ALPHA-1-MICROGLOBULIN FOR USE IN THE PROTECTION OF KIDNEYS IN RADIONUCLIDE THERAPY

Abstract

This invention relates to .sub.1-microglobulin (A1M) for use in the diagnosis or treatment of malignancies requiring radionuclide diagnostics (RD), radionuclide therapy (RNT) or radioimmunotherapy (RIT), respectively, wherein A1M is used as a co-treatment to RD, RNT or RIT.

Claims

1-28. (canceled)

29. A method of treating kidney injuries from a radionuclide or reducing renal side effects of a radionuclide in a subject undergoing radionuclide diagnostics (RD), radionuclide therapy (RNT), or radioimmunotherapy (RIT) for a malignancy, comprising administering .sub.1-microglobulin (A1M) to a subject in need thereof, wherein the A1M has at least 95% sequence identity with SEQ ID NO:1 or SEQ ID NO:2.

30. The method of claim 29, wherein the subject is administered a compound labelled with a radionuclide selected from radionuclide diagnostic agents, radionuclide therapeutic agents, and radioimmunotherapeutic agents.

31. The method of claim 30, wherein the compound is selected from receptor ligands, affibody molecules, diabodies, and antibody fragments.

32. The method of claim 30, wherein the compound labelled with a radionuclide is a peptide coupled to a radiation-emitting radionuclide.

33. The method of claim 30, wherein the compound is a somatostatin analogue.

34. The method of claim 30, wherein the compound is a somatostatin analogue selected from octreotide, lanreotide, Tyr.sup.3-octreotide, Tyr.sup.3-octrotate, DOTADOC, DODATATE, DOTA-lanreotide, pasireotide, dopastatin and octreotide LAR.

35. The method of claim 29, wherein the malignancy is cancer.

36. The method of claim 29, wherein the malignancy is a neuroendocrine tumour.

37. The method of claim 29, wherein the A1M is administered between 60 minutes before and 30 minutes after administration of a compound labelled with a radionuclide.

38. The method of claim 29, wherein the A1M is administered between 30 minutes before and 30 minutes after administration of a compound labelled with a radionuclide.

39. The method of claim 29, wherein the A1M is administered between 10 minutes before and 10 minutes after administration of a compound labelled with a radionuclide.

40. The method of claim 29, wherein the A1M is administered simultaneously with a compound labelled with a radionuclide.

41. The method of claim 29, wherein the A1M is administered in multiple daily doses during the first week after administration of a compound labelled with a radionuclide.

42. A kit for reducing renal side effects of a radionuclide administered for radionuclide diagnostics (RD), radionuclide therapy (RNT), or radioimmunotherapy (RIT) of a malignancy, comprising A1M having at least 95% sequence identity with SEQ ID NO:1 or SEQ ID NO:2 and a compound labelled with a radionuclide, wherein the compound is a radionuclide diagnostic agent, a radionuclide therapeutic agent, or a radioimmunotherapeutic agent for a malignancy.

43. The kit of claim 42, wherein the compound is selected from receptor ligands, affibody molecules, diabodies and antibody fragments.

44. The kit of claim 42, wherein the compound labelled with a radionuclide is a peptide coupled to a radiation-emitting radionuclide.

45. The kit of claim 42, wherein the compound is a somatostatin analogue.

46. The kit of claim 42, wherein the compound is a somatostatin analogue selected from octreotide, lanreotide, Tyr.sup.3-octreotide, Tyr.sup.3-octrotate, DOTADOC, DODATATE, DOTA-lanreotide, pasireotide, dopastatin and octreotide LAR.

47. The kit of claim 42, wherein the malignancy is cancer.

48. The kit of claim 42, wherein the malignancy is a neuroendocrine tumour.

Description

LEGENDS TO FIGURES

[0066] FIG. 1 shows the biodistribution of .sup.125I-A1M (upper left) and .sup.111In-Octreotide (upper right) in normal NMRI mice. Lower left image shows uptake over time for both molecules in the kidneys. Data are presented as % IA/g from 4 animalsSEM.

[0067] FIG. 2 shows the presence of full-length A1M in normal NMRI mice in kidneys and serum at 10, 20 and 60 minutes post-injection. Animals were injected i.v. with 150 g A1M and blood and kidneys collected at the indicated time-points. The blood was allowed to coagulate and serum separated by centrifugation. One kidney was homogenized in 1 ml PBS and centrifuged. 1 l serum and 6 l supernatant from the kidney homogenate were applied to SDS-PAGE, transferred to PVDF-membranes and blotted with anti-A1M. Each lane represents a separate mouse.

[0068] FIG. 3 shows the three-dimensional structure of A1M. The illustration was generated using PyMOL [Molinspiration, M. v. (2014)] and coordinates from the crystal structure of human A1M [Meining, W., and Skerra, A. (2012) The crystal structure of human .sub.1-microglobulin reveals a potential haem-binding site. Biochem J 445, 175-182]. -strands and -helices are shown in green ribbons. Side-chains of C34, K92, K118, K130 and H123, involved in functional activities of A1M, are shown as green sticks with nitrogen atoms in blue. The four lipocalin loops are labeled #1-#4.

[0069] FIG. 4 shows the three-dimensional arrangement of some amino acids (blue ovals, the lysines are depicted by a +), the A1M-framework (barrel), the electron-flow and the radical-trapping.

[0070] FIG. 5 A-D show tubular localization of A1M and Octreotide-A647 at different post-injection times. A1M and Octreotide-A647 conjugate were injected i.v. and animals were terminated at 20 minutes (A, C) and 4 hours (B, D) following injection. The percentage of co-localized A1M (green) and Octreotide-A647 (red) was measured in selected tubular profiles in the cortex, medulla and collecting ducts, in confocal microscopic images (20/0.8 objective). Cell nuclei were visualized using DAPI (blue). Representative profiles (A, B) show different degrees of co-localization (yellow), and the co-localization data from all investigated samples are presented as meanSEM of representative areas (C, D). Scale bar represents 50 m.

[0071] FIGS. 5 E-F show cellular co-localization of A1M and Octreotide-A647 at different post-injection times. A1M and Octreotide-A647 conjugate were injected i.v. and animals were terminated at 20 minutes and 4 hours (E, left and right) following injection. The percentage of co-localized A1M (green) and Octreotide-A647 (red) was measured in selected tubular profiles in the cortex, in high-resolution confocal microscopic images (63/1.4 objective). Cell nuclei were visualized using DAPI (blue). Representative profiles (E) show different degrees of intracellular co-localization (yellow), and the co-localization data from all investigated samples are presented as meanSEM of representative areas (F). Scale bar represents 20 m.

[0072] FIG. 6 show preclinical SPECT/CT images of normal NMRI mice injected with 5 MBq .sup.111In-octreotide (A and B) and 5 MBq .sup.125I-A1M (C and D) and imaged for 40 minutes. A and C show reconstructed three-dimensional views of the whole animal and B and D show planar section through the kidneys. Kidneys show high uptake and a concentration can be seen in the kidney cortex for both molecules. For .sup.125I-A1M, a slight uptake in the thyroids can be observed.

[0073] FIG. 7 show digital autoradiography images of uptake of .sup.111In-octreotide and .sup.125I-A1M in kidneys of normal mice. (A) .sup.125I-A1M, 20 min p.i.; (B) .sup.125I-A1 M, 1 h p.i.; (C) .sup.111In-octreotide, 20 min p.i.; (D) .sup.111In-octreotide, 1 h p.i. All images shows localized uptake in the kidney cortex for both molecules. Note that the scale of each image has been adjusted to optimally illustrate the relative distribution of the radionuclides in each kidney section.

[0074] FIG. 8 shows the distribution of A1M immunoreactivity in the kidney 20 minutes after i.v. injection. A1M was injected i.v., animals were terminated after 20 minutes, and A1M immunoreactivity was detected with the K323 anti-A1M antibody, using immunohistochemistry. The left panel shows representative areas with A1M-immunoreactivity in the cortex (A), medulla (B), and collecting ducts (C); the location of these areas is indicated with A-C and highlighted with boxes in the schematic drawing in the right panel. Scale bar represents 100 m in A-C.

[0075] FIG. 9 shows the distribution of A1M immunoreactivity and Octreotide-A647 in the kidney 20 minutes and 4 hours after i.v. injection. A1M immunoreactivity was detected with the K323 anti-A1M antibody, using immunohistochemistry (left column; bright-field microscopy) or immunofluorescence (middle and right columns; confocal microscopy) in cortex (A), medulla (B), and collecting ducts (C). Distribution of A1M immunofluorescence (green) and Octreotide-A647 (red), and their tubular co-localization (yellow), was investigated at 20 minutes (middle) and 4 hours (right) after injection. Cell nuclei were visualized using DAPI (blue). Scale bar represents 50 m.

[0076] FIG. 10 shows the cellular co-localization of A1M immunofluorescence and Octreotide-A647 following i.v. injections. A1M and Octreotide-A647 conjugate were injected i.v. and animals were terminated at 20 minutes following injection. High-resolution (630.4 objective) confocal microscopic image showing the intracellular distribution of A1M immunofluorescence (green) and Octreotide-A647 fluorescence (red). Cell nuclei were stained with DAPI (blue), and phalloidin-Texas Red labeling (grey) was used to delineate tubular profiles. Resolution of punctuate fluorescence in one cell (A; arrow) was shown by measuring fluorescence intensities along a profile in the cytoplasm just outside the nucleus (B; yellow line), giving the intensities along the profile in the red and green channels (C) as an intensity profile (D) with 8 bits (256 intensity levels) per channel. Scale bar represents 10 m.

[0077] FIG. 11 shows the results of Example 2.

[0078] FIG. 12 show the sequences SEQ ID 1-4.

REFERENCES

[0079] 1. John J Zaknun et al. (2013) The joint IAEA, EANM, and SNMMI practical guidance on peptide receptor radionuclide therapy (PRRNT) in neuroendocrine tumors. Eur J Nucl Med Mol Imaging, 800-16. http://www.ncbi.nlm.nih.gov/pubmed/23389427 [0080] 2. Krenning E P, Bakker W H, Breeman W A, et al (1989) Localisation of endocrine-related tumors with radioiodinated analogue of somatostatin. Lancet 333, 242-244. [0081] 3. de Jong M, Krenning E P. (2002) New advances in peptide receptor radionuclide therapy. J Nucl Med 43, 617-20 [0082] 4. Halliwell B, Gutteridge J M. (1994) The definition and measurement of antioxidants in biological systems. Free Rad Biol Med 16, 135-137 [0083] 5. Prise K M, Folkard M, Michael B D. (2003) A review of the bystander effect and its implications for low-dose exposure. Radiat Prot Dosimetry 104(4):347-55. [0084] 6. Mothersill C, Seymour C. (2001) Radiation-induced bystander effects: past history and future directions. Radiat Res 155(6):759-67. [0085] 7. Little J B, Azzam El, de Toledo S M, Nagasawa H. (2002) Bystander effects: intercellular transmission of radiation damage signals. Radiat Prot Dosimetry 99(1-4):159-62. [0086] 8. Azzam El, de Toledo S M, Little J B. (2003) Oxidative metabolism, gap junctions and the ionizing radiation-induced bystander effect. Oncogene 22(45):7050-7. [0087] 9. Lyng F M, Seymour C B, Mothersill C. (2000) Production of a signal by irradiated cells which leads to a response in unirradiated cells characteristic of initiation of apoptosis. Br J Cancer 83(9):1223-30. [0088] 10. Lyng F M, Seymour C B, Mothersill C. (2002) Initiation of apoptosis in cells exposed to medium from the progeny of irradiated cells: a possible mechanism for bystander-induced genomic instability? Radiat Res 157(4):365-70. [0089] 11. Allhorn M, Berggrd T, Nordberg J, Olsson M L, kerstrm B. (2002) Processing of the lipocalin .sub.1-microglobulin by hemoglobin induces heme-binding and heme-degradation properties. Blood 99: 1894-1901. [0090] 12. Allhorn M, Klapyta A, kerstrm B. (2005) Redox properties of the lipocalin .sub.1-microglobulin: reduction of cytochrome c, hemoglobin, and free iron. Free Radic Biol Med 38(5):557-67. [0091] 13. kerstrm B, Maghzal G, Winterbourn C C, Kettle A J. (2007) The lipocalin .sub.1-microglobulin has radical scavenging activity. J Biol Chem 282: 31493-31503. [0092] 14. Olsson M G, Allhorn M, Blow L, Hansson S R, Ley D, Olsson M L, Schmidtchen A, kerstrm B. (2012) Pathological conditions involving extracellular hemoglobin: Molecular mechanisms, clinical significance and novel therapeutic opportunities for .sub.1-microglobulin. Antiox Redox Signal. 17(5), 813-846. [0093] 15. kerstrm B, Gram M. (2014) A1M, an extravascular tissue cleaning and housekeeping protein. Free Rad Biol Med 74C:274-282. [0094] 16. Olsson M G, Olofsson T, Tapper H, kerstrm B. (2008) The lipocalin .sub.1-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species. Free Rad Res. 42, 725-736. [0095] 17. May K, Rosenlf L, Olsson M G, Centlow M, Mrgelin M, Larsson I, Cederlund M, Rutardottir S, Schneider H, Siegmund W, kerstrm B, Hansson S R. (2011) Perfusion of human placenta with haemoglobin introduces preeclampsia-like injuries that are prevented by .sub.1-microglobulin. Placenta 32(4), 323-332. [0096] 18. Olsson M G, Allhorn M, Larsson J, Cederlund M, Lundqvist, K, Schmidtchen A, Srensen O E, Mrgelin M, kerstrm B. (2011) Up-regulation of A1M/.sub.1-microglobulin in skin by heme and reactive oxygen species gives protection from oxidative damage. PLoS One 6(11):e27505. [0097] 19. Olsson M G, Rosenlf L W, Kotarsky H, Olofsson T, Leanderson T, Mrgelin M, Fellman V, kerstrm B. (2013) The radical-binding lipocalin A1M binds to a Complex I subunit and protects mitochondrial structure and function. Antiox Redox Signal. 18 (16), 2017-2028. [0098] 20. Wester-Rosenlf L, Cassln V, Axelsson J, Edstrm-Hgerwall A, Gram M, Holmqvist M, Johansson M E, Larsson I, Ley D, Marsal K, Mrgelin M, Rippe B, Rutardottir S, Shohani B, kerstrm B, Hansson S R. (2014) A1M/.sub.1-microglobulin protects from heme-induced placental and renal damage in a pregnant sheep model of preeclampsia. PloS One 9(1):e86353. [0099] 21. Sverrison K, Axelsson J, Rippe A, Gram M, kerstrm B, Hansson S R, Rippe B.

[0100] Extracellular fetal hemoglobin induces increases in glomerular permeability: inhibition with .sub.1-microglobulin and Tempol. Am J Physiol Renal Physiol 306(4):F442-448. [0101] 22. Olsson M G, Nilsson E J C, Rutardottir S, Paczesny J, Pallon J, kerstrm B. (2010) Bystander cell death and stress response is inhibited by the radical scavenger .sub.1-microglobulin in irradiated cell cultures. Rad Res 174, 590-600. [0102] 23. Rutardottir S, Nilsson E J C, Pallon J, Gram M, kerstrm B. The cysteine 34 residue of A1M/.sub.1-microglobulin is essential for protection of irradiated cell cultures and reduction of carbonyl groups. Free Radic Res 47(6-7):541-550. [0103] 24. Larsson, J., Wingrdh, K., Berggrd, T., Davies, J. R., Lgdberg, L., Strand, S. E. and kerstrm, B. (2001) Distribution of .sup.125I-labelled .sub.1-microglobulin in rats after intravenous injection. J. Lab. Clin. Med. 137, 165-175. [0104] 25. Kwasek A, Osmark P, Allhorn M, Lindqvist A, kerstrm B, Wasylewski Z. (2007) Production of recombinant human .sub.1-microglobulin and mutated forms involved in chromophore formation. Prot Expr Purif 53, 145-152. [0105] 26. Greenwood F C, Hunter W M, Glover J S (1963) The Preparation of I-131-Labelled Human Growth Hormone of High Specific Radioactivity. Biochem J 89: 114-123. [0106] 27. Laemmli U K: Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 1970, 227:680-685 [0107] 28. Wester L, Johansson M U, kerstrm B: Physicochemical and biochemical characterization of human .sub.1-microglobulin expressed in baculovirus-infected insect cells, Protein expression and purification 1997, 11:95-103 [0108] 29. Strober W, Waldmann T A: The role of the kidneys in the metabolism of plasma proteins. Nephron 1974, 13:35-66 [0109] 30. Nordberg J, Allhorn M, Winqvist I, kerstrm B, Olsson M L. Quantitative and qualitative evaluation of plasma and urine .sub.1-microglobulin in healthy donors and patients with different haemolytic disorders and haemochromatosis. Clin Chim Acta 2007, 386:31-37 [0110] 31. Bck T, Haraldsson B, Hulborn R, Jensen H, Johansson M E, Lindegren S, Jacobsson L. Glomerular filtration rate after alpha-radioimmunotherapy with .sup.211At-MX35-F(ab)2: A long-term study of renal function in nude mice. Cancer Biother Radiopharm 2009, 24:649-658

Example 1

Experimental

Materials and Methods

Recombinant Human A1M

[0111] Recombinant human A1M was expressed in E. coli, purified and re-folded as described by Kwasek et al [25] but with an additional ion-exchange chromatography step. This was performed by applying A1M to a column of DEAE-Sephadex A-50 (GE Healthcare, Uppsala, Sweden) equilibrated with 20 mM Tris-HCl, pH8.0. A1M was eluted with a linear salt gradient (from 20 mM Tris-HCl, pH8.0 to 20 mM Tris-HCl, 0.2 M NaCl) at a flow rate of 1 ml/min. A1M-containing fractions, according to absorbance at 280 nm, were pooled and concentrated.

.SUP.125.I-Labelling of A1M

[0112] Radiolabelling of A1M with .sup.125I was done using the chloramine T method [26]. Briefly, A1M and .sup.125I (Perkin-Elmer, NEZ033005MC) were mixed in 0.5 M sodium phosphate, pH 7.5 at final concentrations of 1 mg/ml and 10 mCi/ml, respectively. Chloramine T was added to 0.4 mg/ml and allowed to react on ice for 2 minutes, and the reaction was stopped by adding NaHSO.sub.3 to 0.8 mg/ml. Protein-bound iodine was separated from free iodide by gel-chromatography on a Sephadex G-25 column (PD10, GE Healthcare, Buckinghamshire, UK). A specific activity of around 50-200 kBq/g protein was obtained.

Octreotide

[0113] The somatostatin analogue peptide octreotide was purchased from Mallinckrodt Pharmaceuticals (http://www.mallinckrodt.com/) and labelled with .sup.111In and with Alexa 647 (HiLyte Fluor 647; AnaSpec, Seraing, Belgium) according to instructions from the merchant. These compounds are referred to as .sup.111In-octreotide and octreotide-647, respectively.

Animal Studies

[0114] All animal experiments were conducted in compliance with the national legislation on laboratory animals' protection and with the approval of the Ethics Committee for Animal Research (Lund University, Sweden). Male and female NMRI normal mice of 6-8 weeks old (Taconic, Ry, Denmark) were used.

Biodistribution

[0115] Biodistribution studies were conducted to determine the pharmacokinetics and biodistribution of .sup.125I-A1M and .sup.111In-octreotide. .sup.125I-A1M (100 kBq, 1 g) and .sup.111In-octreotide (100 kBq, 10 g) were administered i.v. through tail vein injection to NMRI mice (n=3 per injected molecule and time point). Animals were termination at 10, 20, 40, 60 minutes (both A1M and octreotide), 4 and 24 hours (octreotide) post-injection and blood and organs were sampled, weighed and measured in a NaI(TI) well counter (Wallac Wizard 1480 Wizard, Perkin Elmer). Organ-specific uptake values were calculated as percent injected activity per gram of tissue (% IA/g) or percent injected activity (% IA).

Digital Autoradiography

[0116] Digital autoradiography was performed using the Biomolex 700 imaging system (Biomolex AS, Norway). Two groups (n=2) of normal mice were i.v. injected with .sup.125I-A1M (0.5 MBq, 5 g) and .sup.111In-octreotide (0.5 MBq, 50 g). Kidneys were frozen in embedding media using dry ice and sectioned in a cryostat (Leica Microsystems AB, Sweden) in 50 m thick sections and imaged in the Biomolex system. Images were reconstructed using in house software.

Western Blotting

[0117] SDS-PAGE analysis was performed on kidneys and serum from animals that had been injected i.v. with non-labeled A1M (100 l/animal, 1.5 mg/ml). Animals were terminated at 10, 20 and 60 minutes post-injection, blood and kidneys were sampled and kidneys were washed and placed in 1 ml PBS. Following mechanical tissue homogenization, tissue was centrifuged at 10,000g for 10 minutes and supernatant was transferred to a new tube and used for further analysis as describe below. Serum was obtained from the blood samples by centrifugation at 1,000g for 10 minutes. SDS-PAGE gels were run under reducing conditions and the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, Mass., USA) using Trans-Blot Turbo transfer system (Bio-Rad, Delaware, USA). PVDF membranes were subsequently blocked and incubated overnight with the IgG-fraction of rabbit polyclonal anti-A1M antiserum (K322, 5 g/ml) as described previously [28], followed by incubation with Alexa Fluor 647 goat anti-rabbit IgG (diluted 3000; Molecular Probes). The membranes were developed using a ChemiDoc MP Imaging system (BioRad).

SPECT Imaging

[0118] Animals were anaesthetized with 2% to 3% isoflurane gas (Baxter; Deerfield, Ill., USA) during imaging in the NanoSPECT/CT (Bioscan, Washington D.C., USA). Animals were i.v. injected with approximately 5 MBq of .sup.125I-A1M (approximately 30 g) and 5 MBq of .sup.111In-octreotide and imaged 20 m p.i. with the NSP-106 multi-pinhole mouse collimator. For .sup.125I imaging energy windows of 20% were centered over the 35 keV photo peak and for .sup.111In over the 175 and 241 photo peaks. SPECT data were reconstructed using HiSPECT software (SciVis; Goettingen, Germany). CT imaging was done before each whole-body SPECT. After SPECT imaging at 1 hour, kidneys were resected and embedded in Tissue-Tek O.C.T compound (Sakura Finetek; Alphen aan den Rijn, The Netherlands) and frozen on dry ice. The frozen samples were cryosectioned with a thickness of 10 m for autoradiography analysis on the Biomolex system. The kidney sections were stained with Mayer's hematoxylin and chromotrope 2R, Ch2R (both from Histolab; Gothenburg, Sweden), and scanned using a light-microscope slide scanner (Mirax Midi, Carl Zeiss; Oberkochen, Germany).

KidneySample Preparation and Immunolabeling of A1M

[0119] Following simultaneous i.v. injection of 150 g A1M (unconjugated) and 100 g Alexa 647-labelled octreotide (octreotide-647) animals were sacrificed after 10, 20, 40, 60 minutes and 4 hours. All time-points were evaluated but only kidneys from 20 minutes and 4 hours, displaying detailed analyses at the cellular level, including laser confocal scanning microscopy and quantitative image analyses, are included. Importantly, all experiments were performed and evaluated on both wild-type and nude mice, and was shown to possess the same labeling pattern. However, only wild-type data are included.

[0120] After euthanization, kidneys were removed directly frozen and embedded in Tissue Tec. The tissue blocks were sectioned in a cryostat (Microm, HM 500OM, Walldorf, GmbH), and sections (10 m) were collected on SuperFrost plus slides (Merck, Darmstadt, Germany). Serial sectioning was performed, collecting 3-4 sections per slide, of which adjacent slides were used for either chromogen immunohistochemistry (IHC) or immunofluorescence (IF) labeling. Sections were post-fixed in 4% paraformaldehyde (PFA, Sigma, St. Louis, Mo., USA, dissolved in PBS, 0.1 M, pH 7.4) for 15 minutes, and rinsed in PBS two times for 5 minutes.

[0121] For single labeling of A1M, sections were incubated with 0.03% hydrogen peroxide (H.sub.2O.sub.2, Merck, Darmstadt, Germany) for five minutes for chromogen visualization (IHC). For both chromogen and fluorescence visualization sections were incubated with 1% bovine serum albumin (BSA, Sigma, St. Louis, Mo., USA; diluted in PBS) for 30 minutes. Sections were then incubated with rabbit anti-human A1M (K:323, IgG), diluted 1:7500 (in PBS containing 1% BSA, 0.02% Triton X-100 (Sigma, St. Louis, Mo., USA) for 16 hours at 4 C.

[0122] For chromogen visualization of A1M, sections were incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP, Dako Glostrup, Denmark) for 20 minutes at RT. The immunoreaction was performed via incubation in a diaminobenzidine (DAB) solution containing 0.03% H.sub.2O.sub.2, for 10 minutes at RT. Sections were rinsed in PBS (210 minutes) and counterstained with hematoxylin (Mayers, Hematoxylin Mayers Htx Histolab Products AB, Gothenburg, Sweden) followed by dehydration in a graded alcohol series and immersion in 100% Xylene. Sections were mounted and cover slipped in Pertex (Histolab Products AB, Gothenburg, Sweden).

[0123] For IF labeling of A1M, used for simultaneous detection of octreotide-647, sections were incubated with primary antibodies, as described above for chromogen detection, followed by secondary goat ant-rabbit IgG conjugated with Alexa Fluor 488 (AF488, Invitrogen, Molecular probes, USA), diluted 1:150 in PBS containing 1% BSA. Incubations were performed for 45 minutes at RT, followed by incubation in 4,6-diamidino-2-phenylindole (DAPI, nuclear labeling, Invitrogen, Molecular probes, USA) for 15 minutes at RT. A subset of sections was also incubated with phalloidin conjugated with Texas Red (binding to F-actin) for 1 hour at RT to morphologically delineate tubular structures. Sections were rinsed in PBS and were mounted and cover-slipped in antifade solution (Prolong Gold, Invitrogen, Molecular probes, USA).

Optical Detection of A1M and Octreotide-647 in Kidney Sections

[0124] Chromogen single labeled A1M was visualized and digitally documented in a bright-field microscope (Leica DMRE). Digital images were collected with a Leica digital camera (DFC 500). Images used for illustrations were corrected for color balance, brightness and contrast.

[0125] For simultaneous visualization of IF-labeled A1M (A1M-AF488) and octreotide-647 at histological and cellular levels a Zeiss confocal laser scanning microscope (CLSM, LSM 510 META, Dept. Biology, Lund University) was used. Sections were inspected via scanning of emission from Alexa Fluor 488 (A1M), HiLyte Fluor 647 (octreotide), Texas Red (F-actin) and DAPI (cell nuclei).

[0126] The digital image data, one or several optical section (Z-stacks), were collected through regions of the cortex, medulla and collecting ducts. The image documentation was used for calculations of the individual presence and co-existence of A1M-AF488 and octreotide-647 fluorescence. For documentation 3 kidney areas from each section were selected as representative regions (x/y stage position marked and stored by the acquisition software ZEN 2009). Scanning (at 10241024 frame size) was performed with a 20/0.8 Plan Apochromat objective (providing 1.8 m thick optical section with about 400 nm x/y optical resolution), and with a 63/1.4 oil immersion Plan Apochromat objective (providing 0.9 m thick optical sections with about 250 nm x/y optical resolution). The choices of optical sections and scan depths were determined from the centre position of the majority of nuclei (DAPI labeling) in the scanned field. Sequential scanning was performed of channels displaying AF488, HiLyte Fluor 647, DAPI and Texas Red fluorescence. For each channel the renal profile with the highest fluorescence intensities was selected as a reference, from which the acquisition settings (laser power, PMT detector gain, digital offset) were optimized and used for all scanned areas. Image illustrations presented here are chosen from the individual optical sections containing areas/structures used for the quantitative measurements.

Quantitative Image Analysis of Octreotide-647 and A1M in Kidney Sections.

[0127] CLSM images of octreotide-647 fluorescence and A1M-AF488 IF were used for the quantitative analysis. The detailed distribution in renal structures (from cortex, medulla and collecting ducts) and within ductular epithelial cells was investigated. Three (3) areas per CLSM image and between 10 and 20 images per animal were analyzed. The analysis protocol was developed to use as a macro-script with ImageJ software (Version 1.49 g, Rasband, W. S. ImageJ, NIH, USA). Briefly, the CLSM images were displayed with Stack to Images presenting composites of all channels merged together (for AF488, HL647, DAPI and Texas Red). The selection of sample areas were made from the morphology of tubular structures and depicted by nuclear and/or phalloidin labelling. Individual regions for measurement were delimited using the lasso function.

[0128] The green (A1M-AF488) and red (octreotide-647) channels were then used as input images and the ratio of separate green or red pixels or their co-localization was investigated using the co-localization plug-in. Statistical analysis was performed using Origin 9.0 software (Microcal, Northampton, Mass., USA). The histogram in FIG. 5 displays representative data obtained from 3 kidneys at each time-point and are plotted as meanSEM.

Results

Biodistribution

[0129] FIG. 1 shows in vivo biodistribution of .sup.111In-Octreotide at 10, 20, 40 and 60 minutes post-injection and the ex vivo biodistribution of .sup.125I-A1M at 10, 20, 40 and 60 minutes post-injection as well as 4 and 24 h post injection. Comparative uptake (% IA/g) of both molecules in the kidneys over time is illustrated as well. High uptake in the kidneys was observed for both .sup.111In-Octreotide and .sup.125I-A1M, with peak values at 10 and 20 minutes post-injection respectively. Size distribution of injected non-labelled A1M was investigated in blood serum and solubilized kidneys by SDS-PAGE and Western blotting. As shown in FIG. 2, A1M migrates as a homogeneous band with an apparent molecular mass around 25 kDa both in kidneys and serum at all times, and a minor, faint band around 50 kDa. The strong band most likely represents monomeric A1M with a theoretical molecular mass of 22.6 kDa and the latter the dimeric form. Highest amounts are seen at 10 minutes, supporting the kinetics of .sup.125I-labelled A1M shown in FIG. 1, lower panel. These results show that the A1M found in blood and kidneys is intact, full-length and that the degradation therefore is negligible.

SPECT/CT Image Analysis

[0130] A qualitative SPECT/CT analysis was performed for both .sup.111In-Octreotide and .sup.125I-A1M and visualizes the activity distribution in the kidneys. The SPECT/CT images in FIG. 6 demonstrate a high uptake in the kidneys for both molecules. While a visibly higher concentration of .sup.125I-A1M (FIGS. 6 C and D) in the peripheral kidney structures can be seen compared to .sup.111In-Octreotide (FIGS. 6 A and B), both molecules seem to colocalize in the kidney cortex. A slight uptake of .sup.125I-A1M in the thyroids can be seen as well. Activity pooled in the bladder has been omitted for .sup.111In-Octroetide since the strong signal makes it difficult to demonstrate distribution and uptake in kidneys.

Digital Autoradiography

[0131] Digital autoradiography results displayed in FIG. 7 clearly illustrates a localization of both molecules in the kidney cortex, mirroring the SPECT/CT results. It can be observed that results are similar at 20 and 60 minutes post-injection, indicating that the localization of the peptide and protein is completed after 20 minutes. No further sub-compartmentalization can be observed in these images; however, activity distribution is not completely homogenous in the cortex. A noticeable difference in contrast and resolution can be observed between the first two images (7A and B) and the second two (7C and D). Apart from its low-energy conversion electron (30.6 keV), .sup.125I also emits low energy x-ray photons at 27.5 and 27.2 keV. These photons contribute to the noise in the image and the proximity in energy to the conversion electrons makes them difficult to exclude from the final image.

Fluorescence Microscopy

[0132] A1M immunoreactivity was predominantly distributed in the cortex, with a decreasing immunoreactivity into the medulla and collecting ducts. Also, the intensities of A1M immunoreactive labelling was highest in the cortex, weaker in the medulla and weakest in the collecting ducts. The distribution of A1M immunoreactivity in selected areas of the kidneys 20 minutes p.i. is shown in FIGS. 8 and 9 (left panel). Strong labelling was present in a subset of tubular structures, morphologically depicted to compromise proximal tubules and subsets of glomeruli. Fluorescence double labelling of A1M and Octreotide-A647 demonstrated their tubular co-existence, as well as a high degree of cellular co-localization (FIGS. 5, 9 and 10). There was a strong labelling of both molecules, including high degree of tubular co-existence and cellular co-localization (see also FIG. 10), in the medulla and cortex 20 minutes p.i. There was a significant decrease in fluorescence detection of both molecules at 4 hours p.i., being very low in the cortex and medulla and absent in the collecting ducts (FIG. 9). A quantitative analysis of the tubular localization of both molecules was performed from CLSM images 20 minutes and 4 hours p.i., and showed a corresponding degree of co-localization 20 minutes p.i. (FIG. 5). When investigating the co-localization in tubular structures on a cellular level, as visualized and quantified in FIG. 5, similar results were seen in the cortex. The intracellular co-localization of A1M and Octreotide-A647 was demonstrated by comparisons of intensities for individual pixels in a designated profile (FIG. 5).

Example 2

[0133] Short-Term Radioprotection of Kidneys with A1M in PRRT

[0134] The materials and methods mentioned under Example 1 are also used in Example 2 unless otherwise stated below. [0135] Purpose: Initial testing of an 8-day .sup.177Lu-octreotide PRRT kidney damage mouse model including treatment with A1M [0136] Radionuclide activity: 150 MBq .sup.177Lu-DOTATATE (.sup.177Lu-DOTA0,Tyr3]octreotate) [0137] Radiation protection and safety: 150 MBq .sup.177Lu-DOTATATE gives 7 mSv/h at a distance of 1 cm. [0138] A1M dose: 7 mg/kg (in vehicle 2: 10 mM Tris-HCl, pH 8.0+0.125 M NaCl) [0139] A1M-therapy timepoint: T=0 and T=+60 min. NB, .sup.177Lu-DOTATATE and A1M are injected separately at T=0.

Analytic Methods:

[0140]

TABLE-US-00001 1. Blood Albumin & Creatinine glomerular filtration rate ELISA NGAL glomerulo-tubular nephritis ELISA Cytokines inflammation - Luminex - Lund KIM1 kidney injury molecule 1 ELISA A1M oxidative stress ELISA 2. Urine NGAL ELISA Albumin & Creatinine glomerulotubular damage ELISA Hepcidin ELISA KIM1 ELISA 3. Gene expression in kidneys A1M NGAL KIM1 Apoptosis/Necrosis specific genes to be determined Inflammation specific genes to be determined Microarray 4. Histochemistry general tissue damage 5. Immunohistochemistry Apoptosis TUNEL, caspase Gamma H2AX double strand breaks 6. Autoradiography 7. Organ activity measurements

Protocol:

[0141] Depending on availability (Lu-177-oct), groups will be prioritized as follows: 1 day, 8 days, 4 days.

TABLE-US-00002 Group n Treatment Time Sampling Analysis 1 6 Controls 0 Blood, urine Everything 2 4 177Lu-oct Day 1 Kidney 3 4 Day 4 (IHC + 4 4 Day 8 PCR) 5 4 Vehicle 2 Day 1 Other organs 6 4 Day 4 7 4 Day 8 8 4 177Lu-oct + A1M Day 1 9 4 Day 4 10 4 Day 8 11 4 A1M Day 1 12 4 Day 4 13 4 Day 8

Sample Handling:

[0142] 1. Blood: [0143] Sample blood in tubes. Centrifuge in accordance with instructions and transfer plasma/serum to new tube. Freeze in 80 C.

[0144] 2. Urine: [0145] Freeze in 80 C. immediately.

[0146] 3. Kidneys: [0147] a. PCRDissect one kidney into 2 pieces. Place in 2 different tubes (one for mRNA and one for protein extraction) and place on dry ice. [0148] b. Microscopy and autoradiography [0149] Some kidneys imaged in Copenhagen [0150] Immediately dry and freeze kidney in liquid nitrogen.

[0151] 4. Organ Activities: [0152] Use one kidney for activity analysis according to same protocol as previous studies. See also under point 6.

[0153] 5. SPECT Analysis: [0154] Is performed on group 4 (at least 3 animals) on day 8

[0155] 6. Additional Organs:

[0156] If possible, liver, spleen, heart, intestines, gut, brain, pancreas, lung and skin is also taken care of (placed on dry ice) and activity is analyzed according to previous protocol. Organs later used for protein analysis.

[0157] Results

[0158] In the first experiment, Balb/c (nu/nu) mice were injected with either 150 MBq 177Lu-octreotide (DOTATATE), 150 MBq 177Lu-octreotide (DOTATATE)+150 g A1M, or buffer only (control). The mice were left for 1 day or 8 days, and then sacrificed with sampling of plasma and urine, as described above. Thus, 6 groups (n=4) of samples were collected:

TABLE-US-00003 1. DOTATATE 1 day plasma urine 2. DOTATATE + A1M 1 day plasma urine 3. Control 1 day plasma urine 4. DOTATATE 8 days plasma urine 5. DOTATATE + A1M 8 days plasma urine 6. Control 8 days plasma urine

[0159] As an initial estimation of kidney functions, creatinine was measured in plasma, and albumin was measured in urine samples (FIG. 11). Plasma creatinine is a marker of glomerular filtration rate (GFR), and the results suggest that DOTATATE injection resulted in significantly increased creatinine levels, i.e. decreased GFR, both after 1 and 8 days. Simultaneous infusion of A1M restored the GFR to control levels, and the effect is highly significant after 8 days (P<0.01). To estimate proteinuria, albumin concentrations in urine were significantly elevated by DOTATATE injections both after 1 and 8 days, and were more pronounced after 1 day. Simultaneous A1M-treatment resulted in significantly reduced proteinuria after 1 day, but not after 8 days.

[0160] To summarize, 177Lu-octreotide injections resulted in compromised kidney functions (reduced glomerular filtration and proteinuria), which could be treated with co-treatment with A1M.