ALPHA-1-MICROGLOBULIN FOR USE IN THE PROTECTION OF KIDNEYS IN CONNECTION WITH USE OF CONTRAST MEDIA

Abstract

This invention relates to an alpha-1-microglobulin for use in prevention of contras medium nephropathy.

Claims

1-16. (canceled)

17. A method for treating or reducing the risks of side effects of medical contrast media, comprising administering an effective amount of alpha-1-microglobulin (A1M) to a subject in need thereof.

18. The method of claim 17, wherein the side effects comprise kidney-associated side effects.

19. The method of claim 17, wherein the side effects comprise medical contrast media-induced nephropathy (CIN).

20. The method of claim 17, wherein the side effects comprise oxidative stress.

21. The method of claim 17, wherein the subject exhibits one or more of the following risk factors: age>75 years, chronic kidney disease (CKD), diabetes mellitus, hypertension, metabolic syndrome, anemia, multiple myeloma, hypoalbuminemia, renal transplant, and hypovolemia and decreased effective circulating volumes as evidenced by one or more of congestive heart failure (CHF), ejection fraction (EF) of less than 40%, hypotension, and intra-aortic balloon counterpulsation.

22. The method of claim 17, wherein the subject exhibits one or more of the following risk factors: CKD and diabetes mellitus.

23. The method of claim 17, wherein the A1M is administered prior to, essentially at the same time as, during, or after administration of a medical contrast medium.

24. The method of claim 17, wherein the A1M is administered at the latest 24 hours before administration of a medical contrast medium.

25. The method of claim 17, wherein the A1M is administered at the most 1 hour before a medical contrast medium is administered and not later than concomitant with the medical contrast medium.

26. The method of claim 17, wherein the A1M is administered when a reliable marker for kidney function indicates kidney damage.

27. The method of claim 17, wherein the A1M is administered within a time period of from about 12 to about 48 hours after administration of a medical contrast medium.

28. The method of claim 17, wherein the A1M has at least 80% sequence identity to SEQ ID NO:1 or SEQ ID NO:2.

29. The method of claim 29, wherein the A1M has an amino acid sequence having the following amino acid residues at the following positions corresponding to the amino acid positions of human wildtype A1M set forth in SEQ ID NO:1: Y22 C34 K69 K92 K118 K130 Y132 L180 I181 P182 and R183.

30. A kit comprising (a) alpha-1-microglobulin (AIM) and (ii) a medical contrast medium.

31. The kit of claim 31, wherein the A1M is provided in a pharmaceutical composition.

32. The kit of claim 31, further containing instructions for use in a method for treating or reducing the risks of side effects of medical contrast media in a subject in need thereof.

33. The kit of claim 31, wherein the A1M has at least 80% sequence identity to SEQ ID NO:1 or SEQ ID NO:2.

34. The kit of claim 34, wherein the A1M has an amino acid sequence having the following amino acid residues at the following positions corresponding to the amino acid positions of human wildtype A1M set forth in SEQ ID NO:1: Y22 C34 K69 K92 K118 K130 Y132 L180 I181 P182 and R183.

Description

LEGENDS TO FIGURES

[0096] FIG. 1 shows the biodistribution of .sup.125I-A1M (upper left) in normal NMRI mice. Lower left image shows uptake over time in the kidneys. Data are presented as % IA/g from 4 animalsSEM.

[0097] 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.

[0098] 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 J445, 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.

[0099] 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.

[0100] FIG. 5 shows qualitative SPECT/CT analysis for .sup.125I-A1M and visualizes a predominat activity distribution in the kidney cortex, seen from sagittal and dorsal views. A slight uptake of .sup.125I-A1M in the thyroids can be seen as well.

[0101] FIG. 6 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.

[0102] FIG. 7 shows the sequences SEQ ID 1-4.

EXPERIMENTAL

Materials and Methods

Recombinant Human A1M

[0103] 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

[0104] 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.

Animal Studies

[0105] 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

[0106] Biodistribution studies were conducted to determine the pharmacokinetics and biodistribution of .sup.125I-A1M. .sup.125I-A1M (100 kBq, 1 g) was 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 post-injection and blood and organs were sampled, weighed and measured in a NaI(Tl) 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).

Western Blotting

[0107] 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 TransBlot 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], 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

[0108] 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 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.

KidneySample Preparation and Immunolabeling of A1M

[0109] Following i.v. injection of 150 g A1M (unconjugated) 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.

[0110] After euthanization, kidneys were removed directly frozen and embedded in Tissue Tec. The tissue blocks were sectioned in a cryostat (Microm, HM 5000M, 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 chromogen immunohistochemistry (IHC). 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.

[0111] For 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), and then 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.

[0112] The sections were then 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).

[0113] 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.

Results

Biodistribution

[0114] FIG. 1 shows 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. High uptake in the kidneys was observed for .sup.125I-A1M, with peak value at 10 minutes post-injection. 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

[0115] A qualitative SPECT/CT analysis was performed for .sup.125I-A1M and visualizes the activity distribution in the kidneys. The SPECT/CT images in FIG. 5 demonstrate a high uptake in the kidneys. .sup.125I-A1M (FIGS. 6 C and D) seems to localize in the kidney cortex. A slight uptake of .sup.125I-A1M in the thyroids can be seen as well.

[0116] IHC microscopical analysis (FIG. 6) shows that the infused A1M is mainly localized to the kidney cortex with gradually decreasing immunoreactivity towards the medulla and collecting ducts. Strong immunostaining of A1M can mainly be seen in the proximal tubular structures and subsets of glomeruli.