CLUSTERIN FOR USE IN THE TREATMENT OF THROMBOTIC MICROANGIOPATHIES

Abstract

The present invention concerns clusterin for use in the treatment of thrombotic microangiopathies, and a pharmaceutical composition comprising clusterin for use in the treatment of thrombotic microangiopathies, said composition not comprising von Willebrand factor protease. The present invention also concerns an ex vivo method for stratifying a patient suffering, or likely to be suffering, from TMA, comprising the following steps: 1) measuring, in a biological sample from said patient, the amount L.sub.C of clusterin, and 2) comparing the amount L.sub.c measured in step 1) with an amount L.sub.ref of clusterin by calculating the score S1=L.sub.C/L.sub.ref, in which: If S11, the patient is considered to be likely to benefit from a treatment of the TMA with clusterin, If S1>1, the patient is not considered to be likely to benefit from treatment of TMA with clusterin.

Claims

1. A method of treating a thrombotic microangiopathy comprising administering a therapeutically effective amount of clusterin to a subject in need of such treatment.

2. The method of claim 1, wherein said clusterin is a human clusterin of sequence SEQ ID No.: 1.

3. The method of claim 1, wherein said clusterin has a peptide sequence which has at least 70% identity with the sequence SEQ ID No.: 1.

4. The method of claim, wherein said clusterin is a recombinant clusterin, a plasma clusterin, or a synthetic clusterin.

5. The method of claim 1, wherein the thrombotic microangiopathy is thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS) associated or not associated with bacteria, HELLP syndrome, or a secondary thrombotic microangiopathy.

6. The method of claim 1, comprising oral or parenteral administration of said clusterin.

7. The method of claim 6, wherein said parenteral administration is intravenous administration, intramuscular administration, or subcutaneous administration.

8. A pharmaceutical composition comprising clusterin, said composition not comprising von Willebrand factor of protease.

9. The pharmaceutical composition as claimed in claim 8, wherein said clusterin is a human clusterin of sequence SEQ ID No.: 1, or a peptide sequence which has at least 70% identity with the sequence SEQ ID No.: 1.

10. The pharmaceutical composition as claimed in claim 8, said pharmaceutical composition being in the form of an injectable solution.

11. An ex vivo method for stratification of a patient who is or who may be suffering from a thrombotic microangiopathy (TMA), comprising the following steps: 1) measuring, in a biological sample from said patient, the clusterin level L.sub.C, and 2) comparing the level L.sub.C measured in step 1) with a clusterin level L.sub.ref by calculating the score S1=L.sub.C/L.sub.ref, wherein: if S11, the patient is considered to be liable to receive a benefit from a TMA treatment with clusterin, if S1>1, the patent is not considered to be liable to receive a benefit from a TMA treatment with clusterin.

12. The method of claim 11, further comprising administering clusterin to the subject.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0060] FIG. 1 represents the clusterin binding to histones. Histones H1, H2A, H2B, H3 and H4 (New England Biolabs, Ipswich, Mass.) or genomic DNA from human cells, were immobilized at the bottom of ELISA plates (1 g/ml) in 10 mM carbonate/bicarbonate buffer, pH=9.6. After saturation, the plates were incubated with 1 g/ml of recombinant human clusterin (R&D Systems, Abingdon, UK) or of human serum albumin (HSA; Sigma, St Louis, Mo.), coupled to biotin. The binding of Clu or of HSA to the histones was demonstrated with peroxidase-coupled streptavidin. The results are expressed as optical density (meanSD, n=5). The HSA molecule was used as control protein. The binding of the genomic DNA in the ELISA plates was verified with a peroxidase-coupled anti-DNA antibody (data not shown).

[0061] FIG. 2 represents the ELISA quantification of IL-6, in pg/ml, in the culture supernatants of human monocytes isolated from the blood of healthy donors by magnetic sorting then incubated for 12 h with 12.5 g/ml of histones, in the presence (diagram 4) or in the absence (diagram 3) of 25 g/ml of clusterin (CLU). Diagram 1 is the control of the culture of monocytes alone, and diagram 2 is the control of the culture of monocytes incubated with 25 g/ml of clusterin.

[0062] FIG. 3 represents the percentage of apoptotic cells measured in the case of human endothelial cells incubated for 15 min either (1) left-hand graph: in the presence of control sera (HNS) in the presence or absence of histones (50 ng/ml) (Histones from Roche), or (2) right-hand graph: with sera from patients containing endogenous histones (SS; n=5, 10% vol/vol) in the presence or absence of 200 ng/ml of Clu.

[0063] FIG. 4 represents the assaying of serum clusterin, in g/ml, in patients suffering from TMA and healthy patients. (A) The serum clusterin is measured in healthy patients (squares) and TMA patients either at the beginning of the TMA (solid circles) or 3 months after diagnosis (empty circles). (B) The change in serum clusterin in TMA patients at diagnosis (points on the left) and 3 months after diagnosis (points on the right). Each point represents one patient. The statistical analysis was carried out by means of nonparametric tests.

[0064] FIG. 5 represents (A) for the patients suffering from typical HUS, the assaying of serum clusterin in g/ml, at TMA diagnosis (left) and after 3 months of treatment (right) with Eculizumab (Soliris); (B) for the patients suffering from typical HUS, the circulating nucleosomes level (AU) at TMA diagnosis (left) and after 3 months of treatment (right) with Eculizumab (Solids). The ST-mediated TMA patients (typical HUS) were treated uniformly with Eculizumab (Soliris) according to the same treatment scheme. (C) and (D) for the patients suffering from complement-mediated TMA, the circulating nucleosome level (AU) at TMA diagnosis (left) and after 3 months of treatment (right) with Eculizumab (Solids); the complement-mediated TMA patients were treated variably according to the clinical context.

[0065] FIG. 6 represents the concentration of platelets, in g/l (dark gray), of serum clusterin in g/ml (light gray) and of circulating nucleosomes in AU (medium gray), in 7 patients suffering from TMA, from the 1.sup.st to the 8.sup.th Eculizumab injection of the patients (tHUS, n=7). The serum clusterin, the circulating nucleosomes and the platelet concentration were measured just before each Eculizumab injection. Eculizumab was injected repeatedly every 2 weeks. The 8.sup.th injection took place after an average period of 2 months from the diagnosis of a HUS.

[0066] FIG. 7 represents A: the LDH concentration (IU/ml), B: the circulating nucleosome concentration (AU), C: the platelet concentration (g/l), D: the hemoglobin concentration (g/dl), E: the ADAMTS13 concentration (% activity) and F: the percentage IECAE (an arbitrary unit measuring the functional activity of the common terminal complement pathway by ELISA assay, the company Diasorin) as a function of the serum clusterin concentration (g/ml) in a patient at the time of diagnosis of a tHUS (shiga toxin-mediated HUS).

[0067] FIG. 8 represents (A): the assaying of the serum clusterin, in g/ml, in patients suffering from shiga toxin-mediated TMA, tHUS (n=8), patients suffering from complement-mediated TMA, aHUS (n=6) and healthy subjects (n=14); (B) the circulating nucleosome level, in AU, in patients suffering from shiga toxin-mediated TMA (n=8), patients suffering from complement-mediated TMA (n=6) and healthy subjects (n=14).

EXAMPLES

Example 1: Clusterin Neutralizes the Actions of Histones by Blocking the Production of Pro-Inflammatory Molecules

[0068] Human monocytes were isolated from the blood of healthy donors by magnetic sorting (positive sorting on the basis of expression of the CD14 marker CD14; Miltenyi Biotech, Bergisch Gladbach, Germany). The monocytes (210.sup.6 cells/ml) were incubated for 12 h with 12.5 g/ml of histones (Sigma), in the presence or absence of 25 g/ml of clusterin (CLU). The IL-6 was quantified by ELISA (Diaclone, Besanon, France) in the culture supernatants. The results are expressed in pg/ml (meanSD, n=6).

[0069] The results are shown in FIG. 2.

Example 2: Clusterin Protects the Endothelial Cells Against Death Induced by Histone-Containing Sera

[0070] Human endothelial cells (HDMECs from Promocell) are incubated for 15 min either in the presence of control sera (HNS; idem) in the presence or absence of histones (50 ng/ml Histones from Roche), or with patient sera containing endogenous histones (SS; n=5, 10% vol/vol) in the presence or absence of 200 ng/ml of Clu. After 15 min, the endothelial cells are labeled with annexin V and propidium iodide (Becton Dickinson kit) in order to evaluate the percentage of apoptotic cells by flow cytometry.

[0071] The results are shown in FIG. 3.

Example 3: The Amount of Clusterin is Associated with the Activity of the TMA

[0072] The serum clusterin levels are lower in the patients suffering from TMA at diagnosis than in the healthy subjects. The serum clusterin level was assayed in 11/14 patients at the time of the 3.sup.rd month after TMA diagnosis, after treatment. It did not differ significantly from that of the population of healthy subjects, but was significantly higher than at diagnosis. The clusterin was assayed by ELISA (R&D Systems) in sera from healthy subjects and from patients suffering from TMA.

[0073] The results are shown in FIG. 4.

Example 4: The Clusterin Deficiency is Observed in Various Types of TMA

[0074] An assay of the clusterin, the histones and a marker for activation of the common terminal pathway in the serum is carried out in patients suffering from typical HUS (20 cases) or atypical HUS (10 cases), from TTP (30 cases) and/or from HELLP syndrome (50 cases), at diagnosis and as follow up. These assays are carried out using commercial ELISA techniques.

[0075] The expected results make it possible to establish statistical associations and/or correlations with the main patient follow-up biological criteria (platelet level, LDH, haptoglobin level, markers for renal damage (creatininemia, albuminuria/creatininuria, etc.) or markers for extrarenal damage if applicable (troponin, ASAT/ALAT, gammaGT etc.).

Example 5: Clusterin is an Endothelial Cytoprotector in TMA Syndromes

[0076] Microvascular endothelial cells are incubated in the presence of sera from patients suffering from HUS, from TTP or from HELLP syndrome with or without addition of recombinant clusterin (target dose 25 g/ml) for 2 h, 6 h or 24 h. The analysis of cell death (apoptotic or necrotic cell death) is carried out by flow cytometry labeling (annexin-5, propidium iodide).

[0077] It is expected that the histones and nucleosomes circulating in the sera of patients suffering from TMA cause endothelial lesions, and that the addition of clusterin protects the endothelial cells against death induced by these toxic elements.

Example 6: Clusterin Demonstrates an In Vivo Effect in a Murine Model of Typical HUS

[0078] Two types of analyses are carried out. [0079] 1) Study of STX-induced renal toxicity in C57BL/6J mice wherein the clusterin gene has been knocked out (KO Clu) so as to demonstrate a worsening of the renal toxicity and of the biological markers for HUS (thrombocytopenia, anemia) in the absence of clusterin

[0080] In a first part of the experiments, a sublethal dose is desired in order to monitor more accurately the HUS markers, namely anemia, thrombocytopenia and renal failure. The doses of 625 pg/g, 300 pg/g and 100 pg/g are tested on wild-type animals (10 mice per dose, plus 10 control mice). The mice are weighed daily, and the weight is used as an indicator of toxicity since, in the murine model, weight loss correlates with the disease.

[0081] Once the dose has been established, WT mice and clusterin-KO mice are injected intraperitoneally with the chosen dose of STX or with PBS and are then monitored by taking a blood sample on which a complete blood count is carried out. Hemoglobin is assayed in parallel in order to calculate an anemia index. The serum is also analyzed for renal failure markers (creatinine and cystatin). The kidneys are removed in order to analyze the deposits of C3 protein, which is a marker for renal lesions. [0082] 2) Study of the protective and therapeutic role of clusterin in TMA in a murine model of HUS in normal C57BL/6J mice (not clusterin deficient)

[0083] The WT mice are injected intraperitoneally with the chosen dose of STX or with PBS. The mice are treated with regular injections of recombinant murine clusterin (1 mg/kg). Monitoring is carried out by taking a blood sample on which a complete blood count is carried out. Hemoglobin is assayed in parallel in order to calculate an anemia index. The serum is also analyzed for the renal failure markers (creatinine and cystatin). The kidneys are removed in order to analyze the deposits of C3 protein, which is a marker for renal lesions.

[0084] It is expected that: [0085] there is a correlation between the absence of clusterin and the worsening of renal toxicity and of the biological markers of HUS (thrombocytopenia, anemia) [0086] the results make it possible to demonstrate the non-redundant role of clusterin in controlling shiga toxin-induced toxicity in a murine model [0087] the results demonstrate a protective and therapeutic role of clusterin in TMA in a murine model of HUS and demonstrate an improvement in renal function and in the hemolysis and thrombocytopenia parameters of the mice receiving supplementation compared with the control group.