USE OF MULTIFUNCTIONAL LIGANDS FOR TREATING THE RESPIRATORY DISTRESS AND CYTOKINE STORM SYNDROMES ASSOCIATED WITH CORONAVIRUS VIRAL INFECTIONS

Abstract

The enantiomers of AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1 METHOXY-8 METHYL-2 METHYLENEDIOXY-6,7 TETRAHYDRO-, 2,3,4 ISOQUINOLINE or tritoqualine and deuterated derivatives thereof, capable of preventing and treating cytokine storm and respiratory distress in coronavirus infections.

Claims

1-5. (canceled)

6. A method of treating coronavirus infection-related cytokine storm in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of 7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)phthalide and deuterated derivatives thereof.

7. A method of preventing coronavirus infection-related cytokine storm in a subject, comprising administering to subject in need thereof a therapeutically effective amount of 7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)phthalide and deuterated derivatives thereof.

8. A method of treating a coronavirus infection-related respiratory distress in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of 7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)phthalide and deuterated derivatives thereof.

9. A method for preventing coronavirus infection-related respiratory distress in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of 7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)phthalide and deuterated derivatives thereof.

10. The method according to claim 6, wherein the 7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)phthalide and deuterated derivatives thereof are packaged in the form of soft gelatin capsules, tablets, capsules, syrup or gel.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0058] FIG. 1 illustrates the presence of the asymmetric carbons, which are noted as A and B.

[0059] FIG. 2 illustrates the form of the RR isomer.

[0060] FIG. 3 illustrates the form of the SS isomer.

[0061] FIG. 4 illustrates the methyls that can be deuterated on tritoqualine

[0062] FIG. 5 illustrates the variation in resistances in treated and untreated mice

[0063] FIG. 6 illustrates the variation in resistances at 2 minutes between the 3 groups of mice

[0064] FIG. 7 illustrates the variation in resistances at 6 minutes between the 3 groups of mice

[0065] FIG. 8 non-degranulated basophils

[0066] FIG. 9 degranulated basophils

[0067] FIG. 10 effect of tritoqualine on the basophil degranulation inhibition.

DETAILED DESCRIPTION

[0068] To demonstrate that tritoqualine does inhibit respiratory distress syndrome via the basophil, a model described in publication of Ramadan 2013 has been used.

[0069] The inventors used a C57/B16J mouse model, hereafter referred to as BL6.

[0070] The protocol simulating a double-stranded RNA viral infection has been performed with 2 groups of 15 mice; these are 8 week old female mice. A control group treated only with saline served as a negative control.

[0071] BL6 mice are treated according to the protocol below. BL6 mice have been sensitised by intraperitoneal injections of 100 mg OVA (ovalbumin) on days 0, 2 and 4. Thereafter, from day 10 to day 15, the mice have received a daily treatment with aerosolized OVA at the concentration of 20 mg/ml or saline for 20 min, using an ultrasound nebulizer (Ultra-Neb99). One hour later, the mice received 50 mg of poly (A:U) or saline intranasally.

[0072] 3 groups were studied:

[0073] Group 1 negative control which only receives saline.

[0074] Group 2 which is sensitised to OVA and also receives double-stranded RNA (Poly U/Poly A) according to the protocol described above.

[0075] Group 3 which is sensitised to OVA and also receives double stranded RNA (Poly U/Poly A) according to the protocol described above but also tritoqualine at 10 mg/kg.

[0076] Each group is analysed by whole body plethysmography and the measurements are expressed in PenH. A measurement is performed every minute until 10 minutes after the introduction of the double-stranded RNA. Tritoqualine treatment is given 1 hour before the introduction of Poly U/Poly A (double-stranded RNA).

[0077] In group 1, which is the negative control group there are the following results at time 1 minute to 10 minutes, expressed in PenH units and averaged over 15 mice:

TABLE-US-00001 TABLE 1 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Group 1 1.83 1.91 2.04 2.08 2.27 2.30 2.25 2.15 2.15 2.08

[0078] For group 2, which is the positive control group (mice with viral infection after sensitisation to ovalbumin) there are have the following results at time 1 minute to 10 minutes (T1 to T10), expressed in PenH units and averaged over 15 mice:

TABLE-US-00002 TABLE 2 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Group 2 5.1 6.5 7.2 8.2 9.5 11.2 10.6 9.6 8.9 8.1

[0079] In group 3, which is the group treated with tritoqualine at a dose of 10 mg/kg (mice with viral infection after sensitisation to ovalbumin as well) there are have the following results at time 1 minute to 10 minutes, expressed in PenH units and averaged over 15 mice:

TABLE-US-00003 TABLE 3 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Group 3 3.05 3.11 3.13 3.97 4.07 4.14 4.02 3.85 3.14 2.74

[0080] If the different groups are compared, the group treated with tritoqualine shows an increase in its airway resistance, but only moderately compared to the untreated group. This difference is maximal at T 6 minutes between the tritoqualine group and the untreated group. The difference is still very significant at T 10 minutes.

[0081] Statistical analysis shows that the difference between these 2 groups is highly significant (p<0.001).

[0082] The conclusion is that in the model of respiratory inflammation by ovalbumin superinfected with the equivalent of a double-stranded RNA viral infection, tritoqualine shows surprising efficacy. It has been demonstrated in this model that it is the basophil that drives respiratory inflammation. (Ramadan 2013)

[0083] Tritoqualine is therefore capable of treating coronavirus infection-related respiratory distress.

[0084] The inventors have also tested tritoqualine directly on the basophil. For this purpose, the inventors have used a commercial test, called Flow Cast from Bülhmann.

[0085] This test uses the CD63 marker. The CD63 marker is considered as a marker of basophil and mast cell activation. Resting basophils express very little of the CD63 antigen because it is bound to intracytoplasmic granules.

[0086] Activation of basophils and mast cells leads to fusion of the granules with the plasma membrane and thus to CD63 expression on the cell surface.

[0087] CD63 therefore appears on the surface of basophils or mast cells only when CD63 is activated.

[0088] Activation of the basophil or mast cell can either be by an allergen or by an anti-IgE (anti-FcεRI, which is the anti-IgE receptor).

[0089] Activation of the basophil means that the basophil degranulates and releases many cytokines that are toxic to the lung. Indeed, it is in the lung that the toxic effect of the basophil is greatest. It is therefore imperative to block basophil degranulation to avoid toxic shock. In the scope of coronavirus infections, this toxic shock can lead to the patient's death.

[0090] Human mast cells and basophils are relatively similar in morphology, deriving from a CD34+ haematopoietic stem cell.

[0091] They differentiate under the influence of different cytokines, the main one being Stem Cell Factor for mast cells and Interleukin-3 for basophils. While mast cells are tissue-resident elements, the basophil is a circulating cell. It should be noted that mast cells represent a heterogeneous population depending on their tissue location, which is not the case for the basophil.

[0092] Both cells are involved in the IgE-dependent allergic reaction because they express the high-affinity IgE receptor. However, mediators released by these cells during this activation are in some cases different. In addition, basophils and especially mast cells are involved in innate immunity. It is in the context of innate immunity that the basophil is involved in coronavirus infection. Indeed, cytokine storm appears within 6 to 10 days after the start of the coronavirus infection. This is too short a time for acquired immunity to take place.

[0093] Basophils and mast cells are involved in many diseases through the secretion of many interleukins.

[0094] Mast cells and basophils secrete common cytokines such as IL 2, IL 3, IL 4, IL 5, IL 6, IL 9, IL 13, IL 15. Some of these interleukins are involved in allergy such as IL-4, others in pulmonary fibrosis such as IL-13, others in cytokine storm such as IL6. Basophils are also responsible for inflammatory reactions when activated. They degranulate to release histamine, proteoglycans such as heparin and chondroitin, and proteases such as elastase and lysophospholipase. They also secrete lipid mediators such as leukotrienes and various cytokines.

[0095] The inventors have highlighted the surprising role of tritoqualine in modulating CD63 and thus basophil degranulation. The inventors have highlighted the astonishing and surprising properties of tritoqualine in a human cell model of CD63 modulation.

[0096] To study this modulating action of CD63 by tritoqualine, the inventors have used the basophil and a modified commercial test, the Flow CAST® kit from BULHMANN Laboratories AG (Switzerland), which is a Basophil Activation Test (BAT) that can be used for the in vitro detection of basophil degranulation as well as for the study of immediate type allergic reactions and hypersensitivities.

[0097] The test is designed for the in vitro diagnosis of CD63 expression as a surface marker of activated basophils. The test is performed on whole blood; flow cytometry is used to quantify CD63 expression on the surface of activated basophils.

[0098] Activation (or degranulation) of basophils by this test can be made in three different ways: [0099] by an allergen, or [0100] by an “anti-IgE” (anti-FcεRI, which is the anti-IgE receptor) or [0101] by a bacterial lipopolysaccharide antigen, called fMLP.

[0102] It is intended for in vitro diagnosis of CD63 expression, all on whole blood by flow cytometry after allergen stimulation.

[0103] Resting basophils express very little CD63 antigen because it is bound to intracytoplasmic granules.

[0104] Activation of basophils (for example by IgE (immunological activation) or fMLP (non-immunological activation)) leads to fusion of the granules with the plasma membrane and thus to CD63 expression on the cell surface.

[0105] To evaluate degranulation via CD63 expression, the Flow CAST® kit has been partially used. This test includes an anti-IgE receptor. The inventors have only used the latter activator.

[0106] On the other hand, the Flow Cast test uses a second membrane marker, CCR3. The protein encoded by this gene is a C—C type chemokine receptor. It belongs to the G-protein coupled receptor family 1.

[0107] It is highly expressed in eosinophils, basophils and is also detected in TH1 and TH2 cells and airway epithelial cells. This receptor may contribute to the accumulation and activation of eosinophils, basophils and other inflammatory cells in the allergic airways. This receptor coupled to cell size allows specific characterisation of the basophil. Analysis of these two receptors allows the basophil and its degranulation to be characterised in a highly specific manner by flow cytometry.

[0108] Flow cytometry is used to characterise the different blood cells.

[0109] Laser beams allow the evaluation and measurement of different cellular parameters: [0110] The frontal measurement of the diffracted light of the laser beam allows evaluation of the cell size: this is the Forward SCatter (FSC). [0111] The measurement of the perpendicularly diffracted light allows evaluation of the cell granularity: it is the Side SCatter (SSC).

[0112] This granularity may be due to irregularities internally to or at the surface of the cells or to the density of the organelles that compose it.

[0113] Fluorescence markers are then used to better characterise the different cell subpopulations (these markers are coupled with differentiation clusters).

[0114] The apparatus that has been used throughout these experiments is a BD FACS Canto flow cytometer with 2 lasers:

[0115] A blue one with the possibility of 3 frequencies: 564-606 nm, 515-545 nm, 750-810 nm.

[0116] A red one with the possibility of 2 frequencies: 750-810 nm and 650-670 nm.

[0117] Several fluorochromes are used: APC Cy 7 (APC-Cy™ 7 is a fluorochrome that combines APC and a cyanine dye) and FITC (fluorescein isothiocyanate) as well as PE (phycoerythrin).

[0118] These different couplings make it possible to characterise cells separately and in particular basophils.

[0119] These different couplings make it possible to characterise cells separately and in particular basophils. 4 windowing zones will be distinguished according to whether or not CD63 labelling is observed and whether or not CCR3 labelling is observed [0120] CD63+ and CCR3− which characterise non-basophilic degranulated cells. [0121] CD63+ and CCR3+ which characterise degranulated basophils. [0122] CD63− and CCR3− which characterise non-degranulated non-basophilic cells. [0123] CD63− and CCR3+ which characterise non-degranulated basophils.

[0124] Thus with this analysis it is possible to analyse degranulated basophils separately from non-degranulated basophils. It is thus possible, by interacting a molecule of therapeutic interest, to know its capacity or not to act on degranulation.

[0125] The degranulation protocol has been carried out using the Flow CAST.

[0126] The Flow CAST Kit is comprised of [0127] A neutral stimulation Buffer with IL-3. [0128] A Stimulation Buffer with anti-FcεRI antibody positive control (Stimulation Control). [0129] A Stimulation Buffer with fMLP positive control (fMLP).

[0130] Not used in this protocol. [0131] A Staining Reagent. [0132] A Wash Buffer. [0133] A Lysing Reagent.

[0134] Reminder: During basophil activation, CD63 markers bound to intra-cytoplasmic granules will fuse with the plasma membrane. They are then expressed on the cell surface: activated basophils hence become CD63+. Further to CD63, another basophil specific marker is CCR3 (chemokine receptor 3). [0135] Activated and degranulated basophils are CD63+ and CCR3+; [0136] non-degranulated basophils are CD63− and CCR3+.

[0137] Three tests have been made to define and refine the final protocol. Firstly, a “negative control” sample, that is containing only the neutral buffer, has been analysed in order to observe the results expected in the absence of stimulation and therefore of degranulation.

[0138] It can be noticed in [FIG. 8], which has 4 zones, that only the CD63− CCR3+ zone contains a scatter chart corresponding to non-degranulated basophils. In the absence of stimulation, the cells are not activated and do not degranulate.

[0139] The second test comprises a “positive control” sample with the FcεRI antibody. On the 4 windowed zones in flow cytometry, a positive control Sample with FcεRI antibody window allows the observation of the scatter chart in the CD63+ CCR3+ zone. This is the zone in the upper right-hand corner of [FIG. 9]. This indicates that the anti-FcεRI antibody does cause degranulation of basophils. However, not all basophils are degranulated, as the CCR3+ and CD63− window contains a few spots corresponding to non-degranulated basophils.

[0140] Finally, the third test involves the use of the anti-FcεRI antibody for the purpose of degranulating the basophil with a prior incubation of tritoqualine at different concentrations. It is with this last analysis that the inventors were able to demonstrate activity of tritoqualine on CD63 modulation.

[0141] Finally, basophils are a very small population in the blood count, often less than 1%. With a goal of obtaining at least 500 basophils for analysis, more than 50,000 white blood cells had to be sorted on each passage in front of the flow cytometer.

[0142] Next, tritoqualine has been added to each sample with the anti-FcεRI antibody. If tritoqualine is able to inhibit degranulation, then the number of non-degranulated basophils should increase.

[0143] Concentrations of tritoqualine used range from 1 μM to 10 μM corresponding to therapeutic doses of 100 mg to 1 gram per day in a man having an average weight of 70 kg.

[0144] The experiments were as follows:

[0145] First, a patient has been sampled and 5 tubes have been prepared. One negative control tube: without incubation with tritoqualine and without the anti-FcεRI antibody (the degranulation product). Four “positive control” tubes: without incubation with tritoqualine and with the basophil degranulant (anti-FcεRI antibody).

[0146] The aim of these experiments was to demonstrate relevance of the test to differentiate degranulated from non-degranulated basophils.

[0147] The experiment confirmed that the anti-FcεRI antibody caused basophils to degranulate as in the series with the anti-FcεRI antibody (positive control), almost 85% of basophils degranulated. In the “negative control” sample, there are almost no degranulated cells (less than 17%).

[0148] Statistical analysis has been performed using GraphPad Prism 7.0.

[0149] The Student's t test of the negative control versus positive control also shows that p is less than 0.0001. This means that degranulated and non-degranulated basophils are highly differentiable in flow cytometry.

[0150] Thus, the study phase has been performed with 4 patients at doses ranging from 2.5 μmol to 10 μmol. By way of example, [FIG. 10] shows the dose effect of tritoqualine from 1 μmol to 10 μmol on the inhibition of the basophil degranulation and thus of the CD63 expression modulation.

[0151] At 5 μmol, CD63 is still expressed on approximately 40% of the cells whereas at 10 μmol, this expression is reduced to less than 2%. Statistical analysis shows a P greater than 0.001 for doses from 2.5 micromol to 10 micromol. Tritoqualine does modulate CD63 expression on basophils in a surprisingly progressive manner. This CD63 modulation makes it possible to contemplate tritoqualine-based therapies for the treatment of respiratory distress syndrome and coronavirus infection-related cytokine storm. This powerful degranulation inhibition action also makes it possible to contemplate the prevention of cytokine storm as well as coronavirus infection-related respiratory distress.

[0152] The treatment of these pathologies can be done at therapeutic doses between 5 mg/day and 700 mg/day.

[0153] Tritoqualine can be used in different forms, apart from its “compressed” form without modifying its efficacy, for example in the form of soft gelatin capsule, syrup or gel. 7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)phthalide can also be used in combination with complementary drugs, such as Nintedanib and Pirfenidone. These drugs acting with different modes of action, allow for lower doses and thus less side effects and toxicity. Nintedanib can be used at a dose of 10 mg to 50 mg/day.

[0154] Pirfenidone can also be used at lower doses such as 100 to 200 mg/day.

[0155] Commercial tritoqualine is a white powder, very sensitive to light which degrades it to Cotarnine and phthalic acid.

[0156] Tritoqualine has two asymmetric carbons, but analysis of the old commercial form shows that tritoqualine is a racemic mixture of 2 enantiomers (R—R and S—S) and not a mixture of 4 diastereomers.

[0157] Tritoqualine is a benzylisoquinoline with a molecular weight of 500. This compound can be modified or substituted with compounds comprising either carbon-14 or deuterated compounds.

[0158] Isotope-labelled compounds and salts can be used in a variety of ways. They may be suitable for drugs and/or different types of tests, such as tissue distribution tests on a substrate. For example, tritium and/or carbon-14 labelled compounds are particularly useful for various types of tests, such as tissue distribution tests on a substrate, due to their relatively simple preparation and excellent detectability. For example, deuterium labelled products are therapeutically useful and have potential therapeutic advantages over non-deuterium labelled compounds. In general, deuterium-labelled compounds and salts can have higher metabolic stability than non-deuterium-labelled compounds due to the isotope kinetic effect. Higher metabolic stability translates directly into an increased half-life in vivo or lower doses, which may be desired. Isotopically labelled compounds and salts can generally be prepared by following the procedures described in known synthesis schemes such as for example in patent EP3352757 filed by concert pharmaceuticals. It is thus easy to replace non-deuterated methyls with deuterated methyls. Thus 5 substitutions of deuterated methyls could be made on tritoqualine. Two on the cotarnine ring and 3 on the nitrophthalide ring.

[0159] Thus, it is possible to use these deuterated compounds to improve the bioavailability of tritoqualine and its efficacy in cytokine storm and the treatment of coronavirus infection-related respiratory distress.