[161Tb]-BASED RADIOPEPTIDES
20240181094 ยท 2024-06-06
Inventors
- Cristina M?LLER (Nussbaumen, CH)
- Roger Schibli (Baden, CH)
- Nicolas VAN DER MEULEN (Schinznach-Dorf, CH)
- Francesca Borgna (Windisch, CH)
- Damian Wild (Basel, CH)
- Fani MELPOMENI (Basel, CH)
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K51/088
HUMAN NECESSITIES
A61K51/083
HUMAN NECESSITIES
A61K47/22
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K51/08
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K51/12
HUMAN NECESSITIES
Abstract
A radiopeptide is provided which comprises (a) a radionuclide, wherein the radionuclide is terbium-161, (b) a chelator coordinating terbium-161, and (c) a peptide or peptide analogue, which is a somatostatin receptor (SSTR) antagonist. The radiopeptide is suitable for use in the treatment of tumor diseases.
Claims
1. A radiopeptide comprising (a) a radionuclide, wherein the radionuclide is terbium-161, (b) a chelator of terbium-161, and (c) a peptide or peptide analogue, which is a somatostatin receptor (SSTR) antagonist.
2. The radiopeptide of claim 1, wherein the somatostatin receptor (SSTR) antagonist is covalently coupled to (b).
3. The radiopeptide of claim 1 or 2, wherein the chelator is a cyclic chelator, in particular a macrocyclic chelator.
4. The radiopeptide of any of the preceding claims, wherein the chelator is a tetradentate chelator.
5. The radiopeptide of any of the preceding claims, wherein the chelator contains four nitrogen atoms.
6. The radiopeptide of any of the preceding claims, wherein the chelator is a 12-membered tetraaza ring system.
7. The radiopeptide of any of the preceding claims, wherein the chelator comprises at least one substituent containing at least one carboxy function.
8. The radiopeptide of any of the preceding claims, wherein the chelator is DOTA or a DOTA derivative.
9. The radiopeptide of claim 8, wherein the DOTA derivative is selected from the group consisting of: ##STR00004##
10. The radiopeptide of claim 9, wherein the chelator is DOTA (dodecane tetraacetic acid).
11. The radiopeptide of any of the preceding claims, wherein the somatostatin receptor antagonist is covalently coupled to the chelator via an amide linkage.
12. The radiopeptide of any of the preceding claims, wherein the somatostatin receptor antagonist binds to SSTR-2 (sst2) or is an SSTR-2 (sst2) selective antagonist.
13. The radiopeptide of any of the preceding claims, wherein less than 20% of the administered somatostatin receptor antagonist is internalized by cells.
14. The radiopeptide of any of the preceding claims, wherein the somatostatin receptor antagonist is a cyclic peptide.
15. The radiopeptide of any of the preceding claims, wherein the somatostatin receptor antagonist contains two cysteine residues, which preferably form a disulfide bridge.
16. The radiopeptide of claim 15, wherein the somatostatin receptor antagonist contains the cysteine residues at peptide positions 2 and 7.
17. The radiopeptide of any of the preceding claims, wherein the somatostatin receptor antagonist comprises 8 to 14 amino acids, preferably 8 to 10, more preferably 8 amino acids.
18. The radiopeptide of any of the preceding claims, wherein the somatostatin receptor antagonist comprises formula I: X1-cyclo[D-Cys-X3-X4-Lys-Thr-Cys]-D-Tyr-NH.sub.2, wherein X1, X3, and X4 may selected from a naturally or a non-naturally occurring D- or L-amino acid.
19. The radiopeptide of claim 18, wherein (i) X1 is selected from the group consisting of naturally occurring Phe or a substituted Phe having one or more substitutions at the phenyl ring system and Cpa, (ii) X3 is selected from the group consisting of Aph(Hor), Leu, L-Agl(NMe.Math.benzoyl), D-Agl(NMe.Math.benzoyl), Aph(Cbm), Tyr, Aph(CONHOCH.sub.3), Tyr, Aph(CONHOH), and/or (iii) X4 is selected from the group consisting of D-Trp and D-Aph(Cbm) (D-4-amino-Phe(carbamoyl)), wherein X1 is preferably selected from the group consisting of pNOs-Phe, pCl-Phe and Cpa, X3 is preferably selected from the group consisting of Tyr, Aph(Cbm), and Aph(Hor), and/or X4 is preferably selected from the group consisting of D-Trp and D-Aph(Cbm).
20. The radiopeptide of claim 18 or 19, wherein the somatostatin receptor antagonist is selected from the group consisting of LM3 ([p-Cl-Phe-cyclo[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]D-Tyr-NH.sub.2]), JR11 (Cpa-cyclo[D-Cys-Aph(Hor)-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH.sub.2), and BASS (pNO.sub.2-Phe-cyclo[D-Cys-Tyr-D-Trp-Lys-Thr-Cys]D-Tyr-NH.sub.2).
21. The radiopeptide of any of the preceding claims, wherein terbium-161 is produced by neutron irradiation of gadolinium-160.
22. The radiopeptide of any of the preceding claims, wherein terbium-161 is a non-carrier-added terbium-161 (n.c.a. terbium-161).
23. The radiopeptide of any of the preceding claims, wherein the antagonist is preferentially taken up by tumors relative to other tissue.
24. The radiopeptide of claim 23, wherein the ratio of radiopeptide uptake in tumor cells to radiopeptide uptake in blood is at least 50.0, the ratio of radiopeptide uptake in tumor cells to radiopeptide uptake in liver cells is at least 10.0 and/or the ratio of radiopeptide uptake in tumor cells to radiopeptide uptake in kidney cells is at least 2.0, preferably measured 2 hours after administration.
25. The radiopeptide of any of the preceding claims, wherein the radiopeptide has the structure of the following formula: ##STR00005##
26. A pharmaceutical composition, comprising the radiopeptide of any of the preceding claims and at least one pharmaceutically acceptable excipient, preferably water.
27. The pharmaceutical composition of claim 26, wherein the composition comprises 0.001 to 1 mg/ml or 0.01 to 1 mg/ml or 0.05 to 0.5 mg/ml radiopeptide.
28. The pharmaceutical composition of claim 26 or 27, wherein the composition contains at least one of the group consisting of gentisic acid, ethanol, acetate, NaCl and ascorbate/ascorbic acid.
29. The pharmaceutical composition of claim 28, wherein the composition contains ascorbate.
30. The pharmaceutical composition any of claim 29, wherein the composition contains 0.5 mM to 0.5 M, in particular 1 mM to 100 mM or 10 mM to 100 mM ascorbate.
31. The pharmaceutical composition of any of claims 26 to 30, wherein the pH value of the composition is from pH 3.5 to pH 6 or from pH 4 to pH 6.
32. A method of treating a disease or a tumor disease, comprising: administering a radiopeptide of any of claims 1 to 25 or a composition of any of claims 24 to 31 to a subject in need of a disease treatment or tumor disease treatment.
33. The method of claim 32, wherein the subject suffers from a neuroendocrine neoplasm and/or metastases thereof, in particular liver metastases.
34. The method of claim 32 or 33, wherein the neoplasm is a neuroendocrine neoplasm in the gastro-pancreatic, bronchopulmonary tract, thyroid, thymus or pituitary gland.
35. The method of claim 32 or 33, wherein the neuroendocrine neoplasm is selected from the group consisting of gastroenteropancreatic neuroendocrine neoplasm, neuroendocrine tumor of the lung, neuroendocrine carcinoma of the lung, in particular small cell lung cancer, thymic neuroendocrine tumor, paraganglioma, pheochromocytoma, e.g. malignant pheochromocytoma, meningioma, medullary thyroid cancer, thyroid cancer, breast cancer, renal cell carcinoma, prostate cancer, and non-Hodgkin lymphoma.
36. The method of claim 35, wherein the neuroendocrine neoplasm is a pancreatic tumor.
37. The method of any of claims 32 to 36, wherein neuroendocrine neoplasm is of Grade 1, Grade 2 or Grade 3.
38. The method of any of claims 32 to 37, wherein the neuroendocrine neoplasm is stable or refractory to a therapy by Lutetium (.sup.177Lu)-Oxodotreotid (Lutathera?) or other radiolabelled somatostatin analogues.
39. The method of any of claims 32 to 38, wherein the radiopeptide of any of claims 1 to 25 or the composition of any of claims 26 to 31 is administered systemically, preferably intravenously.
Description
FIGURE LEGENDS
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[0072] Appendix The two somatostatin receptor (SSTR)-targeting peptides DOTATOC (SSTR agonist) and DOTA-LM3 (SSTR antagonist) were labeled with either .sup.161Tb or .sup.177Lu and evaluated in preclinical experiments. Their behaviour was studied in preclinical studies.
EXAMPLES
[0073] The Examples of the present disclosure show the impact of the cellular localization on the therapeutic effect of terbium-161 vs. lutetium-177 in vitro and in vivo. DOTATOC and DOTA-LM3 were used. They were labeled with .sup.161Tb-labeled and .sup.177Lu, respectively. The labelled .sup.161Tb radiopeptides were compared with their .sup.177Lu-labeled counterparts, in order to study whether the effects of conversion and Auger electrons emitted by terbium-161 may depend on the radionuclide's cellular localization. A one-to-one comparison of the therapeutic effect of terbium-161 and lutetium-177 was feasible due to equal behavior of the .sup.161Tb- and .sup.177Lu-counterparts with regards to the in vitro cell uptake and in vivo biodistribution [44].
[0074] The cellular localization of the radiopeptide was found to have an impact for its therapeutic effect, which in turn, was dependent on the employed radionuclide.
[0075] The difference in efficacy between the .sup.161Tb- and .sup.177Lu-labeled peptides was evidenced. It was most pronounced in the case of DOTA-LM3. [.sup.161Tb]Tb-DOTA-LM3 revealed to be 102-fold more effective to reduce cell viability in vitro than [.sup.177Lu]Lu-DOTA-LM3.
[0076] The preclinical therapy study confirmed that the .sup.161Tb-labeled somatostatin analogues were more effective than their .sup.177Lu-labeled counterparts in vivo. The therapeutic effect of radiolabeled DOTA-LM3 was significantly more pronounced than the effect of radiolabeled DOTATOC, which can be ascribed to the higher tumor uptake of radiolabeled DOTA-LM3 as demonstrated by the biodistribution data. It was found that .sup.161Tb-labeled somatostatin receptor antagonist (DOTA-LM3) that localize on the cell membrane benefit from the use of terbium-161 over lutetium-177, suggesting the cell membrane as a valid target for terbium-161 as an Auger electron emitter.
I. Production
[0077] Terbium-161 and lutetium-177 were used for comparing the effects based on the cellular localization of the targeting agent. In this regard two different peptides were used: (i) DOTATOC, which localizes primarily in the cell cytosol and (ii) DOTA-LM3, which stays mainly at the cell membrane.
A. Production Methods of Radionuclides and Peptides
[0078] Terbium-161 was produced using the .sup.160Gd(n,?).sup.161Gd-+.sup.161Tb nuclear reaction as previously reported [1, 2 ]. The target material was irradiated at the SAFARI-1 reactor at Necsa in Pelindaba, South Africa or at the RHF at Institut Laue-Langevin in Grenoble, France or at the spallation-induced neutron source SINQ, Villigen-PSI, Switzerland. The chemical separation of terbium-161 was performed at PSI as previously reported [2 ]. Terbium-161 was made available as no-carrier-added (n.c.a.) [.sup.161Tb]TbCl.sub.3 in 0.05 M HCl. Lutetium-177 was obtained as n.c.a. [.sup.177Lu] LuCl.sub.3 in 0.04 M HCl from ITM Medical Isotopes GmbH, Germany.
[0079] DOTA-[Tyr.sup.3]-octreotide (DOTATOC) was provided by ITM GmbH, Germany (
[0080] Terbium-161 was produced in high yields (average of >10 GBq up to 15 GBq/production at end of separation), at an activity concentration of 11-21 MBq/?L and ?99% radionuclidic and radiochemical purity [43 ]. The radiolabeling of the peptides of up to 100 MBq/nmol was possible up to two weeks after chemical separation, enabling planning and performing the preclinical studies. The exact product specifications of terbium-161 were comparable to that of n.c.a. lutetium-177 as reported by Gracheva et al. [43].
B. Preparation of the Radiopeptides
[0081] [.sup.161Tb]Tb-DOTATOC and [.sup.161Tb]Tb-DOTA-LM3 as well as [.sup.177Lu] Lu-DOTATOC and [.sup.177Lu] Lu-DOTA-LM3 were prepared at high molar activity for the in vitro and in vivo evaluation and comparison of the radiopeptides.
[0082] The stock solutions of DOTATOC and DOTA-LM3 were prepared in Milli-Q water to obtain a final concentration of 1 mM. The somatostatin analogues were labeled with terbium-161 or lutetium-177 using a 1:5 (v/v) mixture of sodium acetate (0.5 M) and HCl (0.05 M) at pH ?4.5 at a molar activity up to 100 MBq/nmol, as previously reported [5 ]. The reaction mixture was incubated for 10 min at 95? C., followed by a quality control using HPLC. For this purpose, a Merck Hitachi LaChrom HPLC system, equipped with a D-7000 interface, a L-7200 autosampler, a radioactivity detector (LB 506 B; Berthold) and a L-7100 pump connected with a reversed-phase C18 column (Xterra? MS, C18, 5 ?m, 150?4.6 mm; Waters) was used. The mobile phase consisted of 0.1% (v/v) TFA in Milli-Q water (A) and acetonitrile (B). A linear gradient of solution A (95-20%) and solvent B (5-80%) over 15 min was used at a flow rate of 1.0 mL/min. The radiopeptides were diluted in Milli-Q water containing pentasodium diethylenetriaminepentaacetate (Na.sub.5-DTPA; 50 ?M) prior to injection into HPLC.
[0083] [.sup.161Tb]Tb-DOTATOC and [.sup.177Lu]Lu-DOTATOC as well as [.sup.161Tb]Tb-DOTA-LM3 and [.sup.177Lu]Lu-DOTA-LM3 were obtained at radiochemical purity of ?98% up to a molar activity of 100 MBq/nmol (see
C. Radiolytic Stability of the Radiopeptides
[0084] The stability of the radiolabeled somatostatin analogues was tested to confirm their integrity during in vitro and in vivo evaluation.
[0085] Radiolytic stability of the radiolabeled somatostatin analogues was assessed over a period of 24 h (n=2). For this purpose, DOTATOC and DOTA-LM3 were labeled with terbium-161 or lutetium-177 at a molar activity of 50 MBq/nmol. After quality control using HPLC (t=0, radiochemical purity>98%, set as 100%), the labeling solutions were diluted with saline to a final volume of 250 ?l and an activity concentration of 40 MBq/mL and incubated at room temperature (RT) with and without addition of L-ascorbic acid (120 ?g/10 MBq in the final volume of 250 ?L). Potential degradation of the radiopeptides was determined after 1 h, 4 h and 24 h by analyzing a sample using HPLC. A quantitative assessment of the chromatograms was performed by expressing the integrated peak area of the intact product as percentage of the sum of integrated peak areas of the entire chromatogram comprising released terbium-161 and lutetium-177 as well as degradation products of unknown structure.
[0086] All radiopeptides, irrespective of whether labeled with terbium-161 or lutetium-177, were stable (>90% of intact radiopeptide) up to 1 h after preparation (see
[0087] If the radiopeptides were used at concentrations below 40 MBq/mL and/or immediately after preparation, the addition of L-ascorbic acid was not necessary. Dual-isotope SPECT/CT imaging studies were performed with activity concentrations above 40 MBq/mL (>4 MBq in 100 ?l per mouse), which required the addition of L-ascorbic acid (?300 ?g per 30 MBq in 100 ?L) to ensure integrity of the radiopeptides.
D. Determination of n-Octanol/PBS Distribution Coefficients (LogD Values)
[0088] The n-octanol/PBS distribution coefficients (logD values) were assessed for each radiopeptide in order to investigate their hydrophilicity and enable comparison of the three analogues.
[0089] The logD values of the radiolabeled analogues (DOTATOC and DOTA-LM3; 30 MBq/nmol) were determined by a shake-flask method as previously reported [47 ]. An aliquot of the radiolabeled peptide (0.5 MBq, 25 ?L) was added to a mixture of 1475 ?L PBS pH 7.4 and 1500 ?L n-octanol. The respective tubes were vortexed for 60 sec and centrifuged for 6 min at 2500 rpm followed by the measurement of the activity concentration in a defined volume of each layer using a ?-counter (Perkin Elmer, Wallac Wizard 1480). The logD values were calculated as the logarithm of the ratio of counts per minute (cpm) measured in the n-octanol phase relative to the cpm measured in the PBS pH 7.4 phase. The experiments were performed three times with five replicates for each radiopeptide and the logD value was expressed as the average?standard deviation (SD) of the values obtained in each experiment. The data were analyzed for significance using a two-way ANOVA with Tukey's multiple comparisons test. A p value<0.05 was considered statistically significant.
[0090] The radiopeptides showed a hydrophilic character as demonstrated by the low logD values. Importantly, the .sup.161Tb- and .sup.177Lu-labeled counterparts revealed similar logD values (p>0.05)(Table 1).
TABLE-US-00002 TABLE 1 LogD values of the radiopeptids presented as the average of 3 independent experiments performed in quintuplicate Radiopeptide LogD Radiopeptide LogD Significance [.sup.161Tb]Tb-DOTATOC ?3.3 ? 0.2 [.sup.177Lu] Lu-DOTATOC ?3.1 ? 0.2 p > 0.05 [.sup.161Tb]Tb-DOTA-LM3 2.5 ? 0.1 [.sup.177Lu] Lu-DOTA-LM3 ?2.5 ? 0.1 p > 0.05
II. In Vitro Studies
A. Cell Culture
[0091] AR42 J tumor cells, a SSTR-positive exocrine rat pancreatic cancer cell line [48 ], was used for the in vitro and in vivo investigations in this study.
[0092] AR42 J tumor cells (ECACC 93100618; Health Protection Agency Culture Collections, Salisbury, U.K.) were kept in RPMI 1640 culture medium supplemented with glutamine, antibiotics and 20% fetal calf serum as previously reported [44 ]. Cell culture medium containing glutamine and antibiotics but only 1% FCS (referred herein as assay medium) was used for all in vitro assays. Incubation of cells always referred to standard culture conditions of a humidified atmosphere at 37? C. and 5% CO.sub.2 if not otherwise indicated. Phosphate buffered saline (PBS) at the pH of 7.2 was used for the in vitro experiments if not otherwise indicated. Polystyrene well plates were coated with poly-L-lysine (0.5 mg/ml) for all in vitro experiments to facilitate cell adhesion and prevent adherence of the radiopeptides to the well-plate material.
B. Cell Uptake and Internalization: Comparison of .sup.161Tb- and .sup.177Lu-Labeled Peptides
[0093] Cell uptake and internalization studies were performed with the .sup.161Tb- and .sup.177Lu-labeled counterparts of each radiopeptide.
[0094] The cell uptake and internalization studies were performed in AR42 J tumor cells according to a previously published procedure [5 ]. AR42 J tumor cells (10.sup.6 cells/2 mL) were seeded in poly-L-lysine-coated 12-well-plates in RPMI medium with supplements and incubated overnight. After washing the cells with PBS (pH 7.4), assay medium (975 ?L) and 25 ?L radiopeptide solution (?15 kBq, ?0.75 pmol per well) were added to each well resulting in a radioligand concentration of 0.75 nM. The well-plates were incubated under standard cell culture conditions for 0.5 h, 2 h or 4 h. SSTR-blocking experiments were performed using 1 ?M DOTANOC or 1 UM DOTA-LM3. The total uptake and the internalized fractions were determined using a ?-counter (Perkin Elmer, Wallac Wizard 1480). The activity of the samples was standardized to the average protein concentration in each well (?0.3 mg) using a Micro BCA Protein Assay kit (Pierce, Thermo Scientific). Experiments were performed twice or three times in triplicate. Statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparisons post-test. A p value<0.05 was considered statistically significant.
[0095] The AR42 J tumor cell uptake and internalization were equal for the respective .sup.161Tb- and .sup.177Lu-labeled SSTR analogues (p>0.05) (see
C. Cell Uptake and Internalization at Increasing Molar Amounts of Peptide
[0096] The aim of this in vitro studies was to determine the peptide amount which resulted in SSTR saturation in AR42 J tumor cells after addition of the radiopeptides using variable amounts of DOTATOC and DOTA-LM3.
[0097] The uptake and internalized fraction of the peptides in AR42 J tumor cells was determined using the same method described above, but the radiopeptides were applied using variable molar amounts of peptides. After washing the AR42 J tumor cells with PBS (pH 7.4), they were incubated with 0.375-75 pmol of [.sup.177Lu] Lu-DOTATOC or [.sup.177Lu]Lu-DOTA-LM3 (25 ?l, ?15 kBq). The cell uptake and internalized fraction were determined after an incubation period of 2 hours at 37? C. and the activity of the samples standardized to the average protein concentration in each well (?0.3 mg). Experiments were performed twice in triplicate.
[0098] In all instances, the uptake and internalization of the respective radiopeptide decreased while increasing the molar amount of non-labeled peptide (see
D. Nuclear Localization of the Radiopeptides
[0099] AR42 J tumor cells were incubated with the respective radiopeptide followed by isolation of the cellular nucleus in order to determine the fraction of radiopeptides that localized in the nucleus.
[0100] AR42 J tumor cells (10?10.sup.6) were seeded in PLL-coated Petri dishes using 15 mL of cell culture medium with supplements and incubated overnight at 37? C. and 5% CO.sub.2. The next day, the medium was removed, the tumor cells were washed with PBS (pH 7.4) and 19.5 ml of assay medium was added. The radiopeptides were added in a volume of 0.5 ml (2.5 MBq, 125 pmol) and the tumor cells incubated for 2 h at 37? C. Afterwards, the cells were washed several times with PBS (pH 7.4) to completely remove excess of radiopeptides. Subsequently, the cell nuclei and cytoplasm/membrane fractions were harvested according to the manufacturer's protocol using the Nucli EZ Prep Nuclei Isolation Kit (Sigma Aldrich, USA). Ice-cold Nuclei EZ lysis buffer (4 mL) were added to each Petri dish to lyse the tumor cells followed by transfer of the cell suspension into Eppendorf tube for centrifugation for 5 min at 500 rcf at 4? C. The supernatant containing cytoplasm/membrane fractions was transferred to a tube for counting activity in a ?-counter. The pellet was resuspended in 4 ml ice-cold Nuclei EZ lysis buffer followed by additional centrifugation for 5 min at 500 rcf at 4? C. The supernatant containing residual cytoplasm/membrane fractions was collected for counting activity and the pellet was suspended in 200 ?L Nuclei EZ storage buffer before transferring into a tube for activity measurement in the ?-counter. The measured activity of nuclei and cytoplasm/cell membrane fractions was defined as 100% of the cellular uptake. Nuclear localization was expressed as percentage of total cellular uptake. The collected fractions were stained with 0.4% trypan blue solution and analyzed using a microscope in order to confirm that the nuclei were properly separated from other cell fragments.
[0101] The nuclear uptake of [.sup.177Lu]Lu-DOTATOC and [.sup.177Lu]Lu-DOTA-LM3 was <1% and ?2% of total cellular uptake. Combining these results with the uptake and internalization data presented (see
E. Cell Viability Assay
[0102] Cell viability studies were performed using AR42 J tumor cells to assess potentially different effects of .sup.161Tb- and .sup.177Lu-labeled DOTATOC and DOTA-LM3.
[0103] A total of 7500 AR42 J tumor cells were seeded in 200 ?L cell culture medium with supplements in PLL-coated 96-well plates. After incubation overnight to allow cell adhesion, the medium was removed and the cells were incubated with DOTATOC or DOTA-LM3 diluted in assay medium and radiolabeled with either terbium-161 or lutetium-177 at a molar activity of 100 MBq/nmol. The applied activity concentrations per well ranged from 0.001 MBq/mL to 40 MBq/mL (0.01-400 pmol/mL). After an incubation period of 2 h at 37? C., the cells were washed once with PBS followed by addition of fresh cell culture medium with supplements. The tumor cells were allowed to grow for 6 d at 37? C. without changing cell culture medium and the cell viability analyzed as previously described using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [51 ]. After incubation of the cells with the MTT reagent for 2 h, the formed formazan crystals were dissolved in dimethyl sulfoxide. The absorption was measured at 560 nm with a microplate reader (560 nm, Victor? X3, Perkin Elmer, Waltham, MA, U.S.A.). The absorbance measured for untreated control cells was defined as 100% tumor cell viability. The viability of treated tumor cells (n=12 per concentration) was expressed as percentage of the absorbance of control cells. The cell viability was plotted against the concentration of applied activity (transformed in logarithmic scale) and fitted with a dose-response curve. The cell viability inhibition was calculated as the activity concentration which was necessary to reduce AR42 J tumor cell viability to 50% of untreated control cells (EC.sub.50). EC.sub.50 values were determined in at least four independent experiments.
[0104] Reduction of AR42 J cell viability to less than 10% as compared to the viability of untreated control cells (set as 100%) was achieved at variable activity concentrations dependent on the employed somatostatin analogue and radionuclide, respectively. It was observed that in all cases the .sup.161Tb-labeled somatostatin analogue was more potent than the .sup.177Lu-labeled counterpart (see
[0105] Comparison of the EC.sub.50 of each .sup.161Tb-labeled somatostatin analogue revealed a 157-fold higher potency for [.sup.161Tb]Tb-DOTA-LM3 than for [.sup.161Tb]Tb-DOTATOC (Table 2). The situation was different for [.sup.177Lu]Lu-DOTA-LM3 which was 7.7-fold more potent than [.sup.177Lu]Lu-DOTATOC (Table 2).
TABLE-US-00003 TABLE 2 Results of cell viability experiments expressed as half- maximum inhibitory concentration (EC.sub.50 values). EC.sub.50 [MBq/mL] Potency relative (95% Confidence to radiolabeled Radiopeptide Interval) DOTATOC [.sup.161Tb]Tb-DOTATOC 1.6 1.0 (1.4.1.9) [.sup.161Tb]Tb-DOTA-LM3 0.010 157 (0.008-0.014) [.sup.177Lu]Lu-DOTATOC 8.2 1.0 (6.4-10) [.sup.177Lu]Lu-DOTA-LM3 1.1 7.7 (0.8-1.5)
F. Cell Survival (Clonogenic Assay)
[0106] Capability of a single AR42 J tumor cell to grow into a colony upon exposure to .sup.161Tb- and .sup.177Lu-radiolabeled DOTATOC or DOTA-LM3 was determined by performing clonogenic assays [8].
[0107] In order to allow proper formation of colonies, 300 ?L Matrigel (Growth Factor Reduced Basement Membrane Matrix, Corning Inc., New York, U.S.A; 2 mg/mL) diluted in RPMI cell medium without additives were added to each well of PPL-coated 6-well plates. AR42 J tumor cells were seeded on the solidified Matrigel at a density of 2000 cells per well in 2 ml cell culture medium with supplements and incubated overnight. The next day, the medium was removed and the cells were incubated with .sup.161Tb- and .sup.177Lu-radiolabeled somatostatin analogues (30 MBq/nmol) at activity concentrations of 0.01 MBq/ml to 0.5 MBq/mL (0.3-15 pmol/mL) for 2 h at 37? C. and 5% CO.sub.2. The same procedure was applied to untreated control cells. After incubation, the supernatant was discarded and the tumor cells were washed with PBS before fresh cell culture medium was added. After two weeks incubation time at 37? C. and 5% CO.sub.2, the culture medium was removed and the wells were washed once with PBS. The colonies were stained using crystal violet solution (0.5% crystal violet, 6% glutaraldehyde in water, 800 ?L). The number of colonies (>0.1 mm) was determined visually using a grid of 0.5 cm?0.5 cm in five selected squares under the microscope. The plating efficiency (PE) and survived fraction (SF) were calculated according to the following formulas: PE=([number of colonies formed (untreated)]/[number of cells seeded])*100; SF=([number of colonies formed after treatment]/[number of cells seeded*PE])*100 [52].
[0108] The SF upon exposure to various radioactivity concentrations of the radioligands was determined in at least three independent experiments using triplicates in each experiment. The data were analyzed with a two-way ANOVA with Sidak's multiple comparison post-tests. A p value<0.05 was considered statistically significant.
[0109] Colony forming assays confirmed that the .sup.161Tb-labeled peptides were more effective to reduce cell survival than the respective .sup.177Lu-labeled counterparts (see
G. Evaluation of DNA Damage
[0110] Experiments were performed with AR42 J tumor cells treated with [.sup.161Tb]Tb-DOTATOC and [.sup.177Lu] Lu-DOTATOC or [.sup.161Tb]Tb DOTA-LM3 and [.sup.177Lu]Lu-DOTA-LM3 to investigate the number of induced double strand breaks (DSBs) in each case.
[0111] The number of DNA DSBs was assessed by immunostaining of ?-H2 AX in AR42 J cells treated with either 2.5 MBq/mL or 10 MBq/ml of each radiopeptide. The cells seeded (5?10.sup.6 cells/Petri dish) and let grown overnight. The following day, the medium was removed and the cells were treated with the radiopeptides diluted in assay medium for 2 h. The supernatant was removed and the AR42 J tumor cells washed with PBS prior to the addition of fresh culture medium. After 24 h of incubation, cells were washed with PBS and detached by scraping using 1.5 mL PBS, and centrifuged in 1.5-mL Eppendorf tubes. The cell pellets were fixed by addition of 1 mL 4% neutral buffered formalin for 24 h at RT followed by exchanging it with PBS. After paraffin embedding, sections of 4 ?m thickness were prepared. In brief, antigen retrieval was performed after deparaffination using a solution containing EDTA (pH 9) at 98? C. for 20 min and followed by incubation with REAL Antibody Diluent (Agilent) for 30 min at RT and hydrogen peroxide (Agilent) for 10 min at RT. The sections were incubated with the primary antibody (Cell signaling rabbit monoclonal antibody (Ser139); dilution 1:200) for 1 h at RT. Envision horseradish peroxidase rabbit (Agilent Technologies, Inc) detection system was used with DAB substrate buffer (Agilent). Immunostained sections were scanned using a digital slide scanner (NanoZoomer-XR C12000; Hamamatsu, Japan) and the total of positive and negative cells quantified with the pathology image analysis software VIS (Visiopharm Integrator System, Version 208 2019.02.2.6239, Visiopharm, Hoersholm, Denmark). First, the decision forest classification method was used to outline the tissue cell pellets as regions of interest (ROIs). Subsequently, the cell classification method was used for the detection of cell nuclei within each ROI and classify them as positive (brown) and negative (blue). Separation of the nucleus type was done by training the software with the predetermined options Standard positive nuclei and Standard negative nuclei. The results were expressed as total positive cells and total negative cells. Data were analyzed with a one-way ANOVA with Dunnet's multiple comparison post-tests. A p value<0.05 was considered statistically significant.
[0112] At high activity concentration (10 MBq/mL) an increased number of ?-H2 AX-positive foci was determined in all .sup.161Tb-treated samples as compared to controls, but without reaching a significance level (p<0.05). .sup.177Lu-labeled somatostatin analogues led to only modest increase of ?-H2 AX-positive foci, which was only obvious at high activity concentrations (p>0.05). The results are presented in
III. In Vivo Studies
[0113] In vivo studies were performed to evaluate the tissue distribution of the radiopeptides and optimize the molar amount of injected peptide. Moreover, the therapeutic effect of the radiopeptides and early side effects were assessed.
[0114] All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. In particular, all animal experiments were carried out according to the guidelines of Swiss Regulations for Animal Welfare. The preclinical studies were ethically approved by the Cantonal Committee of Animal Experimentation and permitted by the responsible cantonal authorities (license N? 75721 and 79692). Five-week-old female, athymic nude mice (CD-1 Foxn-1/nu) were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice were subcutaneously inoculated with AR42 J tumor cells (5?10.sup.6 cells in 100 ?l PBS) for SPECT/CT imaging, biodistribution and therapy studies. The SPECT/CT and biodistribution studies were performed 10-14 days after tumor cell inoculation when the tumor size reached a volume of ?250 mm.sup.3.
A. Dual-Isotope SPECT/CT Imaging Studies
[0115] The purpose was to demonstrate that the .sup.161Tb- and .sup.177Lu-labeled somatostatin counterparts have the same in vivo distribution and that the tumor uptake is SSTR-specific. A second objective was to compare the tissue distribution of the three somatostatin analogues.
[0116] Dual-isotope SPECT/CT scans were performed with a dedicated small-animal SPECT/CT scanner (NanoSPECT/CT, Mediso Medical Imaging Systems, Budapest, Hungary; Supplementary Material) as previously reported [44 ]. The scans were acquired using Nucline software (version 1.02, Mediso Ltd., Budapest, Hungary). Simultaneous acquisition of counts stemming from terbium-161 and lutetium-177, respectively, was performed by the selection of distinct energy windows for the two radionuclides. The two energy windows chosen for terbium-161 were set at 47.7 keV?10%, which enabled the detection of X-rays and ?-rays (46.0 keV, 48.9 keV and 52.0 keV), and at 74.6 keV?10%, enabling the detection of the ?-rays at 74.6 keV. For lutetium-177, the windows were set at 208.4?10% and 112.9 keV?10% to detect the ?-rays at 208.4 keV and 112.9 keV, respectively. SPECT data were reconstructed iteratively using HiS-PECT software (version 1.4.3049, Scivis GmbH, Gottingen, Germany). The CT was reconstructed in real time using a cone-beam filtered backprojection. The fused datasets of SPECT and CT scans were analyzed using the VivoQuant postprocessing software (version 3.5, inviCRO Imaging Services and Software, Boston, USA). A Gauss post-reconstruction filter (FWHM=1.0 mm) was applied.
[0117] Mice were i.v. injected with a mixture of [.sup.161Tb]Tb-DOTATOC (?15 MBq, 0.5 nmol/mouse) and [.sup.177Lu]Lu-DOTATOC (?15 MBq, 0.5 nmol/mouse) or a mixture of [.sup.161Tb]Tb-DOTA-LM3 and [.sup.177Lu]Lu-DOTA-LM3 in PBS (pH 7.4) containing 0.05% BSA and ascorbic acid (?300 ?g/30 MBq). Blocking studies were performed under the same experimental conditions, however, in this case, an excess (20 nmol/mouse) of unlabeled DOTATOC or DOTA-LM3 was added to the injection solution. SPECT/CT scans were acquired 2 h, 4 h and 24 h after injection of the radiopeptides using the dual-isotope SPECT acquisition protocol with a frame time of 60 sec resulting in a scan time of 45 min. During the in vivo scans, mice were anesthetized by inhalation of a mixture of isoflurane and oxygen.
[0118] The SPECT/CT images of AR42 J tumor-bearing mice demonstrated equal in vivo distribution of simultaneously injected [.sup.161Tb]Tb-DOTATOC and [.sup.177Lu] Lu-DOTATOC (see
[0119] The SPECT/CT images showed activity accumulation in the AR42 J xenografts, which was higher for the [.sup.161Tb]Tb-DOTA-LM3 and [.sup.177Lu] Lu-DOTA-LM3 than for the [.sup.161Tb]Tb-DOTATOC and [.sup.177Lu] Lu-DOTATOC at all investigated time points. The activity was efficiently cleared through the kidneys over time and almost entirely excreted after 24 h. Due to the favorable uptake of radiolabeled DOTA-LM3 in the tumor tissue, the tumor-to-kidney ratio was higher as compared to the ratio obtained after injection of radiolabelled DOTATOC [44].
[0120] Experiments performed by co-injection of excess of the respective unlabeled peptide resulted in blockade of radiopeptide accumulation in AR42 J tumors, proving that the uptake was SSTR-specific are shown in
B. Biodistribution Studies: Comparison of .sup.161Tb- and .sup.177Lu-Labeled Peptides
[0121] Biodistribution studies were performed to assess quantitatively the tissue distribution of .sup.161Tb-labeled peptides and compare them with the .sup.177Lu-labeled counterparts.
[0122] Biodistribution studies were performed after intravenous injection of the mice with [.sup.161Tb]Tb-DOTATOC or [.sup.177Lu]Lu-DOTATOC (5 MBq; 1 nmol in 100 ?L per mouse) diluted in PBS (pH 7.4) containing 0.05% BSA. [.sup.161Tb]Tb-DOTA-LM3 or [.sup.177Lu]Lu-DOTA-LM3 were used under the same conditions. The mice were sacrificed at 2 h and 24 h post injection (p.i.). Selected tissues and organs were collected, weighed, and the accumulated activity was counted in a ?-counter. The decay-corrected data were listed as the percentage of the injected activity per gram of tissue mass (% IA/g). The data were analyzed for significance using a two-way ANOVA with Tukey's multiple comparison post-tests. A p-value of <0.05 was considered statistically significant.
[0123] The biodistribution of .sup.161Tb-labeled DOTATOC was comparable to that of .sup.177Lu-labeled DOTATOC, demonstrated by similar activity accumulation in tissues and organs at both investigated at 2 h and 24 h, respectively (p>0.05). Equal tissue distribution of .sup.161Tb- and .sup.177Lu-labeled counterparts was also observed when using DOTA-LM3 (see
TABLE-US-00004 TABLE A1 Biodistribution data obtained in AR42J tumor-bearing mice at 2 h and 24 h after injection of [.sup.161Tb]Tb-DOTATOC and [.sup.177Lu]Lu-DOTATOC, respectively. Decay-corrected data are shown as [% IA/g] values, representing the average ? SD (n = 3-4). [Tb]Tb-DOTATOC [Lu]Lu-DOTATOC 2 h p.i. 24 h p.i. 2 h p.i. 24 h p.i. Blood ?0.10 ?0.10 ?0.10 ?0.10 Heart ?0.10 ?0.10 ?0.10 ?0.10 Lung 0.27 ? 0.03 0.11 ? 0.01 0.37 ? 0.06 0.12 ? 0.03 Spleen 0.11 ? 0.02 ?0.10 0.15 ? 0.03 ?0.10 Kidneys 9.9 ? 1.3 5.2 ? 0.6 11 ? 1 5.9 ? 2.1 Adrenals 0.25 ? 0.06 0.23 ? 0.20 0.28 ? 0.08 0.18 ? 0.08 Stomach 0.74 ? 0.38 0.29 ? 0.11 0.65 ? 0.11 0.24 ? 0.04 Pancreas 0.71 ? 0.07 0.18 ? 0.01 0.80 ? 0.13 0.21 ? 0.04 Intestines 0.21 ? 0.13 ?0.10 0.20 ? 0.05 ?0.10 Liver 0.17 ? 0.01 ?0.10 0.26 ? 0.07 0.13 ? 0.02 Muscle ?0.10 ?0.10 ?0.10 ?0.10 Femur ?0.10 ?0.10 ?0.10 ?0.10 AR42J Tumor 8.2 ? 0.2 3.8 ? 0.6 8.9 ? 1.5 3.8 ? 0.3 Tumor-to- 260 ? 13 433 ? 71 146 ? 16 456 ? 118 blood Tumor-to- 49 ? 1 41 ? 7 36 ? 7 31 ? 6 liver Tumor-to- 0.84 ? 0.12 0.73 ? 0.10 0.73 ? 0.07 0.72 ? 0.34 kidney
TABLE-US-00005 TABLE A2 Biodistribution data obtained in AR42J tumor-bearing mice at 2 h and 24 h after injection of [.sup.161Tb]Tb-DOTA-LM3 and [.sup.177Lu]Lu-DOTA-LM3, respectively. Decay-corrected data are shown as [% IA/g] values, representing the average ? SD (n = 3-4). [Tb]Tb-DOTA-LM3 [Lu]Lu-DOTA-LM3 2 h p.i. 24 h p.i. 2 h p.i. 24 h p.i. Blood ?0.10 ?0.10 ?0.10 ?0.10 Heart 0.11 ? 0.02 ?0.10 ?0.10 ?0.10 Lung 1.0 ? 0.2 0.36 ? 0.06 0.83 ? 0.14 0.28 ? 0.02 Spleen 0.27 ? 0.06 0.13 ? 0.03 0.23 ? 0.06 ?0.10 Kidneys 12 ? 2 5.8 ? 0.1 11 ? 1 5.4 ? 1.3 Adrenals 0.44 ? 0.06 0.18 ? 0.05 0.37 ? 0.10 0.17 ? 0.04 Stomach 2.2 ? 0.3 0.69 ? 0.14 1.7 ? 0.4 0.86 ? 0.13 Pancreas 4.2 ? 0.3 1.9 ? 0.1 3.4 ? 0.3 1.5 ? 0.1 Intestines 0.43 ? 0.23 0.11 ? 0.01 0.31 ? 0.14 0.14 ? 0.03 Liver 0.45 ? 0.05 0.22 ? 0.05 0.35 ? 0.08 0.15 ? 0.03 Muscle ?0.10 ?0.10 ?0.10 ?0.10 Femur 0.20 ? 0.05 0.11 ? 0.02 0.17 ? 0.04 ?0.10 AR42J Tumor 18 ? 2 14 ? 2 17 ? 2 14 ? 2 Tumor-to- 200 ? 38 >1000 270 ? 92 >1000 blood Tumor-to- 38 ? 7 63 ? 18 51 ? 7 95 ? 16 liver Tumor-to- 1.4 ? 0.2 2.4 ? 0.5 1.9 ? 0.2 2.9 ? 0.9 kidney
C. Biodistribution Studies: Assessment of the Optimum Injected Molar Amount
[0124] The aim was to assess the impact of the injected amount of peptide on the biodistribution of .sup.161Tb- and .sup.177Lu-labeled DOTATOC and DOTA-LM3.
[0125] The mice (n=3 per group) were intravenously injected with the radiolabeled DOTATOC or DOTA-LM3 (3 MBq, 0.04 nmol/mouse; 5 MBq, 0.2 nmol/mouse; 5 MBq, 1.0 nmol/mouse) in 100 ?L PBS (pH 7.4) containing 0.05% BSA. The mice were sacrificed at 2 h p.i, selected tissues and organs were collected, weighed, and the accumulated activity was counted using a ?-counter (Perkin Elmer). The decay-corrected data were listed as % IA/g. The data were analyzed for significance using a two-way ANOVA with Tukey's multiple comparison post-tests. A p-value of <0.05 was considered statistically significant.
[0126] In agreement with previous studies [53 ], the amount of injected peptide had a significant impact on the accumulated activity in different organs and tissues (see
TABLE-US-00006 TABLE A3 Biodistribution data obtained in AR42J tumor-bearing mice at 2 h after injection of [.sup.161Tb]Tb-DOTATOC or [.sup.177Lu]Lu-DOTATOC. Decay-corrected data are shown as % IA/g tissue, representing the average ? SD. [Tb]Tb-DOTATOC [Lu]Lu-DOTATOC 2 h p.i. 2 h p.i. 0.040 nmol 0.20 nmol 1.0 nmol* 0.040 nmol 0.20 nmol 1.0 nmol* Blood ?0.10 ?0.10 ?0.10 ?0.10 ?0.10 ?0.10 Heart ?0.10 ?0.10 ?0.10 ?0.10 ?0.10 ?0.10 Lung 1.2 ? 0.1 0.47 ? 0.05 0.27 ? 0.03 1.1 ? 0.2 0.63 ? 0.03 0.37 ? 0.06 Spleen 0.50 ? 0.11 0.18 ? 0.02 0.11 ? 0.02 0.39 ? 0.11 0.15 ? 0.02 0.15 ? 0.03 Kidneys 9.0 ? 2.0 9.4 ? 0.4 9.9 ? 1.3 10 ? 2 10 ? 3 11 ? 1 Adrenals 0.65 ? 0.08 0.43 ? 0.11 0.25 ? 0.06 0.65 ? 0.10 0.51 ? 0.35 0.28 ? 0.08 Stomach 2.9 ? 1.1 1.2 ? 0.3 0.74 ? 0.38 1.7 ? 0.3 1.1 ? 0.2 0.65 ? 0.11 Pancreas 2.1 ? 0.5 1.5 ? 0.2 0.71 ? 0.07 1.9 ? 0.2 1.7 ? 0.5 0.80 ? 0.13 Intestines 0.52 ? 0.15 0.24 ? 0.01 0.21 ? 0.13 0.33 ? 0.05 0.40 ? 0.25 0.20 ? 0.05 Liver 0.22 ? 0.02 0.19 ? 0.03 0.17 ? 0.01 0.18 ? 0.02 0.15 ? 0.03 0.26 ? 0.07 Muscle ?0.10 ?0.10 ?0.10 ?0.10 ?0.10 ?0.10 Femur 0.23 ? 0.03 0.12 ? 0.03 ?0.10 0.17 ? 0.05 0.10 ? 0.01 ?0.10 AR42J Tumor 16 ? 4 14 ? 1 8.2 ? 0.2 18 ? 3 17 ? 4 8.9 ? 1.5 Tu-to-blood 187 ? 12 221 ? 56 260 ? 13 213 ? 41 235 ? 60 146 ? 16 Tu-to-liver 73 ? 3 76 ? 14 49 ? 1 100 ? 8 118 ? 24 36 ? 7 Tu-to-kidney 1.8 ? 0.2 1.5 ? 0.1 0.84 ? 0.12 1.9 ? 0.3 1.9 ? 0.4 0.73 ? 0.07
TABLE-US-00007 TABLE A4 Biodistribution data obtained in AR42J tumor-bearing mice at 2 h after injection of [.sup.161Tb]Tb-DOTA-LM3 or [.sup.177Lu]Lu-DOTA-LM3. Decay-corrected data are shown as % IA/g tissue, representing the average ? SD. [Tb]Tb-DOTA-LM3 [Lu]Lu-DOTA-LM3 2 h p.i. 2 h p.i. 0.040 nmol 0.20 nmol 1.0 nmol* 0.040 nmol 0.20 nmol 1.0 nmol* Blood 0.12 ? 0.02 ?0.10 ?0.10 0.11 ? 0.01 ?0.10 ?0.10 Heart 0.34 ? 0.06 0.13 ? 0.01 0.11 ? 0.02 0.24 ? 0.04 0.14 ? 0.04 ?0.10 Lung 7.8 ? 0.7 2.9 ? 0.7 1.0 ? 0.2 8.2 ? 0.7 2.6 ? 0.4 0.83 ? 0.14 Spleen 1.8 ? 1.0 0.41 ? 0.08 0.27 ? 0.06 2.3 ? 1.9 0.41 ? 0.13 0.23 ? 0.06 Kidneys 8.8 ? 1.3 9.5 ? 1.0 12 ? 2 7.8 ? 0.5 11 ? 3 11 ? 1 Adrenals 5.9 ? 1.2 1.5 ? 0.2 0.44 ? 0.06 3.6 ? 0.6 1.4 ? 0.5 0.37 ? 0.10 Stomach 29 ? 7 11 ? 1 2.2 ? 0.3 21 ? 3.4 6.0 ? 0.9 1.7 ? 0.4 Pancreas 53 ? 5 17 ? 3 4.2 ? 0.3 49 ? 2.5 14 ? 2 3.4 ? 0.3 Intestines 2.0 ? 0.2 0.74 ? 0.11 0.43 ? 0.23 1.4 ? 0.6 0.53 ? 0.10 0.31 ? 0.14 Liver 2.6 ? 0.9 0.63 ? 0.24 0.45 ? 0.05 1.2 ? 0.2 0.51 ? 0.16 0.35 ? 0.08 Muscle ?0.10 ?0.10 ?0.10 ?0.10 ?0.10 ?0.10 Femur 1.6 ? 0.5 0.53 ? 0.19 0.20 ? 0.05 0.75 ? 0.12 0.36 ? 0.05 0.17 ? 0.04 AR42J Tumor 29 ? 6 33 ? 6 18 ? 2 37 ? 11 44 ? 7 17 ? 2 Tu-to-blood 259 ? 22 461 ? 29 200 ? 38 289 ? 45 492 ? 102 270 ? 92 Tu-to-liver 13 ? 2 53 ? 9 38 ? 7 29 ? 8 90 ? 11 51 ? 7 Tu-to-kidney 3.5 ? 0.3 3.4 ? 0.1 1.4 ? 0.2 4.3 ? 1.2 4.3 ? 0.4 1.9 ? 0.2
[0127] The tumor uptake of [.sup.161Tb]Tb-/[.sup.177Lu]Lu-DOTATOC applied at 0.04 nmol and 0.2 nmol per mouse was similar (?17% IA/g and ?15% IA/g, respectively, p>0.05), but significantly higher than when 1.0 nmol were injected (?8% IA/g; p<0.05). The uptake in the stomach was higher after injection of 0.04 nmol compared to the injection of 0.2 nmol or 1.0 nmol (p<0.05). The uptake in pancreas, adrenals and lungs showed a similar trend, but the difference was not significant among the settings (p>0.05). No difference was observed in the kidney uptake (?10%) and liver uptake (?0.2% IA/g) irrespective of the applied setting. [.sup.161Tb]Tb/[.sup.177Lu]Lu-DOTA-LM3 showed equally high tumor uptake at 0.040 nmol and 0.20 nmol injected peptide per mouse (?34% and ?38% IA/g, p>0.05). The uptake was lower at 1.0 nmol (?18% IA/g, p<0.05). The uptake in the pancreas, adrenals, lungs and stomach was significantly higher at 0.040 nmol injected peptide than at 0.2 nmol per mouse and the lowest uptake was observed at an injected peptide amount of 1.0 nmol per mouse (p<0.05). The uptake in the liver was slightly, but not significantly elevated at the lowest molar amount injected (p>0.05). The kidney uptake of [.sup.161Tb]Tb/[.sup.177Lu]Lu-DOTA-LM3 was in the range of 10% IA/g irrespective of the injected amount of peptide. The results are presented in
[0128] As a result of these findings, the tumor-to-organ ratios varied dependent on the amount of injected radiopeptide (Table 3 and 4). Tumor-to-kidney (tu-to-ki) and tumor-to-liver (tu-to-li) ratios were favorable after injection of low molar amounts of peptide (0.04 nmol or 0.2 nmol) in both cases, for DOTATOC and DOTA-LM3. The tumor-to-pancreas (tu-to-panc), tumor-to-adrenals (tu-to-adr), tumor-to-lungs (tu-to-lung) and tumor-to-stomach (tu-to-sto) ratios were higher after injection of 0.2 nmol or 1.0 nmol of [.sup.161Tb]Tb/[.sup.177Lu]Lu-DOTATOC. These ratios were, however, more favorable after injection of 1.0 nmol peptide of .sup.161Tb- and .sup.177Lu-labeled DOTA-LM3 than after using 0.2 nmol peptide per mouse. The injection of 0.04 nmol peptide per mouse resulted in the least favorable ratios. Considered that the injection of 0.2 nmol peptide per mouse resulted in the highest tumor uptake and in the majority of the cases favorable tumor-to-background ratios, this molar amount of injected peptide was used for further in vivo studies.
TABLE-US-00008 TABLE 3 Effect of the amount of injected radiopeptide on the tumor-to-organs ratios of [.sup.161Tb]Tb-/[.sup.177Lu]Lu-DOTATOC at 2 h p.i. of the radiopeptide. [.sup.161Tb]Tb-DOTATOC [.sup.177Lu]Lu-DOTATOC 0.040 nmol 0.20 nmol 1.0 nmol 0.040 nmol 0.20 nmol 1.0 nmol Tu-to-ki 1.8 ? 0.2 1.5 ? 0.1 0.84 ? 0.12 1.9 ? 0.3 1.9 ? 0.4 0.72 ? 0.34 Tu-to-li 74 ? 3 76 ? 14 49 ? 1 100 ? 8 118 ? 24 31 ? 6 Tu-to-panc 7.8 ? 0.9 9.4 ? 1.3 12 ? 1 9.7 ? 1.8 10 ? 2 13 ? 1 Tu-to-adr 24 ? 1 34 ? 7 36 ? 10 28 ? 2 47 ? 29 29 ? 3 Tu-to-lung 13 ? 1 30 ? 4 33 ? 4 17 ? 3 27 ? 6 25 ? 3 Tu-to-sto 6 ? 2 12 ? 3 15 ? 2 11 ? 2 15 ? 4 15 ? 5
TABLE-US-00009 TABLE 4 Effect of the amount of injected radiopeptide on the tumor-to-organs ratios of [.sup.161Tb]Tb-/[.sup.177Lu]Lu-DOTA-M3 at 2 h p.i. of the radiopeptide. [.sup.161Tb]Tb-DOTA-LM3 [.sup.177Lu]Lu-DOTA-LM3 0.040 nmol 0.20 nmol 1.0 nmol 0.040 nmol 0.20 nmol 1.0 nmol Tu-to-ki 3.5 ? 0.3 3.4 ? 0.1 1.4 ? 0.2 4.3 ? 1.2 4.3 ? 0.4 1.9 ? 0.2 Tu-to-li 13 ? 2 53 ? 9 38 ? 7 29 ? 8 90 ? 11 51 ? 7 Tu-to-panc 0.59 ? 0.05 1.9 ? 0.4 4.2 ? 0.4 0.77 ? 0.20 3.2 ? 0.3 4.2 ? 0.8 Tu-to-adr 5.4 ? 1.0 22 ? 5 36 ? 16 11 ? 4 34 ? 9.2 36 ? 6 Tu-to-lung 4.0 ? 0.6 12 ? 3 16 ? 4 4.3 ? 1.2 17 ? 2 18 ? 1 Tu-to-sto 1.1 ? 0.2 3.0 ? 0.3 8.5 ? 1.5 1.8 ? 0.6 7.4 ? 0.6 11 ? 2
D. Time-Dependent Biodistribution Studies
[0129] Time-dependent biodistribution studies were performed to assess the total uptake of the radiopeptides in the tumor as well as in healthy organs and tissues.
[0130] The mice (n=3 per group) were intravenously injected with the radiolabeled DOTATOC or DOTA-LM3 (5 MBq, 0.2 nmol/mouse) in 100 ?L PBS (pH 7.4) containing 0.05% BSA. The mice were sacrificed at 0.5 h, 2 h, 4 h, 24 h and 48 h p.i, selected tissues and organs were collected, weighed, and the accumulated activity was counted using a ?-counter (Perkin Elmer). The decay-corrected data were listed as % IA/g. The data were analyzed for significance using a two-way ANOVA with Tukey's multiple comparison post-tests. A p-value of <0.05 was considered statistically significant.
[0131] [.sup.161Tb]Tb-DOTATOC reached the highest tumor uptake (15?2%) already 0.5 h after injection. The activity was retained in the tumor tissue up to 4 h but was cleared over the following hours resulting in 6.4?0.5% IA/g and 3.7?0.7% IA/g after 24 h and 48 h p.i. Significant activity accumulation was observed in the lungs, stomach and pancreas, however, it was cleared efficiently resulting in ?1% IA/g after 4 h p.i. The clearance from kidneys was slow and still ?10% IA/g at 4 h p.i., ?4-5% IA/g and 2-3% after 24 h and 48 h p.i. (see
TABLE-US-00010 TABLE A5 Biodistribution data obtained in AR42J tumor-bearing mice at 0.5, 2, 4, 24 and 48 h after injection of 0.2 nmol of [.sup.161Tb]Tb- DOTATOC. Decay-corrected data are shown as % IA/g tissue, representing the average ? SD [.sup.161Tb]Tb-DOTATOC (0.2 nmol/mouse) 0.5 h 2 h 4 h 24 h 48 h Blood 1.0 ? 0.1 ?0.10 ?0.10 ?0.10 ?0.10 Heart 0.45 ? 0.02 ?0.10 ?0.10 ?0.10 ?0.10 Lung 1.5 ? 0.1 0.47 ? 0.05 0.34 ? 0.06 0.15 ? 0.04 0.11 ? 0.02 Spleen 0.40 ? 0.09 0.18 ? 0.02 0.15 ? 0.00 ?0.10 ?0.10 Kidneys 11 ? 1 9.4 ? 0.4 11 ? 1 4.4 ? 0.9 1.8 ? 0.3 Adrenals 0.77 ? 0.18 0.43 ? 0.11 0.43 ? 0.03 0.16 ? 0.02 0.22 ? 0.07 Stomach 2.5? 0.3 1.2 ? 0.3 0.88 ? 0.09 0.78 ? 0.62 0.29 ? 0.05 Pancreas 3.8 ? 0.2 1.5 ? 0.2 1.1 ? 0.1 0.40 ? 0.02 0.23 ? 0.02 Intestines 0.47 ? 0.04 0.24 ? 0.01 0.20 ? 0.03 0.22 ? 0.25 ?0.10 Liver 0.41 ? 0.02 0.19 ? 0.03 0.15 ? 0.01 ?0.10 ?0.10 Muscle 0.24 ? 0.02 ?0.10 ?0.10 ?0.10 ?0.10 Femur 0.42 ? 0.04 0.12 ? 0.03 ?0.10 ?0.10 ?0.10 AR42J Tumor 15 ? 1 14 ? 1 14 ? 1 6.3 ? 0.6 3.7 ? 0.7 Tumor-to- 15 ? 2 221 ? 56 329 ? 17 490 ? 37 622 ? 14 blood Tumor-to- 37 ? 2 76 ? 14 89 ? 3 67 ? 6 53 ? 3 liver Tumor-to- 1.4 ? 0.2 1.5 ? 0.1 1.3 ? 0.1 1.5 ? 0.2 2.1 ? 0.3 kidney
[0132] Based on the fact that the tissue distribution remains the same irrespective of whether a somatostatin analogue is labeled with terbium-161 or lutetium-177, these results can be extrapolated also to the .sup.177Lu-labeled counterparts [44 ]. The tumor uptake of [.sup.161Tb]Tb-DOTA-LM3 was also fast resulting in 35?7% IA/g at 4 h p.i. After 48 h the activity in the tumor (21?4% IA/g) was still high. In other organs such as the lungs, stomach and pancreas, activity retention was seen over the first 4 h but effectively cleared afterwards. The accumulated activity of [.sup.161Tb]Tb-DOTA-LM3 in tumor, pancreas and stomach was significantly higher than in the case of [.sup.161Tb]Tb-DOTATOC (p<0.05) at all investigated time points whereas renal uptake was similar as observed for [.sup.161Tb]Tb-DOTATOC (p>0.05) (see
TABLE-US-00011 TABLE A6 Biodistribution data obtained in AR42J tumor-bearing mice at 0.5, 2, 4, 24 and 48 h after injection of 0.2 nmol of [.sup.161Tb]Tb-DOTA-LM3. Decay-corrected data are shown as % IA/g tissue, representing the average ? SD [.sup.161Tb]Tb-DOTA-LM3 (0.2 nmol/mouse) 0.5 h 2 h 4 h 24 h 48 h Blood 1.3 ? 0.2 ?0.10 ?0.10 ?0.10 ?0.10 Heart 0.83 ? 0.25 0.13 ? 0.01 ?0.10 ?0.10 ?0.10 Lung 4.4 ? 1.4 2.9 ? 0.7 2.3 ? 0.4 0.94 ? 0.45 0.59 ? 0.11 Spleen 0.77 ? 0.09 0.41 ? 0.08 0.38 ? 0.02 0.18 ? 0.02 0.20 ? 0.01 Kidneys 12 ? 1 9.5 ? 1.0 9.0 ? 1.2 5.9 ? 0.8 3.8 ? 0.5 Adrenals 1.6 ? 0.4 1.5 ? 0.2 1.6 ? 0.6 0.73 ? 0.31 0.53 ? 0.08 Stomach 7.7 ? 2.1 11 ? 1 8.3 ? 0.3 3.8 ? 1.2 3.4 ? 0.5 Pancreas 15 ? 2 17 ? 3 16 ? 1 6.1 ? 0.7 3.7 ? 0.5 Intestines 1.0 ? 0.3 0.74 ? 0.11 0.81 ? 0.41 0.39 ? 0.21 0.25 ? 0.07 Liver 0.93 ? 0.23 0.63 ? 0.24 0.51 ? 0.08 0.29 ? 0.08 0.27 ? 0.04 Muscle 0.32 ? 0.03 ?0.10 ?0.10 ?0.10 ?0.10 Femur 0.92 ? 0.29 0.53 ? 0.19 0.54 ? 0.06 0.29 ? 0.15 0.24 ? 0.01 AR42J Tumor 31 ? 7 33 ? 6 35 ? 7 26 ? 4 21 ? 4 Tumor-to- 24 ? 5 461 ? 29 820 ? 47 1371 ? 446 1249 ? 97 blood Tumor-to- 34 ? 7 53 ? 9 69 ? 2 95 ? 30 78 ? 10 liver Tumor-to- 2.5 ? 0.4 3.4 ? 0.1 3.9 ? 0.1 4.5 ? 1.1 5.5 ? 0.8 kidney
E. Preclinical Therapy Study in AR42 J-Tumor-Bearing Mice
[0133] The aim of this preclinical study was to evaluate the therapeutic effect and potential early side effects of DOTATOC and DOTA-LM3 peptides radiolabeled with either terbium-161 or lutetium-177.
[0134] The therapy study was initiated with mice randomly assigned to five groups (n=6) when the AR42 J tumors reached an average volume of 99?16 mm.sup.3. At Day 0 and Day 7 of the study, the mice were intravenously injected with vehicle only (Group A: PBS with 0.05% BSA; sham-treatment), [.sup.161Tb]Tb-DOTATOC (Group B: 10 MBq, 0.2 nmol), [.sup.177Lu]Lu-DOTATOC (Group C: 10 MBq, 0.2 nmol), [.sup.161Tb]Tb-DOTA-LM3 (Group D: 10 MBq, 0.2 nmol) and [.sup.177Lu]Lu-DOTA-LM3 (Group E: 10 MBq, 0.2 nmol) (Table 5).
[0135] The relative body weight (RBW) and the relative tumor volume (RTV) were defined based on the values at therapy start as previously described [54 ]. The RBW was defined as [BW.sub.x/BW.sub.0], where BW.sub.x is the body weight in gram at a given Day x and BW.sub.0 the body weight in gram at Day 0. The tumor dimension was determined by measuring the longest tumor axis (L) and its perpendicular axis (W) with a digital caliper. The tumor volume (TV) was calculated according to the equation [TV=0.5?(L?W.sup.2)]. The relative tumor volume (RTV) was defined as [TV.sub.x/TV.sub.0], where TV.sub.x is the tumor volume in mm.sup.3 at a given Day x and TV.sub.0 the tumor volume in mm.sup.3 at Day 0.
[0136] The endpoint criteria were defined according to a scoring system which required euthanasia of mice with a score?3. Every second day the following criteria were assessed in the mice assigning a score from 0-3 for each criterion: (i) appearance (general status, skin color, etc.), (ii) behavior (vitality, sociality. crouching etc.), (iii) body weight (stable, loss>5?10%, loss>10<15%, loss? 15% compared to initial body weight), (iv) tumor size (<800 mm.sup.3, ?800 and <900 mm.sup.3, ?900 and <1000 mm.sup.3, ?1000 mm.sup.3), (v) tumor ulceration. A score?3 was due for example to: (i) appearance of wrinkled, translucent skin; (ii) mouse in crouching position and/or apathetic (iii) a body weight loss of ?15% of initial body weight, (iv) a tumor volume of ?1000 mm.sup.3, (v) a combination of a tumor size of >800 mm.sup.3 and body weight loss of ?10% and/or (vi) ulceration of the tumor.
[0137] The efficacy of the treatment was assessed by comparison of the RTVs, measured every second day, of mice in each group using a two-way ANOVA with Sidak's multiple comparisons post-test. The average tumor growth delay, herein defined as the time during which the tumors did not grow or even decreased in size, was determined for mice of each group. For the subsequent phase, in which the tumors started to regrow, the doubling time of the tumor volume was calculated based on the fitted exponential tumor growth curve. The average?SD of tumor growth delay and of the doubling time of the tumor volume in single mice, respectively, were compared among groups with a one-way ANOVA with Tukey's multiple comparisons post-test. The survival times of mice were presented by Kaplan-Meier curves and analyzed using a log-rank test (Mantel-Cox).
TABLE-US-00012 TABLE 5 Design of the therapy study including the average tumor volumes and body weights of mice at therapy start. The mice were injected at Day 0 and Day 7 with the respective radiopeptide at 0.2 nmol peptide amount per mouse (n = 6). Tumor volume.sup.2 Body weight.sup.3 (mm.sup.3) (g) Injected (average ? SD) (average ? SD) Group Treatment activity Day 0 Day 0 A Vehicle.sup.1 118 ? 90 23 ? 2 B [.sup.161Tb]Tb-DOTATOC 2 ? 10 MBq 92 ? 48 24 ? 2 C [.sup.177Lu]Lu-DOTATOC 2 ? 10 MBq 102 ? 56 24 ? 2 D [.sup.161Tb]Tb-DOTA-LM3 2 ? 10 MBq 76 ? 32 23 ? 1 E [.sup.177Lu]Lu-DOTA-LM3 2 ? 10 MBq 109 ? 72 24 ? 2 .sup.1Vehicle: 0.05% BSA in PBS (pH 7.4); .sup.2No significant differences determined between the tumor volumes measured for each group (p > 0.05); .sup.3No significant differences determined between the body weights measured for each group (p > 0.05).
[0138] Sham-treated mice of Group A showed an exponential tumor growth so that the endpoint was reached within the first 14 days in all cases. The tumor growth was delayed in treated mice of Groups B-E, resulting in significantly prolonged median survival times as compared to the 9 days in the control group (
[0139] The tumor growth delay for mice treated with [.sup.161Tb]Tb-DOTA-LM3 was 44?5 days, but only 35?7 days in mice treated with [.sup.177Lu]Lu-DOTA-LM3 (p<0.05). Afterwards, the tumors started to regrow exponentially in 5 out of 6 mice of both groups, however, the doubling time was considerably longer for mice treated with [.sup.161Tb]Tb-DOTA-LM3 as compared to the tumor growth in mice treated with [.sup.177Lu]Lu-DOTA-LM3 (7.4?4.6 days vs 3.8?1.1 days, p>0.05) (
TABLE-US-00013 TABLE 6 Data regarding euthanasia period, median survival and tumor growth delay indices of mice. Time frame of Median euthanasia survival Group Treatment [d] [d] A vehicle 6-15 9 B [.sup.161Tb]Tb-DOTATOC.sup.a 20-26 21 C [.sup.177Lu]Lu-DOTATOC.sup.a 12-22 19.5 D [.sup.161Tb]Tb-DOTA-LM3.sup.a end of study.sup.b n.d. E [.sup.177Lu]Lu-DOTA-LM3.sup.a 42-48 (n = 3) 48.5 end of study (n = 3) .sup.a1.sup.st injection: 10 MBq, 0.2 nmol at Day 0; 2.sup.nd Injection: 10 MBq, 0.2 nmol at Day 7. .sup.bend of study = 49 days. .sup.csignificantly different from group A (p < 0.05)
F. Assessment of Early Side Effects
[0140] The aim was to assess signs of early side effects in mice treated with 2 x10 MBq of [.sup.161Tb]Tb-DOTATOC, [.sup.177Lu]Lu-DOTATOC, [.sup.161Tb]Tb-DOTA-LM3 or [.sup.177Lu]Lu-DOTA-LM3.
[0141] Early side effects were assessed based on the RBW of each mouse which was monitored every second day. When an endpoint was reached or at the end of the study (after 49 days), relevant organs and tissues were collected, weighed and put into relation to the brain mass and body weight of the respective mouse. This allowed comparison of organ-to-brain and organ-to-body mass weight ratios. In addition, blood plasma parameters from blood collected immediately after euthanasia were measured.
[0142] RBW, organ mass and mass ratios at the endpoint. The RBW was monitored every second day. Mice were euthanized when a predefined endpoint criterion was reached or when the study was terminated at Day 49. RBWs, organ masses, organ mass ratios (kidney-to-brain, liver-to-brain and spleen-to-brain) and organ mass to body weight ratios (kidney-to-body mass, liver-to-body mass and spleen-to-body mass) were analyzed for significance using a one-way ANOVA test with a Tukey's multiple comparisons post-test. A p-value of <0.05 was considered as statistically significant.
[0143] Blood plasma chemistry: Immediately before euthanasia of the mice that reached the endpoint, blood was sampled from the retrobulbar vein. The values of creatinine (CRE), blood urea nitrogen (BUN), alkaline phosphatase (ALP), total bilirubin (TBIL) and albumin (ALB) were determined in the plasma after centrifugation of the blood using a dry chemistry analyzer (DRI-CHEM 4000 i, FUJIFILM, Japan). The average blood plasma parameters of each group were analyzed for significance using a one-way ANOVA test with a Tukey's multiple comparisons post-test. A p-value of <0.05 was considered as statistically significant.
[0144] No obvious early side effects were observed in treated mice. The relative body weights increased over the course of the study, a sign of the well-being of the mice (see
[0145] At the time of euthanasia, the organ masses, the organ-to-brain ratios and the organ-to-body mass ratios showed no significant difference between treated mice (Groups B-E) and untreated controls (Group A) (p>0.05) (Tables 8 and 9).
[0146] Blood plasma chemistry revealed no difference among treated mice and untreated controls. The BUN levels were in the acceptable range for all mice, however, elevated in mice treated with [.sup.161Tb]Tb-DOTA-LM3 (9.9?1.5 mmol/L; p<0.05) and [.sup.177Lu]Lu-DOTA-LM3 (8.2?1.9 mmol/L; p>0.05) as compared to control mice (6.2?0.7 mmol/L) (Table 10)
TABLE-US-00014 TABLE 7 Relative body weight of mice at Day 6 and at the endpoint of the therapy study. No significant difference among the groups was observed (p > 0.05). Relative body Relative body Group weight at Day 6.sup.1 weight at endpoint.sup.2 (n = 6) (average ? SD) (average ? SD) A 1.03 ? 0.03 1.05 ? 0.03 B 1.02 ? 0.03 1.07 ? 0.02 C 0.99 ? 0.03 1.01 ? 0.07 D 1.04 ? 0.03 1.06 ? 0.07 E 1.01 ? 0.02 1.11 ? 0.06 .sup.1Data obtained at Day 6 when the first control mouse reached an endpoint. .sup.2Data obtained at the day of euthanasia when an endpoint criterion was reached or at the end of the study.
TABLE-US-00015 TABLE 8 Organ mass of mice of the therapy study collected after euthanasia. No significant difference among the groups was observed (p > 0.05). Organ mass.sup.1 (mg) Group (average ? SD) (n = 6) Kidneys Liver Spleen Brain A 327 ? 28 1096 ? 128 90 ? 10 429 ? 23 B 352 ? 27 1195 ? 57 86 ? 19 440 ? 23 C 342 ? 23 1121 ? 123 90 ? 17 453 ? 31 D 332 ? 46 1275 ? 254 85 ? 27 465 ? 30 E 336 ? 13 1254 ? 106 92 ? 17 471 ? 30 .sup.1Data obtained at the day of euthanasia when an endpoint criterion was reached or at the end of the study.
TABLE-US-00016 TABLE 9 Organ mass-to-brain mass and organ mass-to-body weight ratios. No significant difference among the groups was observed (p > 0.05). Organ mass-to-brain mass ratios Group (average ? SD) (n = 6) Kidney-to-brain Liver-to-brain Spleen-to-brain A 0.77 ? 0.09 2.6 ? 0.3 0.21 ? 0.03 B 0.80 ? 0.03 2.7 ? 0.2 0.20 ? 0.05 C 0.76 ? 0.03 2.5 ? 0.2 0.20 ? 0.03 D 0.72 ? 0.12 2.8 ? 0.7 0.19 ? 0.07 E 0.72 ? 0.05 2.7 ? 0.1 0.20 ? 0.04 Organ mass-to-body mass ratios Group (average ? SD) (n = 6) Kidney-to-body Liver-to-body Spleen-to-body A 0.013 ? 0.001 0.047 ? 0.002 0.004 ? 0.000 B 0.013 ? 0.001 0.049 ? 0.004 0.003 ? 0.001 C 0.014 ? 0.001 0.050 ? 0.003 0.004 ? 0.001 D 0.014 ? 0.001 0.050 ? 0.001 0.004 ? 0.001 E 0.014 ? 0.001 0.050 ? 0.003 0.003 ? 0.001
TABLE-US-00017 TABLE 10 Plasma chemistry determined at the endpoint of the therapy (n = 6, if not differently indicated) CRE BUN ALP TBIL ALB Group (?mol/L) (mmol/L) (U/L) (?mol/L) (g/L) A <18 (n = 6) 6.2 ? 0.7 71 ? 15 <3 (n = 2) 22 ? 2 3 ? 1 (n = 4) B <18 (n = 6) 5.8 ? 1.0 76 ? 21 <3 (n = 4) 23 ? 2 4 ? 1 (n = 2) C <18 (n = 5) 6.3 ? 0.9 77 ? 8 <3 (n = 3) 24 ? 5 4 ? 1 (n = 3) D <18 (n = 5) 9.1 ? 1.5* 65 ? 15 <3 (n = 5) 22 ? 1 18 (n = 1) 3 (n = 1) E <18 (n = 5) 8.2 ? 1.8** 66 ? 5 <3 (n = 4) 23 ? 1 19 (n = 1) 4 ? 1 (n = 2) *Significantly different (p < 0.05) from the value of Group A, B, C. **Significantly different (p < 0.05) from the value of Group B.
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