Glyglycerophosphoinositide for preventing and treating COVID-19 infection and method for obtaining same

Abstract

The present invention relates to the use of glycerophosphoinositide (GPI) in the prevention and treatment of COVID-19 infection, and an environmentally sustainable method for obtaining it. In particular, the present invention relates to a method for preparing glycerophosphoinositide from a crude or partially purified phospholipid mixture, comprising the following steps in this order: a) hydrolyzing the crude or partially purified phospholipid mixture by enzyme treatment with PLA1 and PLA2; b) microfiltration of the mixture from step a) and subsequent ultrafiltration and nanofiltration of the microfiltrate to obtain a concentrated aqueous fraction of the reaction product; c) electrodialyzing the aqueous fraction of step b) for separating ionic compounds from neutral compounds; and d) carrying out ion exchange chromatography. The invention further relates to the use of inositol glycerophosphate in the prevention and treatment of COVID-19 syndrome.

Description

Technical Field

[0001] The present invention relates to the use of glycerophosphoinositide (GPI) in the treatment and prevention of COVID-19 infection and similar viruses, and methods of obtaining it. Background Art

[0002] In mammals, mobilization of arachidonic acid from the sn2 ester bonds of phospholipids is largely due to the activation of cytosolic phospholipase A2 (cPLA2) following receptor activation induced by a variety of agonists including hormones, neurotransmitters, neuropeptides and growth factors.

[0003] One substance produced along with the release of arachidonic acid, L-α-glycero-phospho-D-myoinositol (GPI), is an autacoid and is known to have negative cPLA2 regulatory properties, thus acting as an anti-inflammatory substance.

[0004] By way of illustration, the choline salt of GPI is shown in the following structural formula (I):

[0005]

[0006] like Figure 1 As shown, cPLA2 initially hydrolyzes phosphatidylinositol (PI) into arachidonic acid (AA) and lysophosphatidylinositol; the latter is also a substrate for another enzyme (lysophospholipase A1), resulting in the release of sn-1-linked fatty acids (e.g., stearic acid, SA) and GPI.

[0007] GPI negatively regulates cPLA2 through a physiological feedback mechanism, thereby inhibiting the inflammatory process. In fact, due to strong or prolonged pro-inflammatory stimuli, an increase in endogenous AA levels induces an increase in GPI levels that negatively regulate cPLA2 activity. GPI inhibits inflammation at the same level as cortisone drugs, but, as mentioned above, its mechanism of action is completely different, so it does not cause the typical side effects of cortisone drugs, because it does not interfere with the body's hormonal balance and, therefore, has no immunosuppressive effect.

[0008] Sepsis syndromes typically occur in response to lipopolysaccharides (LPS; also called endotoxins) from Gram-negative bacteria. Tissue factor (TF) is a high affinity receptor and cofactor for factor (F) VII / VIIa. The TF-FVIIa complex is the primary initiator of coagulation and plays an important role in hemostasis. TF is expressed on perivascular cells and epithelial cells on the surfaces of organs and the body, where it forms a hemostatic barrier. TF also provides additional hemostatic protection for vital organs such as the brain, lungs, and heart. Under pathological conditions, TF can trigger arterial and venous thrombosis. In sepsis, the inducible expression of TF on monocytes leads to disseminated intravascular coagulation.

[0009] TNFα and IL-1 are typical inflammatory cytokines that mediate many cellular events associated with LPS exposure. They are rapidly released after exposure to LPS (30-90 minutes after the inflammatory stimulus) and in turn amplify the inflammatory response. In addition, many other cytokines, including IL-1 and IL-6, are potent coagulation inducers. Coagulopathy is common in sepsis, and in many cases (30-50% of patients), they also develop into a more severe clinical form, namely disseminated intravascular coagulation. In sepsis, LPS (or other bacterial components) initiates the coagulation cascade by inducing the expression of tissue factor (TF) on monocytes and endothelial cells. TF in turn activates the proteolytic cascade (coagulation cascade), ultimately leading to the conversion of prothrombin to thrombin and subsequent activation of fibrin. The final result of this series of events is the deposition of fibrin clots in small blood vessels, leading to reduced tissue perfusion, multiple organ failure, and in some cases death of the subject.

[0010] Sepsis is the most common complication in patients with COVID-19. As reported in a cohort study of 191 patients, more than half of the patients developed sepsis. It has been found that the molecular mechanism of the observed increase in inflammation caused by the spike (S) protein of SARS-CoV-2 relies on a specific and unique interaction between the S protein and LPS, resulting in changes in the biophysical properties of LPS. It can be assumed that sepsis is directly caused by SARS-CoV-2 infection. Comorbidities such as obesity, type 2 diabetes (T2D), cardiovascular disease (CVD), the advanced age of the patients and their ethnicity are considered important factors that worsen the disease outcome: all these comorbidities have something in common and are linked by viral-bacterial interactions that are triggered by the transfer of bacterial products such as lipopolysaccharide (LPS) from the intestine to the circulation. In fact, increased plasma levels of LPS and LPS-binding proteins (LBPs) are found in obesity and diabetes, and intestinal dysbiosis is implicated in the pathogenesis of insulin resistance. Low-level inflammation induced by the systemic prevalence of bacterial products is associated with vascular abnormalities, and circulating LPS levels are significantly altered in CVD.

[0011] Given the incidence of the aforementioned complications in the most severe cases among patients with COVID-19, there is a need to provide preventive and / or curative treatments for SARS-Cov-2 infection.

[0012] The preparation of salts of L-α-glycerophosphoryl-D-myo-inositol (GPI) by chemical synthesis dates back to the patent of Tronconi, G. (US005306840A) and the literature cited therein; the process starts with an alkaline transesterification reaction of Na or K alcoholates in an alcoholic environment; such a reaction has a GPI yield of about 50-60%. This reaction carried out in an alcoholic environment already allows the partial separation of the GPI salt, which remains insoluble in alcohol relative to triglycerides, fatty acids and other derivatives of the reaction, which are soluble in alcohol. The patent claims a purification method based on chromatography on a weakly basic resin and subsequent selective crystallization.

[0013] In US 6,924,130 B1, Barenholz, Y. and Amselem, S. claim enzymatic transesterification or hydrolysis of phospholipids in an aqueous environment by using silica particles, which form a solid / liquid interface that provides high conversion rates. The patent focuses on the application of PLC and PLD, and also mentions the use of PLA1 and PLA2.

[0014] In CN106459107A, the authors reported a method for purifying L-α-glycerophosphorylcholine using DMSO, ethanol, methanol and isopropanol solvents after the purification process in ion exchange resin chromatography. Summary of the invention

[0015] Therefore, the present invention first relates to glycerophosphoinositide (GPI) for the prevention and treatment of COVID-19 syndrome.

[0016] In particular, the present invention relates to a process for preparing glycerophosphoinositides from a crude or partially purified phospholipid mixture, the process comprising the following steps in sequence:

[0017] a) hydrolyzing a crude or partially purified phospholipid mixture by treating it with PLA1 and PLA2 enzymes;

[0018] b) microfiltration of the mixture from step a), and subsequent ultrafiltration and nanofiltration of the microfiltrate to obtain a concentrated aqueous fraction of the reaction product;

[0019] c) electrodialysis of the aqueous fraction of step b) for separation of ionic compounds from neutral compounds;

[0020] d) performing ion exchange chromatography,

[0021] As claimed in claims 1 to 14.

[0022] The present invention also relates to glycerophosphoinositide (GPI) for the prevention and early treatment of COVID-19 syndrome, in particular COVID-19 sepsis, as described in claims 15 to 16, or the use of GPI as described in claims 17-18 in the preparation of a medicament for the prevention and early treatment of COVID-19 syndrome, or a method for the prevention or early treatment of syndrome caused by COVID-19 or other viral infections as described in claims 19-20, comprising administering an effective amount of glycerophosphoinositide.

[0023] These and further objects will be set out in the following description as outlined in the appended claims.The text of the claims should be considered included in the description in order to assess the adequacy of the description.

[0024] Other characteristics and advantages of the invention will become apparent from the following description of a preferred embodiment given in a non-limiting manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Enzymatic hydrolysis of phosphatidylinositol to glycerophosphoinositide is shown;

[0026] Figure 2 The general structure of a phospholipid molecule is shown;

[0027] Figure 3 Shown are graphs reporting protein expression levels expressed as fold increase in stimulated cells stimulated with LPS and treated with GPI after 5 h of treatment;

[0028] Figure 4 Shown are graphs reporting protein expression levels expressed as fold increase in stimulated cells stimulated with LPS and treated with GPI after 24 h treatment;

[0029] Figure 5 Shown are graphs reporting miRNA expression associated with the M1 (inflammatory, left) or M2 (anti-inflammatory, right) commitment of macrophages after 24 H of treatment;

[0030] Figure 6 Shown are graphs reporting the production of IL12 and 23 during inflammation as indicated by the fold increase in stimulated cells stimulated with LPS and GPI treatment after 24 hours of treatment. DETAILED DESCRIPTION

[0031] In a first aspect, the present invention relates to a process for preparing L-α-glycerylphosphoryl-D-myoinositol (hereinafter also referred to as glycerophosphoinositide or GPI) from a crude or partially purified phospholipid mixture, and to a process for the parallel production of concentrated and purified solutions of L-α-glycerylphosphoryl-D-choline and L-α-glycerylphosphoryl-D-ethanolamine.

[0032] The term "crude or partially purified phospholipid mixture" refers to crude lecithin of plant or animal origin which also comprises triglycerides, purified lecithin which comprises only phospholipids and further purified lecithin which comprises only the precursor phosphatidyl-inositol.

[0033] Figure 1 A scheme is shown for the enzymatic hydrolysis of phosphatidylinositol to produce glycerophosphoinositide. Figure 2 The general structures of some phospholipid molecules are shown, which can generate the respective glyceryl derivatives by deacylation. For the purposes of the present invention, of particular interest are the molecules represented by the abbreviations PI, PE and PC, which generate L-α-glycerylphosphoryl-D-myoinositol (GPI), L-α-glycerylphosphoryl-D-ethanolamine (GPE) and L-α-glycerylphosphoryl-D-choline (GPC), respectively, by deacylation.

[0034] The method according to the present invention comprises the following steps in sequence:

[0035] a) hydrolyzing a crude or partially purified phospholipid mixture by treating it with PLA1 and PLA2 enzymes;

[0036] b) microfiltration of the mixture from step a), and subsequent ultrafiltration and nanofiltration of the microfiltrate to obtain a concentrated aqueous fraction of the reaction product;

[0037] c) electrodialysis of the aqueous fraction of step b) for separation of ionic compounds from neutral compounds;

[0038] d) performing ion exchange chromatography.

[0039] Step a) enzymatic hydrolysis is carried out on a reaction mixture wherein the phospholipid concentration in water is between 10 and 100 g / kg of mixture, preferably between 20 and 80 g / kg of mixture, even more preferably between 40 and 60 g / kg of mixture. In fact, enzymatic hydrolysis requires maximization of the interaction between the enzyme and the phospholipid itself, which, by forming micelles, reduces the interaction between the reaction site of the enzyme and the substrate itself. Operating at the above concentrations results in the formation of less stable micelles, and therefore more susceptible to enzyme attack.

[0040] In a particularly preferred embodiment, step a) enzymatic hydrolysis is carried out on a fine dispersion of lecithin in water, wherein the lecithin has a concentration between 45 and 55 g / kg, or about 50 g / kg.

[0041] The amount of enzymes (PLA1 and PLA2) ranged between 200 and 4000 mg / kg mixture, depending on the reaction temperature and the total reaction completion time.

[0042] The enzymes PLA1 and PLA2 can be used in two consecutive steps, such as Figure 1 As shown, first operate with PLA2 and then operate with PLA1, or use them in a single step.

[0043] In a preferred embodiment, by operating with a mixture of PLA1 and PLA2, the concentration of PLA1 and the concentration of PLA2 are respectively between 800 and 1300 mg / kg aqueous suspension, more preferably between 950 and 1050 mg / kg aqueous suspension.

[0044] The enzymatic hydrolysis reaction is carried out at a pH between 3.5 and 5.5 or between 3.5 and 5 and a temperature between 15°C and 55°C or between 15°C and 45°C, preferably between 25°C and 40°C. The reaction mixture consisting of the suspension has an initial pH of about 6.5, but is preferably acidified to the above range (pH = 3.5-5) by adding concentrated phosphoric acid. The pH of the solution and the reaction temperature must be carefully maintained within the above range, because competitive hydrolysis reactions of phospho-glycerol bonds or phospho-X radical bonds can significantly affect the reaction yield. In a particularly preferred embodiment, a mixture of PLA1 and PLA2 is used, the reaction temperature is between 28°C and 32°C, or about 30°C, and the pH is between 3.8 and 4.2.

[0045] In step a), the conversion of the phospholipids to the glyceryl derivatives is between 60% and 90%, depending on the starting phospholipids, with an average value generally between 75% and 80%, or about 78%.

[0046] Step b) comprises first adjusting the pH to about 7, for example by adding a concentrated aqueous solution of sodium hydroxide or potassium hydroxide. The reaction mixture is then subjected to microfiltration and subsequent ultrafiltration at said pH of about 7.

[0047] Microfiltration is carried out as tangential filtration on polymeric, ceramic or steel membranes, varying according to the phospholipid matrix used and the concentration factor to be obtained for the recirculating solution.

[0048] The steel membrane used is a sintered membrane consisting of a 316L stainless steel porous metal core with a small wall thickness (0.5-1.0 mm), the inner diameter of which is covered by a thin titanium oxide layer with very small pores (up to 20 nm). These characteristics allow the use of metal membranes in microfiltration and ultrafiltration systems for food (soft drinks, dairy products, etc.), pharmaceuticals, chemical and petrochemical industries or wastewater treatment, among other applications. In this particular application, it is preferred to use membranes with pores of about 0.1 micrometers.

[0049] Ceramic membranes can also be used for the microfiltration process, preferably using membranes with pores of 0.1 micrometer.

[0050] Ceramic membranes have a support of mixed oxides of titanium, zirconium and aluminum and an active layer of zirconium oxide or titanium oxide, are made of the purest materials and can have a pore size range from narrow ultrafiltration to wide microfiltration. The standard pore size for microfiltration is: 0.14-0.2-0.45-0.8-1.4 microns.

[0051] Tubular ceramic membranes can be used under very harsh conditions, maximum temperature = 350°C, maximum pressure = 10 bar, washing with NaOH (pH = 14) at 85°C, washing with HNO3 (pH = 0) at 50°C, heat sterilization (water or steam). There are many configurations available, with the ability to vary the number and diameter of channels and the length of the element.

[0052] All of the above membranes are commercially available.

[0053] The pore size of the membrane may be in the range of 0.45 to 0.1 micron. Filtration, followed by diafiltration to maximize product recovery, is preferably performed with a ceramic or steel membrane having a pore size of about 0.1 micron.

[0054] The purpose of microfiltration is to separate the water-soluble components from the amphiphilic substances still present which tend to form micelles.

[0055] The function of the subsequent ultrafiltration and nanofiltration processes is to remove or select various types of materials according to the molecular weight and size of the material in the solution. The first ultrafiltration process is used to remove the part of enzymes and other high molecular weight molecules, such as residual proteins present in the raw material, which are retained in the recirculation, and other components enter the permeate. The function of the subsequent nanofiltration is to remove fatty acids, which are eluted into the permeate at acidic pH, while other molecules include GPI, GPC, GPE and remain in the recirculation. The first ultrafiltration process is carried out using a membrane cut off at 20kDa to 500kDa, preferably 30kDa to 150kDa, more preferably 40kDa to 80kDa.

[0056] Preferably, a second ultrafiltration process can be performed at a lower cut-off to remove other polymers and oligomers with an average molecular weight that remain in the recirculation, while GPI, GPC and GPE are collected in the permeate. The subsequent nanofiltration process removes the fatty acids that are eluted into the permeate at an acidic pH, while GPI, GPC, GPE and other molecules remain in the recirculation. The second ultrafiltration process is performed using a membrane cut-off in the range of 1 kDa to 10 kDa, preferably 2 kDa to 8 kDa, more preferably 4 kDa to 6 kDa.

[0057] Nanofiltration is performed on a wound spiral polymer membrane with a cut-off ranging from 150 Da to 3000 Da, preferably from 150 Da to 1000 Da, more preferably from 200 Da to 800 Da or from 150 Da to 300 Da.

[0058] In a preferred embodiment, step b) comprises the following sub-steps:

[0059] b1) ultrafiltration of the microfiltration product using a wound spiral polymer PES membrane with a cut-off value of about 50,000 Da;

[0060] b2) subsequent ultrafiltration using a membrane with a cut-off of about 5000 Da, wherein GPI, GPE, GPC and other low molecular weight molecules permeate, and additional oligomers are recycled;

[0061] b3) membrane nanofiltration with a wound spiral PES membrane with a cut-off of about 1000-150 Da, thereby obtaining a partial separation between the GPI kept in the recirculation and the GPC and GPE passed with the permeate;

[0062] b4) diafiltration with demineralized water having a low electrical conductivity to obtain a recirculating solution comprising mainly GPI and partly relatively lower concentrations of GPC and GPE relative to step b3).

[0063] The GPI-rich fraction, but with the presence of GPC and GPE, has the GPI component being charged, while the GPC and GPE components are predominantly neutral (internal salts).

[0064] The function of step c) electrodialysis is to separate charged molecules from amphiphilic molecules, or charged molecules with relatively high molecular weight and poor ionic mobility from inorganic ions with low molecular weight and high ionic mobility, if separation by tangential microfiltration is not effective enough. The aqueous solution is circulated through the electrodialysis system, thereby obtaining the migration of the GPI salt and the retention of GPC and GPE in the mainstream solution.

[0065] In a preferred embodiment, the permeated solution is further subjected to a second stage of nanofiltration using a membrane in the range of 1000 Da to 150 Da to primarily remove low molecular weight inorganics and concentrate the solution.

[0066] Step d) ion exchange chromatography allows to obtain purities > 90% especially in the production of GPI, but also in the production of other glycerophospholipids (GPE and GPC).

[0067] Ion exchange chromatography involves i) preliminary ion exchange of an aqueous solution containing different concentrations of GPI, GPE and GPC on a strong cation resin in H+ form, followed by ii) passing the eluent solution over a strong or weak anion resin in OH- form, depending on the impurities to be removed.

[0068] For step i), the resin to be used is a strong cationic resin, usually a polystyrene / divinylbenzene gel type resin, which has sulfonic acid groups as functional exchange groups and is in the acid form. + Adjustment; After regeneration and adjustment with 2BV of 1M HCl at a flow rate of 1.5-2BV / h, the load flow rate of the solution is preferably about 1.5BV / h (bed volume per hour).

[0069] For step ii), the resin to be used is a weakly anionic macroreticular resin consisting of a styrene / divinylbenzene copolymer support functionalized by a tertiary amine and conditioned in the OH form; after regeneration and conditioning with 2 BV of 1 M NaOH at a flow rate of 1.5-2 BV / h, the load flow rate of the solution is preferably about 1.5 BV / h (bed volume per hour).

[0070] If a weak anion resin is used, the resin is first filled with the solution from the acid exchange and then washed with water, washed with a dilute solution of formic acid, acetic acid or propionic acid at a concentration of about 1-3% w / v to remove less acidic impurities. The resin is then eluted with a 5-10% w / v solution of formic acid, acetic acid or propionic acid or an ammonium carbonate solution at a concentration of 0.1-0.5 g / l to obtain a pure GPI solution.

[0071] Purification of GPE and GPC from related mixtures (also containing trace amounts of GPI) can be achieved by passing the mixture over strong cation resins and strong anion resins.

[0072] In a particularly preferred embodiment, step d) is carried out with a strong sulfonic acid resin in H+ form and then with a weak resin in OH- form, the weak base resin being first washed with 12 g / l of acetic acid and then eluted with ammonium carbonate having a concentration of 0.03 M.

[0073] In a second aspect, the present invention relates to glycerophosphoinositide (GPI) for use in the prevention and treatment of COVID-19 syndrome.

[0074] In particular, the present invention relates to glycerophosphoinositide (GPI) for use in the prevention and early treatment of COVID-19 sepsis.

[0075] GPE and GPC can be used as medicines, supplements or dietary products, for example for stimulating neurological functions, such as memory stimulation or even for treating degenerative brain syndromes in the elderly.

[0076] Experimental Section

[0077] Example 1

[0078] Enzymatic Hydrolysis of Phospholipids in Defatted Sunflower Lecithin

[0079] 10 g of defatted sunflower lecithin were finely dispersed in 200 g of water using a rotor / stator stirrer for 5 minutes; then enzymes PLA1 and PLA2 were added at a concentration of 1000 mg / kg, respectively, and the suspension was stirred and reacted at a temperature of 30° C. at pH 4 for 24 hours by adding phosphoric acid.

[0080] The initial solution had no hydrolyzed phospholipids, whereas the final suspension after 24 hours had a GPI reaction yield of 80%.

[0081] Example 2

[0082] Microfiltration of liposome suspensions from enzymatic hydrolysis of sunflower lecithin

[0083] 200 liters of suspension obtained by enzymatic hydrolysis of sunflower lecithin by phospholipase PLA1 and PLA2 enzymes (Example 1) were microfiltered in a tangential microfiltration system with a sintered steel membrane with a pore size of 0.1 micron. The temperature was maintained at 30° C. and the pressure was in the range of 2.0 to 2.5 bar. A recycle comprising unreacted lipids and phospholipids, free fatty acids and water and a clarified permeate comprising glycerophospholipids, glycerol, enzymes, salts and free fatty acids were obtained.

[0084] Example 3 (Comparative)

[0085] Ultrafiltration of the microfiltration solution from the enzymatic hydrolysis with a 5 kDa cut-off

[0086] 200 liters of the clarified solution containing 3 g / l of GPI after 0.1 micron microfiltration were filtered with a 4040 membrane of PES with a molecular cutoff of 5 kDa at a pressure of 6.5 bar and a temperature of 25°C, and the permeate and the recycle were collected separately.

[0087] Surprisingly and contrary to expectation, the permeate contained a low concentration of GPI (0.2 g / l), while the recycle was concentrated in GPI, the final situation being a permeate volume of 150 liters and a recycle of 50 liters and a final recycle GPI concentration of 11.2 g / l.

[0088] Example 4

[0089] Ultrafiltration of the microfiltration solution from the enzymatic hydrolysis at 50 kDa

[0090] 200 liters of 0.1 micron microfiltered clarified solution containing 3 g / l of GPI were filtered with a 4040 PVDF membrane having a molecular cutoff of 50 kDa at a pressure of 3.0 bar and a temperature of 25°C, and the permeate and recycle were collected separately.

[0091] The total collected permeate (150 liters) had a GPI concentration of 2.9 g / l, and the recycle had a GPI concentration of 3.0 g / l. The recycle was then diluted 1:1 v / v with deionized water and filtered again; the permeate obtained had a GPI concentration of 1.5 g / l, as did the recycle (1.5 g / l).

[0092] Example 5

[0093] The solution filtered at 50 kDa was filtered at 5 kDa for the second time.

[0094] 200 liters of the clarified solution from the 50 kDa ultrafiltration permeate containing 2.5 g / l of GPI (Example 4) were filtered at 6.0 bar and 25°C with a 4040 membrane of PES with a molecular cutoff of 5 kDa, and the permeate and recycle were collected separately.

[0095] The permeate had a GPI concentration of 2.3 g / l, while during the permeation, the GPI concentration of the recycle was maintained at 2.5-2.6 g / l; the recycle volume obtained at the end of the filtration (40 liters) was diluted with deionized water at 1:1 v / v and filtered again at 5 kDa under the same conditions as the previous filtration (6.0 bar and T=25°C), obtaining 40 liters of permeate with a GPI of 1.3 g / l and 40 liters of recycle with a GPI of 1.3 g / l.

[0096] Example 6

[0097] The solution ultrafiltered at 5000Da was nanofiltered at 150-300Da.

[0098] 200 liters of clarified solution from the ultrafiltration solution containing 1.5 g / l of GPI (Example 5) were filtered with a 4040 membrane at a molecular cutoff of 150-300 Da at a pressure of 35.0 bar and a temperature of 25° C., and the permeate and recycle were collected separately.

[0099] The final recycle (40 litres) had a GPI concentration of 7.2 g / l, while the permeate had a GPI concentration of < 0.05 g / l.

[0100] Example 7

[0101] Electrodialysis

[0102] The fluid from the membrane filtration process (Example 6) is treated in an electrodialysis system, preferably in an AEM-CEM-BPM or AEM-BPM configuration. The fluid typically has a GPI concentration of 0.5-5.0 g / l; the product is processed and the GPI is concentrated X2-X4, while it is separated from the uncharged components and the GPC and GPE fractions. The recovery % of GPI appears to be 70-90%.

[0103] Example 8

[0104] Ion exchange chromatography

[0105] As before, the product obtained from Example 7 was subjected to ion exchange chromatography to obtain pure GPI.

[0106] The purity obtained by the above method, expressed as GPI-H or GPI-X (where X is a cation neutralizing the GPI-anion), is >95% w / w.

[0107] Biological experiments

[0108] The purpose of the experiments performed was to evaluate in vitro the anti-inflammatory activity and immunomodulatory capacity of monocytes activated and treated with GPI in response to bacterial or viral activation. The results of the measurement of protein production or miRNA levels are expressed as an increase relative to the corresponding control (unstimulated) cells.

[0109] The inhibitory effect of GPI was observed after 5 h of treatment ( Figure 3 ) and 24 hours ( Figure 4 ) and the miRNA level after 24 hours of treatment ( Figure 5 ) is visible.

[0110] As for the antiviral effect, it is evaluated by focusing on interleukin (IL) 12 and 23. The interleukin (IL)-12 family consists of pro-inflammatory and anti-inflammatory cytokines that are able to send signals to activate host antiviral immunity while preventing the hyperresponsive immune response caused by active viral replication and viral clearance. IL-12 and IL-23 are produced and released by immune cells such as monocytes and macrophages to signal T cell proliferation and release of effector cytokines, which subsequently activate the host's defense against viral infection (1-8).

[0111] Our results ( Figure 6 ) demonstrated that monocytes, once activated and treated with GPI, showed a reduction in these ILs after GPI treatment.

[0112] Monocytes

[0113] By Ficoll-Paque TM PLUS gradient then Percoll TM Human mononuclear cells were isolated from fresh buffy coats of healthy blood donors and provided by CentroImmunotrasfusionale, Hospital of Ferrara, Italy. The cells were firstly purified by 1.077 g / mL Fico ll-PaqueTM PBMCs were obtained by density gradient separation using Ficoll Plus (GE health care Life Sciences, Little Chalfont, UK). After collecting mononuclear cells at the interface between the Ficoll and plasma medium layers, the cells were washed with PBS and then separated by 1.130 g / mL Percoll. TM (GE health care Life Sciences) solution was layered and then centrifuged at 1650rpm for 30min at 4°C. The mononuclear cell layer showed turbidity and occasional red spots, which were carefully aspirated and washed with PBS to remove trace amounts of percollTM. The harvested mononuclear cells were then suspended in RPMI 1640 medium (EuroClone) containing 2% FBS and incubated at 37°C and 5% CO2. After 1h, non-adherent cells including residual lymphocytes were removed and monocytes were separated using a cell scraper and then inoculated into plastic culture wells.

[0114] To simulate the inflammatory environment in vitro, cDMEM was supplemented with 10 ng / mL recombinant human IL-1β (PeproTech, London, UK) and 10 ng / mL recombinant human TNF-α (PeproTech) to generate inflammation-cDMEM (i-cDMEM).

[0115] To stimulate a bacteria-like environment in vitro, cDMEM was supplemented with 0.1 ug / ml of LPS (Sigma).

[0116] Treatment with GPI: 300uM.

[0117] Elisa test analysis

[0118] The culture supernatants after 5 and 24 h were collected, centrifuged at 400×g for 10 min at 4°C, and then analyzed for TF HGF, PGE-2, and IL-10 production using commercial ELISA kits (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0119] RNA extraction and first-strand cDNA synthesis

[0120] Total RNA was extracted from the culture medium 12 and 24 h later using the Total RNA Purification Plus Kit and purified using NanoDrop TM The RNA quality and concentration of the samples were measured by ND-1000 (Thermo Fisher Scientific). 2500 ng of total RNA was reverse transcribed using the First Strand Kit (Qiagen, Hilden, Germany) in a final reaction volume of 20 μL.

[0121] Real-time PCR

[0122] According to inflammation RT 2 Profiler PCR Array (Qiagen) User Manual, using StepOnePlus TM Real-time PCR system (Applied Biosystems TM , Foster City, California, United States) and used RT 2 Real-time PCR was performed using SYBRGreen ROX FAST Master Mix (Qiagen). Thermal cycling and fluorescence detection were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. At the end of each run, melting curve analysis was performed using the following program: 95°C for 1 min, 65°C for 2 min with optics off, and 65°C to 95°C at 2°C / min with optics on. Data were analyzed using web-based PCR array data analysis software, available from https: / / www.qiagen.com / it / shop / genes-and-pathways / data-analysis-center-overview-page / .

[0123] Statistical analysis

[0124] All results are expressed as mean ± standard deviation (SD) obtained from at least three independent experiments. Significant differences between groups were determined by analysis of variance (ANOVA). Student's t-test was performed to determine statistical significance between two samples. Different labels indicate p<0.05, p<0.01, and p<0.001.

[0125] result

[0126] GPI reduces the production of inflammatory cytokines.

[0127] We cultured monocytes in the following medium:

[0128] - In the presence or absence of 300uM GPI, the environment of inflammation was simulated.

[0129] - LPS was used to simulate a sepsis environment in the presence or absence of GPI at a concentration of 300 uM.

[0130] - In the presence or absence of 300uM GPI, the virus infection environment was simulated.

[0131] We tested:

[0132] -Inflammatory cytokine production: IL1, IL2, IL4, IL6, INF G, TNFα, TF

[0133] - Anti-inflammatory cytokine production: IL10

[0134] - miRNAs associated with the M1 (inflammatory) phenotype: miR-181a, miR-155-5p, miR-204-5p, miR-451, miR-125b-5p, miR-181a-5p, miR-193b-3p, miR-125a-5p, Akt2, p110d, PTEN, TSC1, p85a

[0135] - miRNAs associated with M2 (anti-inflammatory) phenotype: Akt1, p110a, p110b, p110g, TSC1, Rictor / mTORC2

[0136] -ILs associated with viral infection: IL12 and IL23 are activated in the presence of viral infection.

[0137] Measurements of protein production or miRNA levels are expressed as fold increase relative to corresponding control (unstimulated) cells and are means (SD) of triplicates from one representative experiment out of four performed with cells from different donors.

[0138] The inhibitory effect of GPI was observed after 5 h of treatment ( Figure 3 ) and 24 hours ( Figure 5 ) and miRNA levels after 24 h of treatment ( Figure 5 ) is obvious.

[0139] The interleukin (IL)-12 family consists of pro-inflammatory and anti-inflammatory cytokines that are able to send signals to activate host antiviral immunity while preventing overly reactive immune responses due to active viral replication and viral clearance. Among them, IL-12 and IL-23 are produced and released by innate immune cells such as monocytes and macrophages to signal T cell proliferation and release of effector cytokines, which subsequently activate host defense against viral infection. To this end, we investigated the production of IL12 and 23 during inflammation. Our results ( Figure 6 ) demonstrated a decrease in these ILs after treatment with GPI.

[0140] Pharmaceutical preparations

[0141] The preparations according to the invention can be prepared according to conventional methods, such as those described in: Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co., New York, USA, 17th edition, 1985, or Remington, The Science and Practice of Pharmacy, edited by Allen, Loyd V., Jr, 22nd edition, 2012.

[0142] The present invention will be further described below by the following formulation examples.

[0143] Preparation Examples

[0144] Example 1 -tablet

[0145]

[0146] Example 2 -Injectable preparations

[0147] Vial 1

[0148] GPI 50mg

[0149] Vial 2

[0150]

Claims

1. A method for preparing glycerophosphoinositide from a crude or partially purified phospholipid mixture, comprising the following steps in order: a) hydrolyzing a crude or partially purified phospholipid mixture by treating it with PLA1 and PLA2 enzymes; b) subjecting the mixture from step a) to microfiltration, and subsequently subjecting the microfiltrate to ultrafiltration and nanofiltration to obtain a concentrated aqueous fraction of the reaction product; c) electrodialyzing the aqueous portion of step b) for separation of ionic compounds from neutral compounds; d) performing ion exchange chromatography.

2. The method according to claim 1, wherein: The crude or partially purified phospholipid mixture of step a) has a phospholipid concentration in water of 10 to 100 g / kg of mixture, preferably 20 to 80 g / kg of mixture, even more preferably 40 to 60 g / kg of mixture.

3. The method according to claim 2, wherein: The phospholipid mixture is a fine dispersion of lecithin in water, wherein the lecithin has a concentration of 45 to 55 g / kg, or about 50 g / kg.

4. The method according to any one of claims 1 to 3, wherein: The amount of PLA1 and PLA2 enzymes in step a) ranges between 200 and 4000 mg / kg of the mixture.

5. The method according to claim 4, wherein: Step a) is carried out with a mixture of PLA1 and PLA2, wherein the concentration of PLA1 and the concentration of PLA2 are 800 to 1300 mg / kg aqueous suspension, or 950 to 1050 mg / kg aqueous suspension, respectively.

6. The method according to any one of claims 1 to 5, wherein: Step a) is carried out at a pH between 3.5 and 5.5 or between 3.5 and 5 and at a temperature between 15°C and 55°C or between 15°C and 45°C, preferably between 25°C and 40°C or between 28°C and 32°C, and wherein the initial pH of the reaction mixture of about 6.5 is preferably brought to a pH range of 3.5-5, preferably by adding concentrated phosphoric acid.

7. The method according to any one of claims 1 to 6, wherein: In step b), the pH is adjusted to about 7 by adding a base, preferably concentrated NaOH or KOH solution.

8. The method according to any one of claims 1 to 7, wherein: In step b), the microfiltration is performed by tangential filtration on a polymeric, ceramic or steel membrane having a pore size of 0.45 to 0.1 μm, and wherein the microfiltration is preferably followed in sequence by diafiltration.

9. The method according to any one of claims 1 to 8, comprising a first ultrafiltration and preferably a second ultrafiltration, wherein: The first ultrafiltration and the second ultrafiltration are performed on a wound spiral polymer membrane, and wherein: - said first ultrafiltration is performed using a membrane with a cut-off ranging from 20 kDa to 500 kDa, preferably from 30 kDa to 150 kDa, more preferably from 40 kDa to 80 kDa, - the second ultrafiltration is performed using a membrane with a cut-off ranging from 1 kDa to 10 kDa, preferably from 2 kDa to 8 kDa, more preferably from 4 kDa to 6 kDa, And wherein the nanofiltration is performed on a wound spiral polymer membrane with a cut-off ranging from 150 Da to 3000 Da, preferably from 150 Da to 1000 Da, more preferably from 200 Da to 800 Da or from 150 Da to 300 Da.

10. The method according to claim 9, wherein: Step b) comprises the following sub-steps: b1) ultrafiltration of the microfiltration product using a wound spiral polymer PES membrane with a cut-off of about 50,000 Da; b2) subsequent ultrafiltration using a membrane with a cut-off of about 5000 Da, wherein GPI, GPE, GPC and other low molecular weight molecules permeate, and additional oligomers are recycled; b3) membrane nanofiltration with a wound spiral PES membrane with a cut-off of about 1000-150 Da, thereby obtaining a partial separation between the GPI kept in the recirculation and the GPC and GPE passed with the permeate; b4) diafiltration with demineralized water having a low electrical conductivity to obtain a recirculating solution comprising mainly GPI and partly relatively lower concentrations of GPC and GPE relative to step b3).

11. The method according to any one of claims 1 to 10, wherein: In step c), the aqueous solution is circulated through an electrodialysis system, thereby obtaining the migration of the GPI salt and the retention of GPC and GPE in the mainstream solution.

12. The method according to any one of claims 1 to 11, wherein: The permeated solution was further subjected to a second step of nanofiltration using a membrane ranging from 1000 Da to 150 Da to remove mainly low molecular weight inorganic substances and concentrate the solution.

13. The method according to any one of claims 1 to 12, wherein: Step d) ion exchange chromatography comprises i) preliminary ion exchange of an aqueous solution containing GPI, GPE and GPC in different concentrations on a strong cationic resin in H+ form, and then ii) passing the elution solution on a strong or weak anionic resin in OH- form, and wherein, if a weak anionic resin is used, the resin is first filled with the solution from the acid exchange, then washed with water, a dilute solution of formic acid, acetic acid or propionic acid at a concentration of about 1-3% w / v to remove less acidic impurities, and then eluted with a 5-10% w / v formic acid, acetic acid or propionic acid solution to obtain a pure GPI solution.

14. The method according to claim 13, wherein: Step d) is carried out with a strong sulfonic acid resin in the H+ form and then with a weak resin in the OH- form, wherein the weak base resin is first washed with 12 g / l acetic acid and then eluted with ammonium carbonate having a concentration of 0.03 M.

15. Use of glycerophosphoinositide obtainable by the method according to any one of claims 1 to 14 for the prevention and early treatment of syndromes caused by COVID-19 or other viral infections.

16. The glycerophosphoinositide according to claim 15, for use in the prevention and early treatment of sepsis caused by COVID-19 or other viral infections.

17. Use of glycerophosphoinositide obtainable by the method according to any one of claims 1 to 14 in the preparation of a medicament for the prevention and early treatment of syndromes caused by COVID-19 or other viral infections.

18. Use of the glycerophosphoinositide according to claim 17 in the preparation of a medicament for the prevention and early treatment of sepsis caused by COVID-19 or other viral infections.

19. A method for preventing or early treating a syndrome due to COVID-19 or other viral infections, comprising administering an effective amount of glycerophosphoinositide obtainable by the method according to any one of claims 1 to 14.

20. The method according to claim 19, wherein: The syndrome is sepsis due to COVID-19 or other viral infection.

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