable vehicle as described above. The present invention may also be used to prepare radiopharmaceuticals comprising at least - a first container containing the conjugate or a salt thereof in unlabeled form; and a second container containing the radionuclide, typically in the form of a salt (chloride, acetate, ...) Parts kit including:

[0029] In the kit-of-parts, the conjugate or salt thereof and the radionuclide may be in any suitable form, for example, in dry form (e.g., powder), in liquid form, i.e., in solution in a pharmaceutically acceptable medium as described above, or in frozen form.

[0030] As is known per se, the kit comprises: one or more reagents and / or one or more solvents or diluents, such as saline, metal-free water, biological buffers, etc.; and / or - A booklet containing instructions for preparing and / or using the radiopharmaceutical It may further include.

[0031] The present invention further relates to the use of the unlabeled conjugate, its salt, or kit-of-parts for preparing a radiopharmaceutical, which comprises chelation of a radionuclide by a chelating agent of the conjugate or its salt.

[0032] In the foregoing, the radionuclide is preferably a lead radionuclide, especially if the radiopharmaceutical is intended for use for in vivo imaging purposes. 203 Pb, or if the radiopharmaceutical is intended for therapeutic use 212 It is Pb.

[0033] The invention further relates to radiopharmaceuticals for use in in vivo imaging, for example by single photon emission computed tomography (SPECT), or in the treatment of cancers which overexpress the gastrin-releasing peptide receptor.

[0034] Such use involves administering an appropriate dose of the radiopharmaceutical, typically intravenously, to the patient to be imaged or treated, and, in the case of in vivo imaging, imaging the patient.

[0035] Preferably, the cancer is prostate cancer, breast cancer, or lung cancer, with or without metastasis, especially prostate cancer.

[0036] Other characteristics and advantages of the invention will become more apparent upon reading the supplement to the following description.

[0037] Obviously, this supplement to the description is set forth merely to illustrate the object of the invention and does not constitute in any way a limitation of said object. [Brief explanation of the drawings]

[0038] [Figure 1]1 shows the results of a biodistribution study performed with a conjugate of the invention labeled with Pb at a specific activity of 10 μCi per 14 ng in subcutaneous PC-3 tumor-bearing athymic nude mice. The results are presented in terms of percent injected dose per gram of organ, expressed as %ID / g, found in organs of mice 1 hour, 4 hours, and 24 hours after injection of the Pb-conjugate dose into the mice. [Figure 2A] 2A-2C show the results of a biodistribution study performed with a Pb-labeled conjugate of the invention at a specific activity of 10 μCi per 28 ng in tumor-non-bearing, immunocompetent mice. The results are presented in terms of percent injected dose per gram of organ, expressed as %ID / g, found in the organs of mice 5 minutes after injection of the Pb-conjugate dose (FIG. 2A). [Figure 2B] 2B shows the results of a biodistribution study performed with a Pb-labeled conjugate of the invention at a specific activity of 10 μCi per 28 ng in tumor-non-bearing, immunocompetent mice. The results are presented in terms of percent injected dose per gram of organ, shown as %ID / g, found in the organs of mice 30 minutes after injection of the Pb-conjugate dose (FIG. 2B). [Figure 2C] 2C shows the results of a biodistribution study performed with a Pb-labeled conjugate of the invention at a specific activity of 10 μCi per 28 ng in tumor-non-bearing, immunocompetent mice. The results are presented in terms of percent injected dose per gram of organ, shown as %ID / g, found in the organs of mice 1 hour after injection of the Pb-conjugate dose (FIG. 2C). [Figure 2D]2D shows the results of a biodistribution study performed with a Pb-labeled conjugate of the invention at a specific activity of 10 μCi per 28 ng in tumor-non-bearing, immunocompetent mice. The results are presented in terms of percent injected dose per gram of organ, shown as %ID / g, found in the organs of mice 4 hours after injection of the Pb-conjugate dose (FIG. 2D). [Figure 2E] 2E shows the results of a biodistribution study performed with a Pb-labeled conjugate of the invention at a specific activity of 10 μCi per 28 ng in tumor-non-bearing, immunocompetent mice. The results are presented in terms of percent injected dose per gram of organ, shown as %ID / g, found in the organs of mice 24 hours after injection of the Pb-conjugate dose (FIG. 2E). [Figure 2F] 2F shows the results of a biodistribution study performed with a Pb-labeled conjugate of the invention at a specific activity of 10 μCi per 28 ng in tumor-non-bearing, immunocompetent mice. The results are presented in terms of percent injected dose per gram of organ, shown as %ID / g, found in the organs of mice 48 hours after injection of the Pb-conjugate dose (FIG. 2F). [Figure 2G] Figure 1 shows the urine, feces and total excretion of 203Pb-labeled conjugates of the present invention in terms of percent injected dose, shown as %ID, in tumor-non-bearing immune-responsive mice as a function of time, shown as t and expressed in hours, after injecting the mice with a dose of 203Pb-conjugate. [Figure 3A] Figure 3A shows the results of a biodistribution study performed with Pb-labeled conjugates of the invention at different specific activities in subcutaneous PC-3 tumor-bearing athymic nude mice. Figure 3A corresponds to a first group of mice, designated as group A, that received a single Pb-conjugate dose of specific activity equal to 10 μCi per 28 ng. [Figure 3B]The figure corresponds to a second group of mice, designated as group B, which received a single dose of 212Pb-conjugate with a specific activity equal to 10 μCi per 140 ng. [Figure 3C] Figure 1 corresponds to a third group of mice, designated as Group C, that received a single Pb-conjugate dose of specific activity equivalent to 10 μCi per 280 ng. In each figure, results are presented in terms of percent injected dose per gram of organ, expressed as %ID / g found in the organs of mice 1 and 4 hours after injection of the Pb-conjugate dose. [Figure 4A] FIG. 1 shows the survival rate, in %, of athymic nude mice as a function of time after injection of cancer cells, indicated as t and expressed in weeks, bearing subcutaneous PC-3 tumors and given either only one dose of a conjugate of the invention labeled with 212Pb at a specific activity of 10 μCi per 14 ng (1 cycle), or three doses of the same conjugate at intervals of 14 days (3 cycles), or sterile saline (control). [Figure 4B] FIG. 1 shows the mean tumor volume, shown as V and expressed in mm, observed in athymic nude mice as a function of time after injection of cancer cells, shown as t and expressed in weeks, bearing subcutaneous PC-3 tumors and given either only one dose (1 cycle) of a conjugate of the invention labeled with 212Pb at a specific activity of 10 μCi per 14 ng, or three doses of the same conjugate at 14 day intervals (3 cycles), or sterile saline (control). [Figure 5]1 shows the results of a comparative study aimed at comparing the biodistribution of a Pb-labeled conjugate of the present invention at a specific activity of 10 μCi per 280 ng in subcutaneous PC-3 tumor-bearing athymic nude mice with that of a Pb-labeled conjugate at the same specific activity, which differs only in that it also contains DOTA as a chelator. The results are presented in terms of the percent injected dose per gram of organ, expressed as %ID / g, found in the organs of mice 1, 4, and 24 hours after injection of the Pb-conjugate dose into the mice; in this figure, the conjugate of the present invention is designated as Pb-DOTAM-conjugate, while the comparative conjugate is designated as Pb-DOTA-conjugate. [Figure 6] 1 shows the results of a comparative study aimed at assessing the biodistribution in subcutaneous PC-3 tumor-bearing athymic nude mice of a Pb-labeled conjugate with a specific activity of 10 μCi per 10 ng, which differs from the conjugate of the present invention only in that it contains a linker composed of a chain of three glutamic acid residues. The results are presented in terms of percent injected dose per gram of organ, expressed as %ID / g, found in the organs of mice 4 hours after injection of the conjugate dose into the mice. [Figure 7] 1 shows the results of a comparative study aimed at assessing the biodistribution in tumor-bearing, immunocompetent mice of a Pb-labeled conjugate with a specific activity of 10 μCi per 4.1 ng, which differs from the conjugate of the present invention only in that it contains a linker comprised of a 4-amino-(1-carboxymethyl)piperidinyl group. The results are presented in terms of percent injected dose per gram of organ, expressed as %ID / g, found in the organs of mice 4 hours after injection of the conjugate dose into the mice. DETAILED DESCRIPTION OF THE INVENTION

[0039] I- Preparation of Unlabeled Conjugates of the Invention : I.1- Peptide sequence β-Ala-β-Ala-DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 Preparation of (SEQ ID NO: 2) : The peptide sequence: β-Ala-β-Ala-DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) was synthesized on a 0.1 mmol scale using an automated microwave peptide synthesizer (Biotage™ Initiator+Alstra™-BIOTAGE™).

[0040] Standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry was used with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N-hydroxylbenzotriazole (HOBt) as activators.

[0041] Rink amide resin was used to obtain the amidated C-terminus.

[0042] The amino acids leucine, valine, and β-alanine were double coupled. In addition to the double coupling, the two β-alanines were double deprotected.

[0043] All amino acids were coupled at 75° C. except histidine and statins, which were coupled at 48° C. to avoid racemization of histidine and O-acylation of statins.

[0044] I.2- Conjugation of DOTAM to peptide sequences : DOTAM may be reacted with a compound having the formula:

[0045] TIFF0007824281000004.tif75115 was conjugated to the resin-bound peptide sequence by using DOTAM monoacid.

[0046] To do this, DOTAM monoacid was first pre-reacted by dissolving 2.25 equivalents of DOTAM monoacid (0.225 mmol; 90.5 mg), 2.25 equivalents of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5,-b]pyridinium 3-oxide hexafluorophosphate (HATU; 0.225 mmol; 85.5 mg), and 6.75 equivalents of diisopropylethylamine (DIEA; 0.675 mmol; 120 μL) in 3 mL of dimethylformamide (DMF) in a round-bottom flask, and the mixture was stirred for 30 min.

[0047] The resin-bound peptide sequence was then added to the mixture and the reaction was rotated overnight.

[0048] After the conjugation was complete, the reaction medium was filtered through a coarse fritted funnel to remove excess reagents, and the residue was washed three times with DMF, three times with methanol, and three times with DCM.

[0049] I.3- Cleavage of the conjugate from the resin : The conjugate was cleaved from the resin by suspending it in a cocktail consisting of 95% (v / v) trifluoroacetic acid (TFA), 2.5% (v / v) triisopropylsilane (TIPS), and 2.5% (v / v) HO to a final volume of 3 mL.

[0050] The reaction mixture was rotated in a round-bottom flask for 3 hours, after which the reaction mixture was filtered through a large-pore fritted funnel. The TFA was evaporated using nitrogen gas, and the conjugate was precipitated using cold ethyl ether. The flask was then centrifuged at 4500 rpm for 10 minutes, and the ethyl ether supernatant was removed. The pellet was then freeze-dried overnight to remove excess ethyl ether.

[0051] I.4- Conjugate purification : The conjugate was purified using reverse-phase HPLC using a PHENOMENEX™ Luna™ 10 μm C18(2) preparative column (250×50 mm) with the following gradient: - t = 0–5 min: Eluent A (0.1% TFA in water) containing 1% Eluent B (0.1% TFA in acetonitrile (ACN); - t=5-45 min: Eluent B linearly increased from 1% to 75% in Eluent A.

[0052] The pure conjugate had a retention time of approximately 24 minutes. The collected peak was rotary evaporated to remove the organic solvent and freeze-dried.

[0053] Thus, 13 mg of conjugate was obtained with a purity of >95% as determined using an AGILENT™ 1100 Series LC-MS using a RESTREK™ Ultra IBD 3 μm analytical column (150×2.1 mm) with the following gradient: - t=0~2 min: Eluent B (100%H2O); - t = 2-17 min: Eluent B linearly decreased from 100% to 0% in eluent A (0.1% TFA in ACN).

[0054] The mass of the pure peptide was confirmed using an AGILENT™ 1100 Series LC-MS coupled with a HEWLETT PACKARD™ 1100 Series MSD: expected 1638.91; observed 1638.7.

[0055] The conjugate was stored at −80° C. prior to lead labeling.

[0056] II- Radiolabeling of the conjugates of the present invention : For in vivo distribution and efficacy studies in mice, 212 Pb or 203 Pb-labeled conjugates are hereafter referred to as 212 Pb-conjugates" and " 203 Each was labeled as "Pb-conjugate" and was prepared on the day of injection into mice based on the specific radioactivity at the time of conjugation and diluted to the specific activity required at the time of injection.

[0057] To do so, the conjugate obtained in item I above was thawed and diluted with metal-free water. An appropriate volume of the conjugate solution thus obtained was then added to a cryogenic vial optionally containing appropriate volumes of 0.4 M ammonium acetate, ascorbic acid, ethanol, and tween solution. Subsequently, an appropriate volume of NaOH / 0.4 M ammonium acetate solution was added to the vial, optionally containing appropriate volumes of 0.4 M ammonium acetate, ascorbic acid, ethanol, and tween solution. 212 Pb-acetic acid solution (ORANO MED) or 203 Pb-chloride solution (LANTHEUS) was added.

[0058] The sample was incubated at 50°C for 10 minutes to allow for the conjugate to react with the sample. 212 Pb or 203 The chelation of Pb remaining free in the sample was confirmed using instant thin layer chromatography (iTLC). 212 Pb or 203 This was verified by measuring Pb.

[0059] III- IN VIVO STUDIES USING THE LEAD-LABELED CONJUGATES OF THE INVENTION : In the following: *Athymic nude mice used were Hsd:Athymic Nude-Foxn1 from ENVIGO™ nu It is a mouse; *The immune-competent mice used were Hsd:ICR (CD-1™) mice from ENVIGO™; *The PC-3 human prostate cancer cells used were ATCC™ CRL-1435™ cells from ATCC™; *The automatic gamma counter used was a Wizard manufactured by PERKIN ELMER (trademark) 2 (trademark) counter; *"Buffer 1" refers to a mixture of saline, 23 mM ascorbic acid, 0.08% (v / v) Tween™ 20, and 5% (v / v) ethanol; *"Buffer 2" refers to a mixture of saline, 20 mM ascorbic acid, 0.02% (v / v) Tween™ 80 and 5% (v / v) ethanol.

[0060] III.1- in xenograft-bearing mice 212 Pb-conjugate biodistribution studies : This study demonstrated that the specific activity of 10 μCi per 14 ng of human prostate cancer cells was 10 μCi in athymic nude mice bearing human prostate cancer cell tumors. 212 We aimed to evaluate the biodistribution of the Pb-labeled conjugates.

[0061] * of conjugate at 10 μCi per 14 ng of conjugate 212 Pb-labeling :

[0062] TIFF0007824281000005.tif18144

[0063] *Approx. 10μCi / 100μL 212 Preparation of Pb-conjugate dose:

[0064] TIFF0007824281000006.tif31166

[0065] 212 The solution obtained from the Pb-conjugate / buffer mixture and a single dose of approximately 10 μCi / 100 μL 212 Insulin syringes containing 100 μL of the solution corresponding to the Pb-conjugate dose were prepared and injected into the mice.

[0066] * research design : At the start of the study, 15 male athymic nude mice, 7-8 weeks old and weighing 27.74±1.87 g, received 10 sucrose in 100 μL of RPMI-1640 medium / Matrigel™ (v / v: 1 / 1). 6 PC-3 human prostate cancer cells were injected subcutaneously into the right flank. 3 The mixture was grown until it reached a volume of 0.5 × length × width (equation: volume = 0.5 × length × width). 2 (Determined by:

[0067] Each mouse was then given a single intravenous injection (into the tail vein). 212 Pb-conjugate doses were given.

[0068] The mice were then divided into three groups of five mice each, designated as "Group A," "Group B," and "Group C," respectively.

[0069] Mice in group A were sacrificed 1 hour after the injection of the dose; mice in group B were sacrificed 4 hours after the injection of the dose, while mice in group C were sacrificed 24 hours after the injection of the dose.

[0070] Blood, reproductive organs, small intestine, colon including cecum, spleen, pancreas, kidneys, stomach, liver, lungs, heart, brain, femur, abdominal fat, skeletal muscle, tail (as injection site), and PC-3 tumor were collected from each sacrificed mouse, weighed, and transferred to individual tubes for an automated gamma counter.

[0071] The tubes were counted for 2 minutes. A standard consisting of 5 μL of the solution injected into the mice was also counted for each group of mice. The background was automatically subtracted from the counts. The standard was also used for decay correction.

[0072] The percent injected dose per gram, expressed as %ID / g, was calculated for each organ collected (mean±standard deviation).

[0073] * result : The results are shown in Figure 1.

[0074] As shown in this figure, 1 hour after the dose injection, there was a significant increase in the number of GRPRs present in the pancreas, likely due to the well-known expression of GRPR in the pancreas. 212 The highest uptake of Pb-conjugates (approximately 12% ID / g) was observed in the pancreas. 212 The uptake of the Pb conjugates was also high (approximately 6% ID / g) with only a small decrease at 4 and 24 hours after the dose was injected.

[0075] 212 Pb-conjugates have a rapid clearance, resulting in a high tumor / blood ratio.

[0076] III.2- in tumor-non-bearing immune-responsive mice 203 Pb-conjugate biodistribution studies : This study demonstrated that 28ng of IgG4 was detected in non-tumor-bearing immunocompetent mice at a specific activity of 10 μCi per 28ng of IgG4. 203 We aimed to evaluate the biodistribution of the Pb-labeled conjugates.

[0077] * of conjugate at 10 μCi per 28 ng of conjugate 203 Pb-labeling :

[0078] TIFF0007824281000007.tif35140

[0079] * Approximately 10μCi / 100μL 203 Preparation of Pb-conjugate doses :

[0080] TIFF0007824281000008.tif18141

[0081] 203 The solution obtained from the Pb-conjugate / buffer mixture and a single dose of approximately 10 μCi / 100 μL 203 Insulin syringes containing 100 μL of the solution corresponding to the Pb-conjugate dose were prepared and injected into the mice.

[0082] * research design : At the start of the study, 30 male and 30 female immunocompetent CD1 mice, 7-8 weeks old and weighing 27.75±2.52 g for males and 25.91±2.75 g for females, were given a single intravenous injection. 203 Pb-conjugate doses were given.

[0083] The mice were then divided into six groups of 10 mice each, designated as groups A, B, C, D, E and F, which contained five males and five females each.

[0084] Mice in group A were killed 5 minutes after the dose was injected; mice in group B were killed 30 minutes after the dose was injected; mice in group C were killed 1 hour after the dose was injected; mice in group D were killed 4 hours after the dose was injected, while mice in group E were killed 24 hours after the dose was injected.

[0085] Mice in group F were placed in metabolic cages and their urine and fecal excretion were collected 4 hours, 24 hours and 48 hours after the dose injection; the mice were sacrificed 48 hours after the dose injection.

[0086] Blood, bladder, reproductive organs, small intestine, colon including cecum, spleen, pancreas, kidneys, stomach, liver, lungs, heart, brain, femur, abdominal fat, skeletal muscle, salivary glands, and tail were collected from each sacrificed mouse, weighed, and transferred to individual tubes for an automated gamma counter.

[0087] The tubes were counted for 2 minutes. A standard consisting of 5 μL of the solution injected into the mice was also counted for each group of mice. The background was automatically subtracted from the counts. The standard was also used for decay correction.

[0088] The feces of mice in group F were also counted.

[0089] The percent injected dose per gram, expressed as %ID / g, was calculated for each organ collected (mean ± standard deviation), and simultaneously the percent injected dose, expressed as %ID, was calculated for the feces of mice in group F (mean ± standard deviation).

[0090] * result : The results are shown in Figures 2A to 2G.

[0091] As shown in Figures 2A to 2F,203 The Pb-conjugate has a safe biodistribution profile in both male and female mice.

[0092] In fact, in the pancreas 203 Although there is an initial high uptake of Pb-conjugates (>30% ID / g 5 min after dose injection), the %ID / g is well below 10 for all organs just 4 h after dose injection.

[0093] No significant differences in %ID / g were observed between male and female mice, except for a higher kidney uptake at 5 min in female mice, which is likely due to the kidneys of female mice being smaller than those of male mice, resulting in a higher %ID per gram of organ.

[0094] Furthermore, Figure 2G shows 203 This shows that Pb-conjugates are eliminated primarily by renal excretion.

[0095] III.3- in xenograft-bearing mice at different specific activities 212 Pb-conjugate biodistribution studies : This study demonstrated that specific radioactivity varied from 10 μCi per 28 ng to 10 μCi per 280 ng in athymic nude mice bearing human prostate cancer cell tumors. 212 We aimed to evaluate the biodistribution of the Pb-labeled conjugates.

[0096] * of conjugate at 10 μCi per 28 ng of conjugate 212 Pb-labeling :

[0097] TIFF0007824281000009.tif24137

[0098] * of conjugate at 10 μCi per 140 ng of conjugate 212 Pb-labeling :

[0099] TIFF0007824281000010.tif24137

[0100] * of conjugate at 10 μCi per 280 ng of conjugate 212 Pb-labeling :

[0101] TIFF0007824281000011.tif24137

[0102] * Approximately 10μCi / 100μL 212 Preparation of Pb-conjugate doses :

[0103] TIFF0007824281000012.tif58145

[0104] 212 One of the solutions obtained from the Pb-conjugate / buffer mixture and one dose of approximately 10 μCi / 100 μL 212 Insulin syringes each containing 100 μL of one of the solutions corresponding to the Pb-conjugate dose were prepared and injected into the mice.

[0105] * research design : At the start of the study, 30 male athymic nude mice, 7-8 weeks old and weighing 27.89 ± 2.27 g, were administered 10 sera in 100 μL of RPMI-1640 medium / Matrigel™ (v / v: 1 / 1). 6 PC-3 human prostate cancer cells were injected subcutaneously into the right flank. 3 was grown until it reached

[0106] The mice were divided into three groups of 10 mice each, designated as groups A, B, and C, respectively.

[0107] Each mouse in group A received a single dose of specific radioactivity equivalent to 10 μCi per 28 ng intravenously. 212 Each mouse in group B was given a single dose of Pb-conjugate with a specific activity equivalent to 10 μCi per 140 ng intravenously. 212 Pb-conjugate dose and at the same time each mouse in group C was given a single intravenous dose of specific radioactivity equal to 10 μCi per 280 ng. 212 Pb-conjugate doses were given.

[0108] Five mice from each of groups A, B, and C were sacrificed 1 hour after the dose was injected, and five mice from each of groups A, B, and C were sacrificed 4 hours after the dose was injected.

[0109] Blood, reproductive organs, small intestine, colon including cecum, spleen, pancreas, kidney, stomach, liver, lung, heart, brain, femur, abdominal fat, skeletal muscle, tail, and PC-3 tumor were collected from each sacrificed mouse, weighed, and transferred to individual tubes for an automated gamma counter.

[0110] The tubes were counted for 2 minutes. A standard consisting of 5 μL of the solution injected into the mice was also counted. The background was automatically subtracted from the counts. The standard was also used for decay correction.

[0111] The percent injected dose per gram, expressed as %ID / g, was calculated for each organ collected (mean±standard deviation).

[0112] * result : The results are shown in Figures 3A to 3C.

[0113] As shown in these figures, 212 Lower specific activity Pb-conjugates result in lower uptake in healthy organs but do not affect tumor uptake.

[0114] III.4- in xenograft-bearing mice 212 Efficacy study of Pb-conjugates : This study demonstrated that the specific activity of 10 μCi per 14 ng of human prostate cancer cells was 10 μCi in athymic nude mice bearing human prostate cancer cell tumors. 212 The aim was to evaluate the efficacy of one treatment cycle (cycle 1) or three treatment cycles (cycles 1, 2 and 3) using a Pb-labeled conjugate of the invention.

[0115] * of conjugate at 10 μCi per 14 ng of conjugate 212 Pb-labeling:

[0116] TIFF0007824281000013.tif63146

[0117] * Approximately 10μCi / 100μL 212 Preparation of Pb-conjugate doses :

[0118] TIFF0007824281000014.tif47146

[0119] 212 One of the solutions obtained from the Pb-conjugate / saline mixture and one dose of approximately 10 μCi / 100 μL 212 Insulin syringes each containing 100 μL of one of the solutions corresponding to the Pb-conjugate dose were prepared and injected into the mice.

[0120] * research design : At the start of the study, 40 male athymic nude mice, 7-8 weeks old and weighing 28.58±1.97 g, were administered 10 sera in 100 μL of RPMI-1640 medium / Matrigel™ (v / v: 1 / 1). 6 PC-3 human prostate cancer cells were injected subcutaneously into the right flank. 3 was grown until it reached

[0121] Twenty mice received a single dose of cycle 1 intravenously 10 days after cancer cell injection, and simultaneously 10 mice received a single dose of 100 μL of sterile saline intravenously (control).

[0122] Ten of the 20 mice received the cycle 1 dose, one cycle 2 dose 24 days after cancer cell injection, and one cycle 3 dose 38 days after cancer cell injection.

[0123] During the study, tumor volume was 2000 mm 3Mice were euthanized immediately after reaching 0.05%. Additionally, mice were euthanized before the scheduled endpoint if they showed unacceptable signs of distress or pain due to tumor burden, adverse effects of the injection, or a combination of two or more of the following termination criteria: severe weight loss (e.g., 15% weight loss over two consecutive days); poor tumor condition (e.g., ulcers, teeth marks, or open wounds); soiling / lack of grooming for 5 days; lethargy or decreased activity for 3 days; weakness / balance problems for 5 days; hunchback appearance; diarrhea; paralysis; or severe anemia and hypothermia.

[0124] * result : The results are shown in Figures 4A and 4B.

[0125] As shown in Figure 4A, one or three treatment cycles using the conjugates of the invention increase the median survival time from 7.9 weeks (control) to 13.9 weeks (3 cycles).

[0126] There is no significant difference between one treatment cycle and three treatment cycles. The time interval between two consecutive doses in three treatment cycles is suboptimal, and the effectiveness of treatment with multiple doses may be increased by optimizing the time interval between two consecutive doses.

[0127] IV- comparative study : IV.1- Effect of varying chelators on biodistribution in xenograft-bearing mice : This study was conducted in athymic nude mice bearing human prostate cancer cell tumors. 212 The biodistribution of the Pb-labeled conjugates of the present invention was also investigated. 212 Pb-labeled, the chelator corresponds to DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), with the formula:

[0128] TIFF0007824281000015.tif5995 where the dotted line represents a covalent bond to the linker. The purpose of this study was to compare the conjugates of the present invention with those of conjugates that differ only in that they have

[0129] For clarity, the conjugate of the present invention will be designated hereinafter as the "DOTAM-conjugate" and the comparative conjugate will be designated hereinafter as the "DOTA-conjugate."

[0130] * Preparation of unlabeled DOTA-conjugates : Unlabeled DOTA conjugates were prepared following a similar protocol described in section I above, except that DOTAM was replaced with DOTA in the step of conjugating the chelator to the peptide sequence.

[0131] * of DOTAM- and DOTA-conjugates at 10 μCi per 280 ng of conjugate 212 Pb-labeling :

[0132] TIFF0007824281000016.tif100146 * Approximately 10μCi / 100μL 212 Pb-DOTAM-conjugates and 212 Preparation of Pb-DOTA-conjugate dose :

[0133] TIFF0007824281000017.tif35159

[0134] 212 Pb-DOTAM-conjugate / buffer 2 mixture and 212 Insulin syringes containing 100 μL each of one of the solutions obtained from Pb-DOTA-conjugate / buffer 2 and one of the solutions corresponding to a single dose of approximately 10 μCi / 100 μL were prepared and injected into mice.

[0135] * research design : At the start of the study, 30 male athymic nude mice, 7-8 weeks old and weighing 27.90±1.9 g, were administered 10 sera in 100 μL of RPMI-1640 medium / Matrigel™ (v / v: 1 / 1). 6PC-3 human prostate cancer cells were injected subcutaneously into the right flank. 3 was grown until it reached

[0136] The mice were divided into six groups of five mice each, designated as groups A, B, C, D, E, and F, respectively.

[0137] Each mouse in groups A, B, and C was given a single intravenous 212 Pb-DOTAM-conjugate dose was given intravenously to each mouse in groups D, E, and F at the same time. 212 Pb-DOTA-conjugate doses were given.

[0138] Mice in groups A and D were sacrificed 1 hour after the dose was injected; mice in groups B and E were sacrificed 4 hours after the dose was injected, while mice in groups C and F were sacrificed 24 hours after the dose was injected.

[0139] Blood, reproductive organs, small intestine, colon including cecum, spleen, pancreas, kidneys, stomach, liver, lungs, heart, brain, femur, abdominal fat, skeletal muscle, tail, salivary glands, and PC-3 tumor were collected from each sacrificed mouse, weighed, and transferred to individual tubes for an automated gamma counter.

[0140] The tubes were counted for 2 minutes. A standard consisting of 5 μL of the solution injected into the mice was also counted. The background was automatically subtracted from the counts. The standard was also used for decay correction.

[0141] The percent injected dose per gram, expressed as %ID / g, was calculated for each organ collected (mean±standard deviation).

[0142] * result : The results are shown in Figure 5.

[0143] As shown in this figure, 212 Pb-DOTAM-conjugates 212It has a better biodistribution profile than the Pb-DOTA-conjugate, with better tumor retention over the first 24 hours.

[0144] 212 For the Pb-DOTAM-conjugate, an initial high uptake was observed in the pancreas 1 hour after the dose injection.

[0145] As mentioned above, GRPR is known to be expressed in the pancreas, and this initial high uptake is due to high binding affinity for cells expressing GRPR. 212 than Pb-DOTA-conjugates 212 It is possible that it was transferred to the Pb-DOTAM-conjugate.

[0146] IV.2- Effect of linker modification on biodistribution in mice : Studies in xenograft-bearing mice : This study demonstrated that the specific activity of 10 μCi per 10 ng of human prostate cancer cells was 10 μCi / 10 ng in athymic nude mice bearing human prostate cancer cells. 212 The aim was to assess the biodistribution of a conjugate that was Pb-labeled and differed from the conjugates of the present invention only in that it contained a linker composed of a chain of three glutamic acid residues.

[0147] This conjugate is hereafter referred to as " 212 Pb-3Glu-conjugate”.

[0148] * Preparation of unlabeled 3Glu-conjugate : The unlabeled 3Glu-conjugate was prepared according to the same protocol described in section I above, except that in the step of preparing the peptide sequence, two β-alanine residues were replaced with three glutamic acid residues.

[0149] * of 3Glu-conjugate at 10 μCi per 10 ng of conjugate 212 Pb-labeling:

[0150] TIFF0007824281000018.tif23140

[0151] * Approximately 10μCi / 100μL 212 Preparation of Pb-3Glu-conjugate dose :

[0152] TIFF0007824281000019.tif23140

[0153] 212 The solution obtained from the Pb-3Glu-conjugate / saline mixture and a single dose of approximately 10 μCi / 100 μL 212 Insulin syringes containing 100 μL of the solution corresponding to the Pb-3Glu-conjugate dose were prepared and injected into the mice.

[0154] * research design : At the start of the study, five male athymic nude mice, 7-8 weeks old and weighing 21.25±0.9 g, received 10 sucrose in 100 μL of RPMI-1640 medium / Matrigel™ (v / v: 1 / 1). 6 PC-3 human prostate cancer cells were injected subcutaneously into the right flank. 3 was grown until it reached

[0155] Each mouse received one intravenous 212 Pb-3Glu-conjugate doses were given.

[0156] Mice were sacrificed 4 hours after the dose was injected.

[0157] Blood, bladder, reproductive organs, small intestine, colon including cecum, spleen, pancreas, kidneys, stomach, liver, lungs, heart, brain, femur, abdominal fat, skeletal muscle, tail, and PC-3 tumor were collected from each sacrificed mouse, weighed, and transferred to individual tubes for an automated gamma counter.

[0158] The tubes were counted for 2 minutes. A standard consisting of 5 μL of the solution injected into the mice was also counted. The background was automatically subtracted from the counts. The standard was also used for decay correction.

[0159] The percent injected dose per gram, expressed as %ID / g, was calculated for each organ collected (mean±standard deviation).

[0160] * result : The results are shown in Figure 6.

[0161] As shown in this figure, simply replacing the -β-Ala-β-Ala- linker with a -Glu-Glu-Glu- linker results in 212 No significant initial uptake in the pancreas or significant tumor uptake was observed for the Pb-3Glu-conjugate, resulting in a completely different biodistribution profile.

[0162] Studies in non-tumor-bearing immune-competent mice : The study demonstrated a specific activity of 10 mCi per 4.1 ng in tumor-nonbearing, immunocompetent mice. 212 Pb-labeled 4-amino-(1-carboxymethyl)piperidinyl group, formula:

[0163] TIFF0007824281000020.tif3583 The aim of the present invention was to evaluate the biodistribution of conjugates that differ from the conjugates of the present invention only in that they contain a linker composed of:

[0164] This conjugate is hereafter referred to as " 212 Pb-ACMP-conjugate”.

[0165] * Preparation of unlabeled ACMP-conjugates : Unlabeled ACMP-conjugates were prepared following a similar protocol as described in Section I above, except that peptide synthesis was terminated after coupling of DPhe, and 4-amino-(1-carboxymethyl)piperidine was conjugated to the peptide sequence prior to conjugation of DOTAM.

[0166] * of ACMP-conjugate at 10 μCi per 4.1 ng of conjugate 212 Pb-labeling :

[0167] TIFF0007824281000021.tif18140 *Chelation took 30 minutes

[0168] * Approximately 10μCi / 100μL 212 Preparation of Pb-ACMP-conjugate dose:

[0169] TIFF0007824281000022.tif23140

[0170] 212 The solution obtained from the Pb-ACMP-conjugate / saline mixture and a single dose of approximately 10 μCi / 100 μL 212 Insulin syringes containing 100 μL of the solution corresponding to the Pb-ACMP-conjugate dose were prepared and injected into the mice.

[0171] * research design : Five immune-competent CD1 female mice, 7–8 weeks old, were given a single intravenous injection. 212 Pb-ACMP-conjugate dose was injected.

[0172] Mice were sacrificed 4 hours after the dose was injected.

[0173] Blood, bladder, reproductive organs, small intestine, colon including cecum, spleen, pancreas, kidneys, stomach, liver, lungs, heart, brain, femur, abdominal fat, and skeletal muscle were collected from each sacrificed mouse, weighed, and transferred to individual tubes for an automated gamma counter.

[0174] The tubes were counted for 2 minutes. A standard consisting of 5 μL of the solution injected into the mice was also counted. The background was automatically subtracted from the counts. The standard was also used for decay correction.

[0175] The percent injected dose per gram, expressed as %ID / g, was calculated for each organ collected (mean±standard deviation).

[0176] * result : The results are shown in Figure 7.

[0177] As shown in this figure, simple replacement of the -β-Ala-β-Ala- linker with a 4-amino-(1-carboxymethyl)piperidinyl linker resulted in a significantly reduced safety profile and a five-fold increase in renal uptake.

[0178] Cited References EP-A-2252628

Claims

1. A conjugate having the formula: C-L-A, or a pharmaceutically acceptable salt thereof, wherein C is a chelator, L is a linker covalently attached to the chelator, and A is a gastrin-releasing peptide receptor antagonist covalently attached to the linker; The chelating agent has the formula: characterized in that it has The dotted line represents the covalent bond to the linker; the linker has the formula: -β-Ala-β-Ala-; Gastrin-releasing peptide receptor antagonists have the amino acid sequence: DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH ２ (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof.

2. 10. The conjugate or salt of claim 1, further comprising a radionuclide chelated by a chelating agent.

3. 3. The conjugate or salt of claim 2, wherein the radionuclide is a lead radionuclide.

4. Lead radionuclides, ２０３ Pb or ２１２ 4. The conjugate or salt of claim 3, wherein Pb is Pb.

5. A composition comprising the conjugate or salt of claim 1 in a pharmaceutically acceptable medium.

6. A radiopharmaceutical comprising a conjugate or salt according to any one of claims 2 to 4 in a pharmaceutically acceptable medium.

7. at least, - a first container containing the conjugate or salt of claim 1; and - a second container containing the radionuclide; A parts kit comprising:

8. 8. The kit of parts according to claim 7, wherein the radionuclide is a lead radionuclide.

9. Lead radionuclides, ２０３ Pb or ２１２ 9. The kit of parts of claim 8, wherein the metal is Pb.

10. 10. Use of the conjugate or salt of claim 1 for preparing a radiopharmaceutical, comprising chelating a radionuclide with a chelating agent of the conjugate or salt thereof.

11. 11. The use according to claim 10, wherein the radionuclide is a lead radionuclide.

12. Lead radionuclides, ２０３ Pb or ２１２ The use according to claim 11, wherein the metal is Pb.

13. 13. The use according to any one of claims 10 to 12, wherein the radiopharmaceutical is for in vivo imaging or treatment of cancers that overexpress gastrin-releasing peptide receptors.

14. 14. The use according to claim 13, wherein the cancer is prostate cancer, breast cancer, or lung cancer.

15. The use according to claim 14, wherein the cancer is prostate cancer.

16. 10. Use of the kit of parts according to any one of claims 7 to 9 for preparing a radiopharmaceutical, comprising chelating a radionuclide with the chelating agent of the conjugate or its salt.

17. 16. The use of claim 15, wherein the radiopharmaceutical is for in vivo imaging or treatment of cancers that overexpress gastrin-releasing peptide receptors.

18. 18. The use according to claim 17, wherein the cancer is prostate cancer, breast cancer, or lung cancer.

19. 19. The use according to claim 18, wherein the cancer is prostate cancer.

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