TREM-1 INHIBITORS FOR THE TREATMENT OF VASO-OCCLUSIONS AND TISSUE INJURIES IN PATIENTS SUFFERING FROM SICKLE CELL DISEASE

Abstract

Sickle cell disease (SCD) is a single gene disorder characterized by mutant hemoglobin-S(HbS) and chronic intravascular haemolysis. Painful vaso-occlusive crises (VOC) are typical of SCD and often associated to a further rise in hemolysis. VOC is the clinically painful form of vaso-occlusion, that is due to the aggregation of red blood cells in the capillaries and venules. Such event is promoted or aggravated by adhesion of polymorphonuclear neutrophils (PMNs) to red blood cells and the endothelium leading to tissue ischemia, inflammation and imperfect repair. Repeated vaso-occlusion and PMNs interactions with the vascular endothelium are thought to promote microvascular injuries in SCD patients. The inventors tested the effect of pharmacological inhibition of TREM-1 with LR12 peptide in two experimental vaso-occlusive crisis models. Additional validation of TREM-1 involvement in vaso-occlusion was verified using mice with sickle cell disease and Trem-1 gene deficiency. In particular, the inventors showed that TREM-1 inhibition is particular suitable for limiting the severity of vaso-occlusions. The results obtained by the inventors also suggest that plasmatic concentration of sTREM-1 could be a reliable biomarker for predicting vaso-occlusions and/or SCD-associated organ dysfunction and end-organ damage.

Claims

1. A method of treating a vaso-occlusion in a patient suffering from sickle cell disease comprising administering to the patient a therapeutically effective amount of a TREM-1 inhibitor.

2. The method of claim 1 wherein the TREM-1 inhibitor is a peptide selected from the group consisting of 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 and SEQ ID NO:11.

3. A method of determining whether a patient suffering from sickle cell disease has or is at risk of having vaso-occlusion comprising determining the level of sTREM-1 in a blood sample obtained from the patient and administering a TREM-1 inhibitor to the patient when the level of sTREM-1 in the blood sample is higher than the level in healthy control subjects.

4. The method according to claim 3 wherein vaso-occlusion includes vaso-occlusive crisis or acute chest syndrome.

5. A method of determining whether a patient suffering from sickle cell disease has or is at risk of having chronic vascular and tissue injury comprising determining the level of sTREM-1 in a blood sample obtained from the patient and administering a TREM-1 inhibitor to the patient when the level of sTREM-1 in the blood sample is higher than the level in healthy control subjects.

6. The method according to claim 5 wherein the chronic vascular and tissue injury include sickle cell nephropathy, progressive cerebrovascular remodeling, cognitive impairment, osteonecrosis, leg ulcers, retinopathy, and pulmonary arterial hypertension.

Description

FIGURES

[0062] FIG. 1: Results of intravital microscopy obtained by observation of neutrophils in the post-capillary venous microcirculation of the cremaster of HbSS knock-in sickle cell mice, after injection of TNFα. (A-C) The LR12 allows a significant reduction in neutrophil rolling between 3 h and 4 h30 post-injection of TNFα (A, 2way ANOVA P value=0.0041), their adhesion throughout the experiment (B, 2way ANOVA P value <0.0001), and their transmigration from 3 h to 5 h post-TNFα (C, 2way ANOVA P value <0.0001). (D-I) Post-microscopy blood count. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. SS (n=5), SS LR12 (n=6), AA (n=5).

[0063] FIG. 2: Results of intravital microscopy obtained by observation of neutrophils in the post-capillary venous microcirculation of cremaster of Trem-1 gene knock-out (TREM-1 KO) sickle cell mice (SAD background), after injection of TNFα at TO. (A-C) The genetic deletion of Trem-1 gene allows a significant reduction in neutrophil adhesion throughout the experiment (B, 2way ANOVA P value <0.0001), and their transmigration (C, 2way ANOVA P value <0.0021). *=SAD TREM-1+/+vs SAD TREM-1−/−. * P<0.05, ** p<0.01, **** p<0.0001. SAD TREM-1+/+(n=5), SAD TREM-1−/−(n=5), wild-type (WT) (C57BL/6J) TREM-1+/+(n=4), WT TREM-1−/−(n=4)).

[0064] FIG. 3: LR12 administration promoted a significant reduction vascular congestion with Ter 119+erythrocytes in sickle cell mice as observed in histological sections of liver (A), spleen (B) and renal medulla (D), but not in lung sections (C). * p<0.05, **** p<0.0001. Scale=50 μm. SS PBS (n=12), SS LR12 (n=14).

[0065] FIG. 4: LR12 administration induced a significant reduction in the abundance of neutrophils on histological sections of liver (A) and lung (B) from sickle cell mice. The amount of PMNs is expressed as % green fluorescent surface. * p<0.05, ** p<0.01. SS PBS (n=8), SS LR12 (n=7).

[0066] FIG. 5: Plasma concentrations of sTREM-1 are significantly higher in SS sickle cell mice than in mice carrying normal AA hemoglobin. *: p<0.05.

EXAMPLE

[0067] Methods:

[0068] Mouse Model and Study Design.

[0069] Experiments were performed on 4-6 week-old sex matched healthy control (Hba.sup.tm1(HBA)Tow Hbb.sup.tm3(HBG1,HBB) Tow) (HbAA (h.sub.α/hα::β.sup.A/β.sup.A)) and SCD (Hba.sup.tm1(HBA)Tow Hbb.sup.tm2(HBG1,HBB*)Tow) (HbSS (hα/hα::βS/βS)) mice as well as SAD transgenic sickle mice with or without Trem-1 gene deletion (TREM-1 KO). The animal protocol was approved by the Paris Descartes University Animal Care and Use Committee and the French Ministry of Agriculture. Animals were anesthetized with isoflurane, and whole blood was collected from each mouse via retro-orbital venipuncture by heparinized microcapillaries. In anesthetized animals, organs were immediately removed and divided into two and either immediately frozen in liquid nitrogen or fixed in 10% formalin and embedded in paraffin for histology.

[0070] In order to test the pharmacological inhibition of TREM-1 on erythrocyte vascular congestion and neutrophils infiltrates, homozygous HbS (SS) mice were subjected to 14 hours hypoxia (8% oxygen) and then 1 hours reoxygenation (21% oxygen) to trigger acute vaso-occlusive crisis as previously described (Sabaa N et al. J Clin Invest. 2008 May; 118(5):1924-33). The TREM-1 inhibitory peptide for each of the hypoxia-reoxygenation experiments was administered by intraperitoneal injection just prior to the onset of hypoxia, as well as at the end of hypoxia, at the beginning of the reoxygenation phase. These conditions reproduce a situation of severe hypoxia for SCD, provoking crises and damage related to ischemia-reperfusion. After this experiment, the tissues were analyzed in the same way as for the SS mice in the base state.

[0071] Intravital Video Microscopy Experimental Protocol:

[0072] To characterize in vivo the effects of the TREM-1 pathway on neutrophils recruitment, we used a protocol as previously described (Koehl B et al. Haematologica. 2017 Jul; 102(7):1161-1172). Male mice were anaesthetized by spontaneous inhalation of isoflurane and maintained at 37° C. with a heating pad. The left jugular vein was cannulated to administer drugs and antibodies. The left cremaster muscle was exposed and mounted for intravital microscopic observations of the cremasteric microcirculation and adjacent tissue. The muscle was superfused with 36° C. warmed bicarbonate-buffered saline pH 7.4. LR12 peptide, or equivalent volume of vehicle (isotonic saline) were injected 10 min before (6 mg/kg) and 3 h15 after (3 mg/kg) intrascrotal injection of 0.5 μg tumor necrosis factor (TNFα, R&D Systems). 2 h after TNFα injection, the cremaster muscle was incised and neutrophils were monitored by injection of labeled Phycoerythrin-conjugated anti-Ly6G antibody (0.05 μg/g body weight, clone RB6-8C5, BD Biosciences Pharmingen). Venules were visualized with an intravital microscope (Zeiss Examiner D1) equipped with a water-immersion objective (Zeiss Plan-Apochromat 20×/1.0 NA) and fluorescence excitation was made through a Lambda DG-4 high-speed wavelength switcher (Sutter Instrument). Images were collected with a 512×512 pixel back-thinned EMCCD camera (Evolve, Photometrics). Images were analyzed using SlideBook 6.0 software (Intelligent Imaging Innovations). The neutrophil rolling flux fraction, adhesion density, adhesion efficiency and transmigration were measured using playback assessment of 3-min digital time-lapse videos and 3D z-stack recordings of single unbranched venules (3 venules per mouse, 20-40 μm) from 2.5 hours to 5 hours after the TNFα challenge at 30-min intervals. Vessel diameter and wall shear rate measure methods are described in (Koehl B et al. Haematologica. 2017 July; 102(7):1161-1172). Blood samples used to determine blood counts were taken immediately after recording (Hemavet, Drew scientific).

[0073] Assessment of TREM-1 Plasma Concentrations:

[0074] The animals' blood was collected in EDTA anticoagulated tubes (Minicollect, BD). After centrifugation (5000 g, 10 min, 4° C.), plasma samples were aliquoted and stored at −80° C. An ELISA kit was used to perform the TREM-1 plasma assay (DuoSet Mouse TREM-1, R & D Systems, # DY1187) and the results are extracted from the reading of the optical density by a spectrophotometer as recommended by the manufacturer.

[0075] Results:

[0076] We showed that LR12 reduces the rolling, adhesion and transmigration of neutrophils in post-capillary venules after challenge with TNFα (FIGS. 1A-1C). Likewise, genetic deletion of Trem-1 did not decrease rolling, but decreases the adhesion and transmigration of neutrophils after challenge with TNFα (FIGS. 2A-2C). LR12 administration promoted a significant reduction vascular congestion of tissues of sickle cell mice (FIGS. 3A-3D). LR12 administration induced a significant reduction in the abundance of neutrophils in tissues of sickle cell mice during experimental vaso-occlusive crisis (VOC) (FIGS. 4A and 4B). Plasma concentrations of sTREM-1 are significantly higher in SS sickle cell mice than in mice carrying normal AA hemoglobin (FIG. 5).

REFERENCES

[0077] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.