Compositions for treating (therapy) or preventing vasculitis and diseases associated with vasculitis

Abstract

The present invention relates to a composition for use in the treatment or prevention of vasculitis, comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture, the PBMC cell culture comprising 1x10 5 ~1x10 8 The cells contain 10 PBMC / ml and are irradiated before or during culture and incubated for at least 4 hours.

Description

Technical Field

[0001] The present invention relates to compositions and methods for treating (therapeutically) and preventing vasculitis and diseases associated with vasculitis.

Background Art

[0002] Vasculitis is a rare group of diseases of blood vessels, including arteries, veins, and lymphatic vessels, which cause their destruction. Neutrophils are one of the important drivers of vasculitis that affect endothelial cells by the release of neutrophil extracellular traps (NETs). There are many types of vasculitis, and the symptoms, severity, and duration can vary widely. Most types of vasculitis are rare, and their causes are generally not well known. Vasculitis can occur regardless of gender and age.

[0003] Vasculitis is a systemic disease that affects substantially all organ systems, including the skin, lungs, joints, kidneys, gastrointestinal tract, blood, eyes, brain, nerves, sinuses, nose, and throat. The pattern of organ involvement is specific to the individual and to the type of vasculitis.

[0004] The treatment (therapy) of vasculitis mainly focuses on controlling inflammation and managing any underlying conditions that can induce vasculitis. Current treatment methods typically are based on corticosteroids such as prednisone. The side effects of corticosteroids can be severe, especially when taken over a long period. Possible side effects include weight gain, diabetes, and bone weakness. Other drugs can be prescribed together with corticosteroids to control inflammation so that the dosage of corticosteroids can be tapered more rapidly. The drugs used depend on the type of vasculitis present. These drugs can include methotrexate, azathioprine, mycophenolate, cyclophosphamide, tocilizumab, or rituximab. Occasionally, vasculitis causes aneurysms. Aneurysms may require surgery to reduce the risk of rupture. Occluded arteries may also require surgical procedures to restore blood flow to the affected area.

[0005] All of these medical interventions have side effects and / or pose specific risks to the patient's health. Furthermore, they are all known to target tissue inflammation mainly by affecting T cell function, thereby inhibiting neutrophil recruitment to the endothelium. Treatment options to prevent NET release or protect the endothelium have not been available until now. Accordingly, it is an object of the present invention to provide alternative means and methods for treating (therapy) or preventing vasculitis and related diseases. Summary of the Invention

[0006] Surprisingly, a composition comprising the supernatant of a PBMC cell culture, wherein the supernatant can be obtained by culturing the PBMC cell culture for at least 4 hours, and the PBMC cell culture is irradiated before or during culture with 1x10 5 ~1x10 8 cells of PBMC / ml was found to be useful for treating (therapy) or preventing vasculitis.

[0007] In recent years, cell-based therapies have made visible progress in modern regenerative medicine, either to repair damaged tissues and organs, restore their functions, or replace damaged cells. Due to their self-renewal ability, the potential to differentiate into several different cell types, and numerous successful preclinical studies, regenerative therapies using mesenchymal stem cells (MSCs) are considered a very promising approach. Despite promising preclinical data, most first-in-man clinical trials have not been able to demonstrate efficacy in patients. Regarding the mode of action (MOA) of MSCs, it has become increasingly clear that paracrine factors released from the injected cells are involved in many of the beneficial biological effects and thus significantly contribute to tissue regeneration. There are significant limitations to stem cell secretome-based therapies, namely, the low abundance of cells, the invasive procedures for obtaining cells, and the high cost. Therefore, alternative sources of regenerative cell secretomes are highly sought after.

[0008] In the past few years, several studies have suggested that the secretome of irradiated peripheral blood mononuclear cells (APOSEC) is a beneficial alternative to MSCs. APOSEC exhibits equivalent tissue regeneration properties compared to paracrine factors released from stem cells and can be obtained more easily (Ankersmit, H.J. et al. Eur. J. Clin. Invest. 39 (2009): 445 - 456). Detailed functional analysis of APOSEC has revealed proteins, lipids, and extracellular vesicles as the three major biological components. Their MOA is very diverse and has been tested in many pre - clinical experimental settings of induced tissue injury.

[0009] Cells, particularly mammalian cells, are known to secrete numerous substances into the cell culture medium during culturing. The conditioned culture medium thus obtained can be used for the treatment and / or prevention of various diseases and disorders. For example, International Publication Nos. WO 2010 / 070105 and WO 2010 / 079086 disclose conditioned culture media ("supernatants") obtained by culturing PBMCs and which can be used for the treatment of various inflammatory conditions. Thus, as used herein, "supernatant of a peripheral blood mononuclear cell (PBMC) cell culture" refers to any supernatant that can be obtained by culturing PBMCs in vitro in a culture medium. After the culturing step, the cultured PBMCs are removed from the culture medium to obtain a substantially cell - free, preferably completely cell - free supernatant. The supernatant of a PBMC culture contains substances produced and secreted by PBMCs and / or even lysed PBMCs alongside the components of the culture medium. The "supernatant" can be used interchangeably with the conditioned culture medium obtainable by culturing PBMCs.

[0010] It is a surprising discovery that the supernatant of the present invention can inhibit or even prevent the release of NETs involved in the formation of vasculitis. It is also surprising that the supernatant of the present invention exhibits an endothelial - protective effect due to its protease - inhibitory activity. This helps to maintain the integrity of blood vessels and the function of preventing and / or treating (treating) vasculitis.

[0011] Another aspect of the present invention is a method for treating vasculitis in a subject in need thereof, the method comprising administering to the subject a composition comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture, the supernatant being obtainable by culturing the PBMC cell culture for at least 4 hours, the PBMC cell culture being irradiated before or during culture with 1 x 10 5 ~1 x 10 8 PBMCs / ml, relates to a method.

[0012] Yet another aspect of the present invention is the use of a composition comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture for the preparation of a medicament for use in the treatment or prevention of vasculitis, the supernatant being obtainable by culturing the PBMC cell culture for at least 4 hours, the PBMC cell culture being irradiated before or during culture with 1 x 10 5 ~1 x 10 8 PBMCs / ml, relates to a use. BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

Figure 1

Figure 2

[0014] As used herein, the terms "prevent" and "prevention" refer to the prevention or inhibition of the recurrence, occurrence, and onset of vasculitis in the human and mammalian body resulting from the administration of the supernatant according to the present invention. In some embodiments, "prevent" and "prevention" refer to a reduction in the risk of developing vasculitis. The term "prevent" encompasses not only preventing the occurrence of vasculitis, but also stopping its progression and taking measures to reduce the consequences once established.

[0015] As used herein, the terms "treating" and "treatment" refer to reducing or inhibiting the progression and duration of vasculitis, reducing or ameliorating the severity of vasculitis, and ameliorating one or more of its symptoms. "Treatment" also encompasses amelioration and / or recovery of the symptoms of vasculitis. The term "treatment" refers to both therapeutic treatment and prophylactic treatment. For example, those who benefit from treatment by the compositions and methods of the invention include those who already have vasculitis and those in whom vasculitis is to be prevented.

[0016] As used herein, the term "vasculitis" refers to inflammation of blood vessels (i.e., veins or arteries). Vasculitis can be acute or chronic, hypoimmune or immune, large ( "large-vessel vasculitis") or small-vessel ( "small-vessel vasculitis"), and can be primary or secondary to another disorder. Examples of vasculitis include, for example, Behçet's syndrome, Buerger's disease (thromboangiitis obliterans), antineutrophil cytoplasmic autoantibody (ANCA)-associated systemic vasculitis (AASV) (including Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg-Strauss syndrome (CSS)), cryoglobulinemia, giant cell arteritis (GCA), Henoch-Schönlein purpura, hypersensitivity vasculitis, Kawasaki disease (mucocutaneous lymph node syndrome), polyarteritis nodosa, rheumatoid vasculitis, Takayasu arteritis, and polymyalgia rheumatica (PMR). Vasculitis, including autoimmune vasculitis, can be acute or chronic and can be primary or secondary to another disorder. As used herein, the phrase "autoimmune vasculitis" refers to vasculitis arising from the immune system of a subject that reacts against one or more autoantigens.

[0017] Vasculitis can affect small, medium, and large blood vessels and can occur in otorhinolaryngology (ENT) vasculitis, preferably granulomatous and / or necrotic lesions of the ENT tract, pulmonary hemorrhage, hemoptysis, cutaneous vasculitis, glomerulonephritis, proteinuria, hematuria, nodular vasculitis, eosinophilic myocarditis, cardiomyopathy, and virus-related vasculitis. Vasculitis also plays a role in acute respiratory distress syndrome (ARDS) caused by viral infections such as, for example, SARS-CoV2. Therefore, it is particularly preferred to use the compositions of the present invention for the treatment (therapy) and / or prevention of ARDS, particularly ARDS caused by SARS-CoV2.

[0018] According to a further preferred embodiment of the present invention, the PBMCs are cultured in a cell culture medium selected from the group consisting of a cell growth medium, preferably CellGro medium, more preferably Cellgro GMP DC medium, RPMI, DMEM, X-vivo, and Ultraculture.

[0019] According to the present invention, the PBMCs of the PBMC cell culture are subjected to stress-inducing conditions before or during culturing.

[0020] As used herein, the term "stress-inducing conditions" refers to culture conditions that result in stressed cells. Conditions that cause stress to the cells include at least any kind of radiation (e.g., ionizing radiation such as UV radiation, gamma rays), preferably ionizing radiation or UV radiation. However, the cells can also be further subjected to heat, chemicals, hypoxia, osmotic pressure, pH shift, etc., which can cause additional stress to the cells.

[0021] Therefore, stress-inducing conditions can also include hypoxia, ozone, heat (e.g., a temperature higher than the optimal culture temperature of PBMCs, i.e., more than 2°C, preferably more than 5°C, more preferably more than 10°C higher than 37°C), chemicals, osmotic pressure (i.e., an osmotic pressure condition that is at least 10% lower compared to the osmotic pressure conditions regularly occurring in body fluids, particularly in blood), or combinations thereof.

[0022] According to another preferred embodiment of the present invention, the PBMCs are exposed to ionizing radiation, preferably gamma rays, at a dose of at least 10 Gy, preferably at least 20 Gy, more preferably at least 30 Gy, more preferably at least 40 Gy, and even more preferably at least 50 Gy.

[0023] According to a preferred embodiment of the present invention, the PBMCs are cultured for at least 6 hours, preferably at least 12 hours, before isolating their supernatant.

[0024] According to a preferred embodiment of the present invention, the PBMC cell culture contains 1x10 6 ~1x10 7 PBMCs / ml, preferably 2x10 6 ~25x10 6 PBMCs / ml.

[0025] The composition of the supernatant obtained by culturing PBMCs has been found to exhibit advantageous properties when culturing a specific amount of PBMCs per 1 ml of cell culture medium.

[0026] According to another preferred embodiment of the present invention, 0.1 to 5 ml of the supernatant / kg body weight, preferably 0.3 to 3 ml / kg body weight, more preferably 0.5 to 2 ml / kg body weight, and even more preferably 0.8 to 1.2 ml / kg body weight, is administered to the body of a human or mammalian animal.

[0027] The composition of the present invention contains a sufficient amount of the supernatant to treat (cure) or prevent vasculitis and diseases associated with vasculitis. The volume of the supernatant administered per kg body weight to a human or mammal as described above directly refers to the supernatant. If the volume is too large to be administered to the body of a human or mammal, this volume can be reduced, for example, by lyophilization. Therefore, the volume of the composition of the present invention to be administered may be lower than the volume indicated for the supernatant. Those skilled in the art know how much volume can be administered using a specific administration route.

[0028] According to a preferred embodiment of the present invention, the composition is administered by inhalation, topically, orally, sublingually, buccally, subcutaneously or intravenously, whereby it is most preferred to administer the composition of the present invention intravenously.

[0029] The composition of the present invention may contain pharmaceutically acceptable excipients such as diluents, stabilizers, carriers and the like. Depending on the dosage form, the preparations according to the present invention contain the respective components. The method for preparing this is well known to those skilled in the art.

[0030] In order to extend the shelf life of the composition according to the present invention, even the supernatant or the complete composition can be lyophilized. The method for lyophilizing such preparations is well known to those skilled in the art.

[0031] Before its use, the lyophilized preparation can be brought into contact with water or an aqueous solution containing buffers, stabilizers, salts and the like.

[0032] According to another preferred embodiment of the present invention, the mammal is a horse, dog, cat or camel.

[0033] The composition of the present invention can be used to treat all kinds of mammals. However, the aforementioned mammals are most preferred.

[0034] Another aspect of the present invention relates to a method for treating (treating) or preventing vasculitis, which includes the step of administering the composition defined above.

Example

[0035] This example shows that APOSEC protects against vascular inflammation and injury by a dual mechanism: 1) APOSEC inhibits the induction of NETs, thereby preventing NET-induced vasculitis, and 2) APOSEC directly prevents vascular leakage.

[0036] Materials and methods Isolation of PBMCs and generation of PBMC secretome APOSEC was manufactured by the Austrian Red Cross, Transfusion Service Oberösterreich (Austria), in compliance with Good Manufacturing Practice (GMP). Briefly, PBMC were obtained by Ficoll-Paque PLUS (GE Healthcare, USA) supported density gradient centrifugation and exposed to 60 Gy of caesium 137 gamma irradiation (IBL 437C, Isotopen Diagnostik CIS GmbH, Germany). Cells were adjusted to a concentration of 2.5×10 7 cells / mL (equivalent to 25 U / ml) and cultured for 24 h in phenol red-free CellGenix GMP DC medium (CellGenix GmbH, Germany). Cells and cell debris were removed by centrifugation and the supernatant was passed through a 0.2 μm filter. Methylene blue treatment was performed for virus clearance. The secretome was lyophilized, terminally sterilized by high-dose gamma irradiation and stored frozen.

[0037] Protease activity assay To test the inhibitory effect of APOSEC on protease activity, a fluorescence enzyme activity assay (Enzcheck) using non-selective serine protease trypsin (Gibco) at a concentration of 0.05% was performed. The PBMCsec stock concentration was set to 12.5 U / ml according to the manufacturer's instructions.

[0038] Transendothelial electrical resistance (TEER) Changes in the electrical impedance of dermal microvascular endothelial cells (DMEC) were measured using an ECIS system (Applied Biophysics). A total of 4000 cells / cm were seeded into each well of an ECIS chamber (ibidi GmbH, Germany). 2Cells were seeded. Once complete confluence was reached, the cells were treated with thrombin and thrombin combined with either control medium (concentrated at 12.5 U / ml) or APOSEC (concentrated at 12.5 U / ml). Endothelial resistance was monitored at 250 Hz for 2 hours after the addition of the treatment according to a previously published protocol (Schossleitner, K. et al. Arter. Thromb Vasc Biol 36 (2016): 647 - 654).

[0039] Co - culture of endothelial cells and neutrophils Neutrophils were isolated from healthy donors and stimulated with ionomycin to induce NET formation in the presence or absence of APOSEC. The cells were incubated together at 37°C for 1 hour with each treatment and then washed with PBS before adding to primary human umbilical vein endothelial cells (HUVEC). Co - culture was performed either by directly adding neutrophils to a confluent HUVEC monolayer or by separating the two cell populations with a transwell insert providing a physical barrier with a 0.4 μm mesh / filter. After 2 hours, the neutrophils were removed and the HUVEC were cultured under standard conditions for 24 hours. The cell culture supernatant of HUVEC was obtained and stored at - 20°C until further processing.

[0040] ELISA Enzyme - linked immunosorbent assays (ELISA) were performed according to the manufacturer's instructions for common pro - inflammatory cytokines and activation markers related to injured or damaged endothelial tissue (all from R&D Systems, USA). The citH3 ELISA (clone 11D3, Cay501620 - 96, Caymen) was performed as recommended by the manufacturer.

[0041] Flow cytometry Flow cytometric analysis was performed daily on a FACSCalibur (BD Biosciences, USA) as recommended by the manufacturer. For the evaluation of apoptosis, triple staining with CD66b-Pacific Blue (Biolegend, USA), CD15-PE-Cy7 (Biolegend) and citH3-FITC (Abcam, UK) was carried out.

[0042] Results APOSEC inhibits serine proteases and blocks protease-induced vascular leakage To examine the efficacy of APOSEC in inhibiting serine protease activity, a protease activity assay against the serine protease trypsin was performed. The control medium showed a negligible inhibitory effect on protease activity (3.72%), while the addition of APOSEC resulted in a 41.96% inhibition of the enzymatic activity of trypsin (Figure 1A). Next, it was also investigated whether APOSEC interferes with the thrombin-induced disruption of the endothelial barrier measured by electrical resistance. No effect was shown with the basal medium alone, but the addition of thrombin resulted in a strong decrease in endothelial resistance. This decrease was significantly reduced by the addition of APOSEC. The addition of the control medium showed only a weak effect on endothelial resistance (Figure 1B).

[0043] Specifically, Figure 1 shows that APOSEC inhibits serine protease and blocks the thrombin-induced decrease in endothelial barrier function in vitro. According to Figure 1A, APOSEC significantly inhibits the enzymatic activity of trypsin after 60 minutes when compared to the control medium (relative inhibition of trypsin activity of 3.72% in the control medium vs. 41.96%, p-value = 0.0002). According to Figure 1B, dermal microvascular endothelial cells (DMECs) showed a decrease in barrier function after treatment with the serine protease thrombin when compared to the basal medium (1 in the basal medium vs. 0.29 thrombin). The combination of thrombin and the control medium resulted in a comparable decrease of APOSEC to thrombin (1 in the basal medium vs. 0.38 thrombin + control medium), but the addition of APOSEC to thrombin showed a significantly lower reduction (1 in the basal medium vs. 87.5 thrombin + Aposec), preventing the disruption of the endothelial barrier. The barrier function of thrombin + Aposec was significantly higher compared to thrombin (p-value = 0.0071) and thrombin + control medium (p-value = 0.0126)

[0044] APOSEC inhibits NET formation and NET-induced activation of endothelial cells To examine whether APOSEC affects ionomycin-induced NET formation, whole blood was stimulated with ionomycin in the absence or presence of APOSEC. The induction of citrullinated histone-3 (CitH3), a specific marker of NETosis, was measured by flow cytometry (Figure 2A) and ELISA (Figure 2B). A strong reduction in ionomycin-induced histone citrullination was observed in both assays (Figure 2A and B). Next, the effect of APOSEC on NET-induced endothelial tissue damage in an in vitro co-culture model was investigated. To evaluate whether direct contact of activated neutrophils is required for endothelial cell activation and damage, co-cultures were established either with direct contact between neutrophils and endothelial cells (Figure 2D) or with a physical barrier between them in the form of a transwell insert (Figure 2C). The physical barrier appeared to be sufficient to block the pro-inflammatory effect of neutrophils on endothelial cells as well as their tissue damaging ability, as shown in Figure 2C. However, direct contact between ionomycin-activated neutrophils and endothelial cells significantly increased the production of pro-inflammatory cytokines, including interleukin (IL)-6, IL-8 and tumor necrosis factor alpha (TNFα), as well as endothelial activation markers such as intercellular adhesion molecule 1 (ICAM-1) (Figure 2D). Treatment of activated neutrophils with APOSEC prior to addition to endothelial cells almost completely abolished the inflammatory response of endothelial cells and resembled cytokine levels similar to untreated cells (Figure 2D).

[0045] Figure 2 shows that Aposec prevents NET-induced endothelial tissue damage. In Figure 2A, flow cytometry analysis (n = 3) of whole blood samples stimulated with ionomycin and treated with APOSEC is shown. Figure 2B shows an ELISA for citrullinated histone h3 (citH3) of whole blood samples stimulated with ionomycin and treated with APOSEC (n = 2). Figures 2C and 2D show ELISAs of pro-inflammatory cytokines and activation markers of HUVECs derived from neutrophil-HUVEC co-cultures using either a transwell insert (Figure 2C) or direct contact (Figure 2D) as a barrier between cell populations. The treatment conditions are indicated by - and + below each graph. IL6, interleukin-6; IL8, interleukin-8; TNF-α, tumor necrosis factor α; ICAM-1, intracellular adhesion molecule 1; -, absent; +, present.

[0046] Conclusion In summary, the above data indicate that APOSEC can reduce vascular damage by inhibiting the enzymatic disruption of vascular integrity and by inhibiting NET release by neutrophils. These mechanisms are involved in various severe diseases including all types of vasculitis, granulomatous or necrotic lesions of the ENT-tract, pulmonary hemorrhage and hemoptysis, petechiae or eruptions associated with necrotizing cutaneous vasculitis, glomerulonephritis and proteinuria, hematuria, localized vasculitis of the vasa nervorum affecting the peripheral nervous system, eosinophilic myocarditis and cardiomyopathy, and Covid-19, all of which may benefit from the application of APOSEC.

Claims

1. A composition for use in the treatment or prevention of vasculitis, comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture, wherein the supernatant can be obtained by culturing the PBMC cell culture for at least 4 hours, and the PBMC cell culture is subjected to ionizing radiation at a dose of at least 10 Gy before or during culturing, and contains 1x10 ５ to 1x10 ８ PBMC / ml.

2. The composition for use according to claim 1, which is administered subcutaneously, intramuscularly, subdermally, intradermally or intravenously.

3. The composition for use according to claim 1 or 2, wherein the PBMC cell culture contains monocytes, T cells, B cells and / or NK cells.

4. The composition for use according to claim 1 or 2, wherein the PBMC is cultured in a cell culture medium containing a cell growth medium.

5. The composition for use according to claim 4, wherein the cell growth medium is selected from the group consisting of CellGro medium, Cellgro GMP DC medium, RPMI, DMEM, X-vivo and Ultraculture.

6. The composition for use according to claim 1 or 2, wherein the PBMC is subjected to ionizing radiation at a dose of at least 20 Gy, at least 30 Gy, at least 40 Gy, or at least 50 Gy.

7. The composition for use according to claim 1 or 2, wherein the PBMC is cultured for at least 6 hours, at least 12 hours, or at least 24 hours before isolating its supernatant.

8. The PBMC cell culture contains 1x10 ６ to 1x10 ７ PBMC / ml, or 2x10 ６ to 25x10 ６ PBMC / ml. The composition for use according to claim 1 or 2.

Images