METHOD FOR TREATING BLOOD, BLOOD PRODUCTS AND ORGANS

Abstract

The invention relates to the treatment of blood, blood products and organs for the removal and/or detoxification of amyloid-beta oligomers.

Claims

1. A method for treating blood, a blood product and/or an organ in vitro or ex vivo, wherein the method comprises removing the blood, blood product and/or organ from the human or animal body and removing and/or detoxifying amyloid-beta oligomers from the removed blood, blood product and/or organ, and wherein the method comprises contacting the blood, blood product and/or organ with one or more compounds which comprise one or more of: a) rprtrlhthrnr (D3 peptide, SEQ ID NO:73), kqhhveygsdhrfead (retro-inverso-A-beta(1-16), SEQ ID NO:2) or an antibody which aa) binds to a retro-inverse sequence of an amyloid beta-peptide or amyloid beta-peptide part fragments and/or bb) binds to a multimerization domain of an amyloid beta-peptide and also to the amyloid beta-peptide and/or cc) binds to a sequence according to the invention or homologous sequences thereof; b) a hybrid compound of formula A-B, where A is an aminopyrazole or a derivative thereof, and B is a peptide selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64. SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79 and A and B are covalently bonded to one another directly or by a linker; c) a standard molecule, which is a polymer of a polypeptide sequence according to the invention and/or is composed of copies of an amino-terminal part of an A-beta peptide; d) a standard molecule comprising or composed of copies of an amino-terminal part of an A-beta peptide selected from part regions A-beta 1-11, A-beta 3-11, or pyroGluA-beta 3-11; e) a dimer, polymer and/or multimer comprising at least two monomers which bind to amyloid-beta oligomers; f) rprtrlhthrnrrprtrlhthrnr (D3D3, SEQ ID NO:13), rprtrlhthrnrnwnrprtrlhthrnr (D3nwnD3, SEQ ID NO:14), (rprtrlhthrnr).sub.2-PEG3 (double-D3-free-Ntermini, SEQ ID NO:15), and/or PEG5-(rprtrlhthrnr).sub.2 (double-D3-free-Ctermini, SEQ ID NO:16), double-D3-free-Ntermini: (rprtrlhthrnr)2- (SEQ ID NO:63), double-D3-free-Ctermini: (rprtrlhthrnr)2 (SEQ ID NO:64), DB 3: rpitrlrthqnr (SEQ ID NO: 65),RD 2: ptlhthnrrrrr (SEQ ID NO: 66), RD 1: pnhhrrrrrrtl (SEQ ID NO: 67), RD 3: rrptlrhthnrr (SEQ ID NO: 68), D3-delta-hth: rprtrlrnr (SEQ ID NO:69), NT-D3: rprtrl (SEQ ID NO: 70), DB 1: rpitrlhtnrnr (SEQ ID NO: 71), DB 2:rpittlqthqnr (SEQ ID NO: 72), DB 5: rpitrlqtheqr (SEQ ID NO. 74), D3-delta-hth D3-delta-hth: rprtrlrnrrprtrlrnr (SEQ ID NO:75), RD 2- RD 2: ptlhthnrrrrrptlhthnrrrrr (SEQ ID NO: 76), DO 3: sgwhynwqywwk (SEQ ID NO:77), rprtrsgwhynwqywwkrnr (SEQ ID NO:78) and ptlsgwhynwqywwkrrrrr (SEQ ID NO:79), and homologs thereof; g) SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:73, and homologs thereof.

2. The method of claim 1, wherein the method further comprises examining the blood, blood product and/or organ for the presence of amyloid-beta oligomers prior to removing the blood, blood product and/or organ from the human or animal body.

3. The method of claim 1, wherein the one or more compounds are arranged as capture molecules on a support, via which a sample containing the blood, blood product and/or organ is passed.

4. The method of claim 3, wherein the capture molecules are fixed onto beads.

5. The method of claim 3, wherein the capture molecules are immobilized on nanomagnets.

6. The method of claim 3, wherein the capture molecules are arranged in a dialysis system.

7. The method of claim 3, wherein the support for the capture molecules is made of a biocompatible material.

8. The method of claim 3, wherein the support for the capture molecules is selected from one or more of membranes, filters, filter sponges, beads, rods, cords, column, hollow fibers.

9. The method of claim 1, wherein a kit is used for a selective quantification of A-beta aggregates and/or for treating (in vitro) blood, a blood product and/or an organ, the kit comprising one or more of: a substrate made of glass which is coated with a hydrophobic material; a standard; a capture molecule; a probe; a substrate with capture molecule; one or more solutions; a buffer.

10. The method of claim 1, wherein the one or more compounds are passed as capture molecules ex vivo over and/or through an organ.

11. The method of claim 1, wherein the method comprises removing amyloid-beta oligomers from blood, a blood product and/or an organ.

12. The method of claim 11, wherein Alzheimer's disease is treated.

13. The method of claim 1, wherein the method comprises detoxifying amyloid-beta oligomers present in blood, a blood product and/or an organ.

14. The method of claim 13, wherein Alzheimer's disease is treated.

15. The method of claim 1, wherein the one or more compounds comprise ptlhthnrrrrr (RD 2; SEQ ID NO: 66) and/or a homolog thereof.

16. The method of claim 15, wherein the one or more compounds further comprise one or more hybrid compounds of formula A-B, where A is an aminopyrazole or a derivative thereof, and B is a peptide selected from SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, and A and B are covalently bonded to one another directly or by a linker.

17. The method of claim 15, wherein the one or more compounds further comprise one or more linkers.

18. The method of claim 15, wherein the method further comprises examining the blood, blood product and/or organ for the presence of amyloid beta oligomers prior to removing the blood, blood product and/or organ from the human or animal body.

19. The method of claim 1, wherein the one or more compounds are arranged as capture molecules on a support, via which a sample containing the blood, blood product and/or organ is passed.

20. The method of claim 15, wherein the method comprises removing amyloid-beta oligomers from blood, a blood product and/or an organ.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0172] In the accompanying drawings,

[0173] FIG. 1 is a diagram showing the relative fluorescence as a function of time in the experiment described below in the absence and presence of preformed Abeta aggregates which have formed without addition of inhibitor substances;

[0174] FIG. 2 is a diagram showing the relative fluorescence as a function of time in the experiment described below in the absence and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of a first inhibitor substance;

[0175] FIG. 3 is a diagram showing the relative fluorescence as a function of time in the experiment described below in the absence and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of a second inhibitor substance;

[0176] FIG. 4 is a diagram showing the relative fluorescence as a function of time in the experiment described below in the absence and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of a third inhibitor substance;

[0177] FIG. 5 is a diagram showing the relative fluorescence as a function of time in the experiment described below in the absence and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of a fourth inhibitor substance;

[0178] FIG. 6 is a diagram showing the relative fluorescence as a function of time in the experiment described below in the absence and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of a fifth inhibitor substance; and

[0179] FIG. 7 is a diagram showing the relative fluorescence as a function of time in the experiment described below in the absence and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of a sixth inhibitor substance.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

EXAMPLES

[0180] 1. ThT Seeding Assay: Principles

[0181] Amyloidogenic peptides have the ability to form amyloid fibrils. This can occur spontaneously with a certain probability. If no amyloid germs are present, it may take some time until the first amyloid fibrils are formed, the formation and replication of which can be quantitatively monitored with the help of the fluorescence from Thioflavin T (ThT). ThT interacts with A-beta fibrils and the fibril-dye complex exhibits increased fluorescence (em: 450 nm, ex: 490 nm). The time until the ThT signal begins to increase is called the lag phase. This lag phase can be avoided or greatly shortened if amyloid germs, also called seeds, are added to the aggregation batch. A known example is the addition of prion-containing brain material to a solution of monomeric recombinant prion protein, which then forms ThT-positive fibrils with a significantly shortened lag phase. A further example consists in the addition of a small amount of Abeta-amyloid fibrils to a solution of nonaggregated A-beta peptide (Abeta). Here too, the lag phase to the formation of ThT-positive Abeta fibrils is significantly shortened.

[0182] Consequently, this test (called ThT seeding assay) allows any desired substances or substance mixtures to be examined as to their content of germ-able amyloids. If the substance mixture added to the aggregation batch contains seed-able amyloids, this results in the lack or shortening of the lag phase. This in vitro property is often considered analogously to the prion-like infectiosity or transmissibility in vivo.

[0183] In the first control experiment it was investigated whether preformed Abeta aggregates, being aggregation germs, actually shorten the lag phase of the aggregation of freshly dissolved Abeta. For this purpose, the ThT fluorescence of freshly dissolved Abeta was monitored in the absence and presence of preformed Abeta aggregates. Then, Abeta aggregates were prepared which were formed from a mixture of Abeta(1-42) and an inhibitor substance (in this example in each case one of the D-peptides D3, DB1, DB2, DB3, DB4, DB5), which should interfere in the formation of amyloid fibrils. The Abeta-inhibitor coaggregates formed in this way were added just like the amyloid Abeta aggregates to a ThT seeding assay in order to determine the remaining amyloid potential of these coaggregates.

[0184] 2. ThT Seeding Assay: Experimental Details

[0185] 20 M of A-beta peptide (1-42) were preincubated together with one of the inhibitor substances (20 M) for 7 days at 37 C. in 10 mM NaPi pH 7.4. The sample was then centrifuged (20 min, 16 100g), the aggregate pellet was washed 3 and resuspended in 10 mM NaPi pH 7.4. In an analogous manner, A fibrils were produced without inhibitor as positive control. Directly before the actual ThT seeding assay, fresh A (20 M in 10 mM NaPi pH 7.4) was mixed (80:20 volume fractions) with the resuspended aggregation germs which had formed in the presence or absence of the inhibitor substances, and 10 M ThT were added thereto. The reference used was fresh A solution which was mixed with buffer solution without aggregation germs (80:20 volume fractions) and comprised 10 M ThT. 50 l of the respective reaction solution were pipetted into an indentation of a black 384-well microtiter plate. The ThT fluorescence was measured every 30 min over 20 h at an excitation wavelength of 440 nm and an emission wavelength of 490 nm. For the evaluation, the fluorescence intensity was corrected by subtraction of the 20% added aggregation germs and the average value was calculated. An eight-fold determination was carried out.

[0186] 3. Procedure:

[0187] FIG. 1 shows the course over time of the ThT fluorescence in the absence (solid line) and presence of preformed Abeta aggregates which have formed without the addition of inhibitor substances.

[0188] It can be clearly seen that these aggregates significantly increase the rate of the aggregation of fresh Abeta (t of about 4 h), i.e. clearly exhibit a seeding effect.

[0189] FIG. 2 shows the course over time of the ThT fluorescence in the absence (solid line) and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of the inhibitor substance D3.

[0190] It can be clearly seen that these aggregates are not able to increase the rate of the aggregation of fresh Abeta, i.e. clearly show no seeding effect.

[0191] FIG. 3 shows the course over time of the ThT fluorescence in the absence (solid line) and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of the inhibitor substance DB1.

[0192] It can be clearly seen that these aggregates are not able to increase the rate of the aggregation of fresh Abeta, i.e. clearly exhibit no seeding effect.

[0193] FIG. 4 shows the course over time of the ThT fluorescence in the absence (solid line) and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of the inhibitor substance DB2.

[0194] It can clearly be seen that these aggregates are not able to increase the rate of the aggregation of fresh Abeta, i.e. clearly exhibit no seeding effect.

[0195] FIG. 5 shows the course over time of the ThT fluorescence in the absence (solid line) and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of the inhibitor substance DB3.

[0196] It can clearly be seen that these aggregates are not able to increase the rate of the aggregation of fresh Abeta, i.e. clearly exhibit no seeding effect.

[0197] FIG. 6 shows the course over time of the ThT fluorescence in the absence (solid line) and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of the inhibitor substance DB4.

[0198] It can clearly be seen that these aggregates are not able to increase the rate of the aggregation of fresh Abeta, i.e. clearly exhibit no seeding effect. Moreover, the DB4-Abeta coaggregates even appear to reduce the formation of ThT-positive Abeta aggregates in the later phase.

[0199] FIG. 7 shows the course over time of the ThT fluorescence in the absence (solid line) and presence of preformed Abeta aggregates which have formed before the start of the experiment with the addition of the inhibitor substance DB5.

[0200] It can be seen that these aggregates are not able to increase the rate of the aggregation of fresh Abeta, i.e. clearly exhibit no seeding effect.

[0201] 4. Summary of the Results:

[0202] All of the tested -peptides formed aggregates with Abeta which were no longer able to shorten the lag phase of the aggregation of freshly dissolved Abeta. These coaggregates are thus not amyloidogenic.