2-Deoxy-D-Glucose for Use in Disease Therapy

Technical Field

The present application relates to the use of 2-DG ( 2 -deoxy-D-glucose ) for the prevention and/or the treatment of a viral disease , collaboratively in the treatment of cancer with other cancer treatments , and in a natural preparation with natural minerals to assist the health and immunity of a subj ect .

In particular , the present application relates to the use of various preparations and forms of 2- DG, particularly encapsulated in liposomes or pro-lipo- somes or not-encapsulated, for use in treating viral diseases and in the treatment of cancer .

Background Art

2-DG is a glucose analogue e . g . as a marker for glucose uptake and hexokinase activity, as an inhibitor of glucose-6-phosphate isomerase and thereby as an inhibitor of glycolysis . Medical use of 2-DG includes ra- dioactively labelled forms 2 -DG for use in diagnostic methods such as autoradiography and further includes therapeutic applications in cancer therapy . Clinical trials revealed a very high tolerance of 2-DG up to 63 mg per kg body weight and day .

Like cancer cells , also virally infected cells exhibit a high rate of glycolysis . Gualdoni , G et al . ( 2018 ) reported glycolysis inhibition by 2-deoxyglu- cose ( 2 -DG) causing glucose deprivation of infected cells resulted in decreasing rhinovirus replication in vitro and inhibited infection and inflammation in a mouse model (Gualdoni , G et al . PNAS 115 , E7158 -E7165 ( 2018 ) . Bo kova et al. reported inhibition of glycolysis on SARS-CoV-2 replication by 2-DG (Bojkova, D. et al. Nature 583, 469- 472 (2020) .

However, while 2-DG has potential in the treatment of such infections and diseases, considerable difficulties have been faced in producing suitable preparations and forms and delivery systems that can be used in treating a subject in need thereof, such that 2-DG can actually reach targeted tissues efficiently and effectively and be maintained in sufficient concentrations and usable forms in order to provide therapeutic benefit.

Thus, there is still a need to provide new preparations and forms of 2-DG for use in treatment of viral infections, diseases, and in the general improvement of the immune health of a subject.

Disclosure of the Application

Hence, it is a general object of the application in a first aspect to provide a preparation of 2-DG that is suitable for treatment of a viral disease caused by an enveloped virus, in particular by SARS-CoV-2, influenza, and Dengue fever.

It is another general object of the application in a second aspect to provide a preparation of 2-DG that is suitable for use in the treatment of cancer, in particular collaboratively in the treatment of cancer with other cancer treatments .

It is another general object of the application in a third aspect to provide a natural preparation of 2-DG with natural minerals for use as a health supplement . These three aspects of the application can be implemented independently of each other or in any combination unless the context clearly dictates the contrary .

In order to implement these and still further obj ects of the application, which will become more readily apparent as the description proceeds , the use of 2-DG in medical applications of the application is described and claimed below in various embodiments :

2-DG Preparations and Forms

Preferred preparations and forms of 2 -DG for use in the first and second aspects of the invention and the embodiments as described herein are provided as follows .

In an embodiment , 2-DG is provided as a micron or a submicron particle in a preparation, wherein in particular said micron or submicron particle is a mechanically micronized particle or is a micronized particle obtained by spray drying .

In an embodiment , 2-DG is provided as a preparation in a liposomal or a proliposomal formulation preferably achieved by a spray-dying method, nebulisation method, and/or other liposomes preparation method .

The preparation preferably comprises an amount of 2-DG in a range of between approximately 1% and 75 % w/w of the total weight of the preparation, in particular an amount of 2-DG in a range with lower limit of approximately 10% or 20% or 30% and an upper limit of approximately 35 % or 45 % to 55 % w/w of the total weight of the preparation, in particular between approximately 10% and 40% w/w or between approximately 15% w/w and 30% w/w of the total weight of the preparation .

The preparation preferably further comprises an excipient comprising a lipid fraction comprising or consisting of a phospholipid fraction in an amount of approximately 5% to 80% w/w, in particular approximately 15 % to 50% w/w of the total weight of the preparation .

The total phospholipid fraction preferably comprises at least approximately 10% w/w up to 60% w/w, preferably in a range between approximately 20% w/w and 50% w/w most preferably in a range between approximately 30% w/w and 40% w/w of a combination of dipalmitoyl phosphatidylcholine ( DPPC ) and dimyristoylphosphatidylcholine ( DMPC ) in any weight ratio .

The preparation comprises DPPC and DMPC in a molar ratio from approximately 50 : 50 to 100 : 0 , preferably a molar ratio of DPPC to DMPC from approximately 100 : 0 to 85 : 15 molar ratio , most preferably from approximately 95 : 5 to 90 : 10 molar ratio .

In an embodiment , the preparation comprises a further excipient selected from the group of excipients comprising :

- an amino acid, in particular leucine or glycine , in particular in an amount of 0% w/w up to approximately 80% w/w of the total weight of the preparation, more particular in an amount of approximately 10% w/w up to approximately 80% w/w, more particular in an amount of approximately 10% w/w up to 50% w/w, more particular in an amount of approximately 10% w/w up to 30% w/w;

- trehalose in an amount of 0% w/w up to approximately 60% w/w of the total weight of the preparation, more particular in an amount of approximately 5% w/w up to 30% w/w;

- mannitol , 0% w/w up to 60% w/w of the total weight of the preparation, more particular in an amount of5 % w/w up to 30% w/w;

-propylene glycol or/and, glycerol , ethyl alcohol in the concentration range from 10 to 80% of the total weight of the liquid preparation;

- one or more further phospholipid, in particular a natural or a semi-synthetic phospholipid, one or more further negatively or a positively charged phospholipid, in particular in an amount of approximately 1% up to 10% of the molar % of the phospholipid fraction, more particular in an amount of approximately

5 molar % up to 10 molar % , wherein in particular the one or more further phospholipid is in particular selected from the group comprising phosphatidylglycerol , dimiristoyl phosphatidylglycerol , dipalmitoylphosphatidylglycerol , hydrogenated soybean phosphatidylcholine ( HSPC ) , soybean phosphatidylcholine ( SPC ) and wherein optionally the phospholipids comprises DPPE or DSPE with covalently attached hydrophilic polymer, in particular a PEG or polyglycerol in a molar ratio of 0 to approximately 10 molar % of the total lipid fraction, more particular in an amount of approximately 5 molar % of total lipid fraction;

- sterol , in particular cholesterol in an amount of

0 molar % up to approximately 55 molar % of the total the lipid fraction, more particular in an amount of approximately 0 molar % up to 30 molar % ;

- antioxidant , in particular ascorbyl palmitate (AP ) in an amount of 0 molar % to 10 molar % of total lipid fraction - nicotinic acid amide , in an amount of approximately 10% w/w up to 80% w/w of the total weight of the preparation, more particular in an amount of approximately 20 to 60% w/w of the preparation; and/or

- urea in an amount of approximately 20% w/w up to

80% w/w of the total weight of the preparation, more particular in an amount of approximately 40 to 60% w/w of the preparation .

In an embodiment , the formulation comprises :

- liposome sizes ranging from approximately 30 nm to 200 nm in particular for intravenous delivery;

- liposome sizes ranging from approximately 50 nm to 5 pm, in particular for pulmonary delivery;

- unilamellar liposomes of sizes ranging from approximately 30 to 120 nm in particular for intravenous delivery;

In an embodiment :

- liposome sizes range from approximately 30 nm to 200 nm in particular for intravenous delivery;

- liposome sizes range from approximately 50 nm to 5 pm, in particular for pulmonary delivery;

- unilamellar liposomes sizes range from approximately 30 to 120 nm.

In an embodiment , the liposomal or a prolipo- somal formulation upon contact with an aqueous environment forms liposomes within a size range selected from group of size ranges comprising :

- liposome sizes ranging from approximately 30 nm to 200 nm in particular for intravenous delivery;

- liposome sizes ranging from approximately 50 nm to 5 pm, in particular for pulmonary delivery; - unilamellar liposomes of sizes ranging from approximately 30 to 120 nm for intravenous delivery .

In an embodiment , the liposomes comprise an amount of encapsulated 2-DG in a range of approximately 1 mg to 1500 mg, in particular approximately 50 mg to 500 mg , preferably, approximately 100-200 mg per unit dosage . Preferably, the amount of 2-DG in the liposomal or pro- liposomal formulation is in a range of approximately 1 mg to 1500 mg, in particular approximately 50 mg to 500 mg, preferably, approximately 100-200 mg per unit dosage .

In an embodiment , 2-DG is provided as a preparation in a liposomal or proliposomal slow release formulation in particular for intravenous administration, and wherein the amount of the active ingredient , 2-DG, released at the time of administration ( t = 0 ) ranges from approximately 10 % to 70 % w/w of the total amount of the active ingredient in the preparation and preferably ranges from approximately 30 % to 50 % w/w .

In an embodiment , 2-DG is provided as a preparation in a liposomal or proliposomal slow release formulation in particular for intravenous administration, and wherein a dosage of 2-DG, in particular one unit dosage , preferably according to dosages described herein is administered at intervals between approximately once in 2 hours to 72 hours , in particular between approximately once in 4 to 24 hours or at intervals of approximately 6 or 12 hours .

In an embodiment , the liposomal or proliposomal formulation obtained by a method of preparation selected from a group of methods comprising :

- lyophilizing a liposomal formulation comprising 2 - DG as active ingredient , wherein in particular the preparation - by spray drying a composition comprising 2 -DG, phospholipids such as DMPC, DPPC or HSPC and optional further excipients , wherein the optional further excipient is preferably selected from the group comprising :

- auxiliary phospholipid for spray drying selected from natural phosphatidylglycerol , DMPG, DPPG, DSPG and natural cardiolipin used at a concentration of 0 to approximately 30 mol% of the total phospholipid content ; and/or

- auxiliary lipids for spray drying selected from the group of sterols , in particular cholesterol ,

- the method of preparation of proliposomes by dehy- dration-rehydration a composition comprising 2 -DG, phospholipids and optional excipients followed by extrusion and spray drying for the formation of unilamellar liposomes .

- by nebulization of the liquid proliposomal formulation containing 2 -DG, lipids (phospholipids , sterols ) and one of the selected solvents such like propylene glycol , glycerol , alcohol and others to form liposomes by micron solution particles upon contact with water;

In an embodiment , 2-DG is provided as a preparation for administration by inhalation, wherein the preparation comprises particles or droplets for inhalation with a diameter of approximately 10 pm or less , in particular less than approximately 5 pm, 3 pm, 1 pm, 0 . 3 pm or 0 . 1 pm, more particular particles with a diameter in a range with a lower limit between approximately 0 . 1 pm and 1 pm and with an upper limit between approximately 0 . 5 and 5 pm.

In an embodiment , 2-DG is provided as a dry preparation for administration by inhalation, wherein the preparation comprises a content of 2-DG as active ingredient of between approximately 5 % to 80 % w/w of the total dry weight of the preparation, preferably between approximately 15 % to 60 % w/w .

In an embodiment , 2-DG is provided is formulated as a micron or a submicron particle or droplet for administration by a nebulizer, wherein in particular 2-DG is dissolved in an isotonic solution, in particular in approximately 0 . 9% saline or in water/organic solution containing lipids .

In an embodiment , 2-DG is provided as a preparation for administration by inhalation, wherein the preparation comprises an amount of 2-DG as active ingredient per unit dosage of between approximately 0 . 1 mg to 20 mg, in particular between approximately 0 . 25 mg to 10 mg , more particularly between approximately 1 to 2 mg .

In an embodiment , the size of the unilamellar liposomes comprise between approximately 30 nm to 250 nm .

2-DG is preferably provided as a preparation for administration by inhalation comprising lipids or surfactants comprising one or more of Tween 20 , Tween 80 , Pluronics , and 2 -DG in solution are encapsulated within liposomes from between approximately 1 to 600 mM, and/or encapsulated in unilamellar liposomes of the size from between approximately 30-250 nm.

In an embodiment , 2-DG is administered with at least one of the group comprising ribavirin, emetine , and NMS-873 .

In an embodiment , the application further provides a process for preparing a liposome-encapsulated pharmaceutical composition comprising at least one active ingredient selected from the group comprising ribavirin, emetine , 2-DG and NMS-873 . More preferably, the liposome- encapsulated pharmaceutical composition comprises 2 -DG and at least one active ingredient selected from the group comprising ribavirin, emetine , and NMS-873 . Even more preferably, the active ingredient comprises 2-DG .

In an embodiment , the process for preparing the liposome-encapsulated pharmaceutical composition comprises the step of spray drying the liposome-encapsulated pharmaceutical composition into particles . Preferably, the spray dried particles are less than approximately 5 pm in diameter .

2-DG is preferably provided as a preparation for administration by inhalation as a slow release formulation, wherein the amount of the active ingredient , 2- DG, released at the time of administration ( t = 0 ) ranges from between approximately 10 % to 70 % w/w of the total amount of the active ingredient in the preparation and preferably ranges from between approximately 30 % to 50 % w/w .

2-DG is preferably provided as a preparation for administration by inhalation, wherein a dosage of 2- DG, in particular one unit dosage , preferably according to dosages described herein, is administered at intervals between approximately once in 0 . 5 hours to 24 hours , in particular between approximately once in 1 to 12 hours and preferably at intervals of approximately up to 2 or 4 or 6 or 8 or 10 or 12 or 24 hours .

In an embodiment , the application provides a method of applying a substance to the body of a subj ect , the method comprising :

- providing the substance , and - delivering the substance in the form of aerosol particles or powder particles to the nose or mouth of a subj ect , wherein the particles comprise at least one active ingredient selected from the group comprising ribavirin, emetine , 2-DG and NMS-873 . More preferably, the particles comprise 2-DG and at least one active ingredient selected from the group comprising ribavirin, emetine , and NMS-873 . Even more preferably, the active ingredient comprises 2-DG .

For the purposes of describing the invention, reference herein to a subj ect comprises reference to a human or an animal .

The particles preferably comprise a carrier material carrying the active ingredient . The carrier material preferably comprises at least one liquid selected from the group comprising : water , alcohol , propylene glycol , glycerol , liquid glucose , and/or aqueous solution . The particles preferably comprise at least a lactose and/or liposome . Delivering of the substance preferably comprises propelling the substance by means of at least one propellant comprising CFG ( chlorofluorocarbon ) and/or HFA (hydro-f luoroalkane ) .

In an embodiment , the application provides a use of a substance for inhalation, wherein the substance is provided in the form of aerosol particles or powder particles , and wherein the particles comprise at least one active ingredient selected from the group comprising ribavirin, emetine , 2 -DG and NMS-873 . More preferably, the particles comprise 2-DG and at least one active ingredient selected from the group comprising ribavirin, emetine , and NMS-873 . In a preferred embodiment , the application provides a use of a substance for inhalation, wherein the substance is provided in the form of aerosol particles or powder particles , and wherein the particles comprise 2-DG .

In an embodiment , the application provides a method of dispensing a substance , the method comprising :

- providing the substance in the form of aerosol particles or powder particles ,

-creating an aerosol with the particles suspended in the aerosol ,

-creating a directed flow of aerosol such that the suspended particles move essentially along the flow direction of the aerosol , wherein the particles comprise at least one active ingredient selected from the group comprising ribavirin, emetine , 2-DG and NMS-873 . More preferably, the particles comprise 2-DG and at least one active ingredient selected from the group comprising ribavirin, emetine , and NMS-873 .

Even more preferably, the active ingredient comprises 2-DG .

In an embodiment , the application provides a device for inhaling a substance in the form of aerosol particles or powder particles , the device comprising : a discharge nozzle for dispensing the substance in the form of aerosol particles or powder particles , a container for receiving and keeping the substance , and

- an actuator for activating the device , the actuator being configured to release a certain amount or dose of the substance kept in the container for conveying the substance through the discharge noz zle of the device , wherein the particles comprise at least one active ingredient selected from the group comprising ribavirin, emetine , 2 -DG and NMS-873 . More preferably, the particles comprise 2-DG and at least one active ingredient selected from the group comprising ribavirin, emetine , and NMS-873 . Even more preferably, the active ingredient comprises 2-DG .

The actuator is preferably a manual actuator which can be activated manually . The device preferably comprises a dosage valve defining the amount of the substance to be released, and wherein the actuator is configured to activate the dosage valve . The dosage valve is preferably an adj ustable valve such that the dosage of the substance can be adj usted prior to dispensing the substance . Upstream to the discharge noz zle , an air flow channel for conveying the substance released by the actuator to the discharge nozzle is preferably arranged . The air flow in the air flow channel is preferably created by the user by inhaling the air while keeping the nozzle of the device in a nostril or in the mouth . The actuator is preferably further configured to provide a pressurized air flow in the airflow channel . The device preferably further comprises a reservoir with a propellant , and wherein the actuator is further configured to release the propellant such that the substance can be conveyed by droplets of propellant along the flow channel .

In an embodiment , the application provides a substance for applying to a human or animal body for the treatment of lung tissue cells by inhalation, the substance comprising at least one active ingredient selected from the group comprising ribavirin, emetine , 2 -DG and NMS-873 . More preferably the substance comprises 2-DG and at least one active ingredient selected from the group comprising ribavirin, emetine , and NMS-873 . In a preferred embodiment , the application provides a substance for applying to a human or animal body for the treatment of lung tissue cells by inhalation, the substance comprising 2 -DG . The substance preferably comprises a carrier material carrying the active ingredient. The carrier material preferably comprises at least one liquid selected from: water, alcohol, liquid glucose, and/or aqueous solution. The substance preferably comprises lactose and/or liposome .

In an embodiment, the application provides a pharmaceutical composition for administration to the airways of a subject, the pharmaceutical composition comprising at least one active ingredient selected from the group comprising: ribavirin, emetine, 2-DG and NMS-873. More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising ribavirin, emetine, and NMS-873. In a preferred embodiment, the application provides a pharmaceutical composition for administration to the airways of a subject, the pharmaceutical composition comprising 2-DG.

In an embodiment, the application provides a method of treating a subject, the method comprising the step of inhalation by a subject of a pharmaceutical composition comprising at least one active ingredient selected from the group comprising: ribavirin, emetine, 2- DG and NMS-873. More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising ribavirin, emetine, and NMS-873. In a preferred embodiment, the application provides a method of treating a subject, the method comprising the step of inhalation by a subject of a pharmaceutical composition comprising 2-DG.

In an embodiment, the application pro-vides a method of producing a pharmaceutical composition for administration to the airways of a subject, the pharmaceutical composition comprising at least one active ingredient selected from the group comprising: ribavirin, emetine, 2-DG and NMS-873. More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising ribavirin, emetine, and NMS-873. In a preferred embodiment, the application provides a method of producing a pharmaceutical composition for administration to the airways of a subject, the pharmaceutical composition comprising 2-DG.

In an embodiment, the application provides a method of producing an inhalable pharmaceutical composition for a subject, the pharmaceutical composition comprising at least one active ingredient selected from the group comprising: ribavirin, emetine, 2-DG and NMS-873. More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising ribavirin, emetine, and NMS-873. In a preferred embodiment, the application provides a method of producing an inhalable pharmaceutical composition for a subject, the pharmaceutical composition comprising 2-DG.

In an embodiment, the application provides for the manufacture of a pharmaceutical composition comprising at least one active ingredient selected from the group comprising: ribavirin, emetine, 2-DG and NMS-873, for administration to the airways of a subject. More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising ribavirin, emetine, and NMS-873. In a preferred embodiment, the application provides for the manufacture of a pharmaceutical composition comprising 2-DG.

In an embodiment, the application provides for the administration to the airways of a subject a pharmaceutical composition comprising at least one active ingredient selected from the group comprising: ribavirin, emetine, 2-DG and NMS-873. More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising ribavirin, emetine, and NMS-873. In a preferred embodiment, the application provides for the administration to the airways of a subject a pharmaceutical composition comprising 2-DG.

In an embodiment, the application provides for the pulmonary delivery of a pharmaceutical composition to a subject, the pharmaceutical composition comprising at least one active ingredient selected from the group comprising: ribavirin, emetine, 2-DG and NMS-873. More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising ribavirin, emetine, and NMS-873. In a preferred embodiment, the application provides for the pulmonary delivery of a pharmaceutical composition to a subject, the pharmaceutical composition comprising 2-DG.

The pharmaceutical composition preferably comprises a powder or an aerosolized form. The pharmaceutical composition preferably comprises a liposome-encapsulated pharmaceutical composition. The pharmaceutical composition is preferably delivered to the respiratory tract of the subject.

Administration or delivery to the airways of a subject preferably comprises inhalation by the subject. Inhalation is preferably through the mouth and/or the nose of the subject.

An inhalation device is preferably used to dispense the pharmaceutical composition for inhalation by the subject. The inhalation device preferably comprises a pressurized meter dose inhaler (pMDIs) , nebulizer, or a dry powder inhaler (DPIs) . The pharmaceutical composition preferably enters cells of the respiratory tract of the subject. In an embodiment of the application, a method of treating a human or non-human animal in need thereof is provided comprising the administration of 2-DG according to one or more embodiment of the application as described herein or any combination thereof .

In an embodiment of the application, a method of treating a human or non-human animal in need thereof is provided comprising the administration of 2-DG according to one or more embodiment described herein or any combination thereof .

In an embodiment of the application, a use of 2-DG for the manufacture of a medicament is provided according to one or more embodiment described herein or any combination thereof .

In an embodiment of the application, a pharmaceutical formulation is provided comprising 2 -DG according to one or more embodiment described herein or any combination thereof .

In an embodiment of the application, a device for inhaling a preparation ( substance ) comprising 2 -DG in the form of aerosol particles or powder particles is provided, in particular according to one or more embodiment described herein or any combination thereof .

In an embodiment of the application, a kit is provided comprising at least 2 -DG, wherein 2 -DG is provided as a micron or a submicron particle in a preparation, wherein in particular said micron or submicron particle is preferably a mechanically micronized particle or is a micronized particle obtained by spray drying , or 2- DG is provided as a preparation in a liposomal or a pro- liposomal formulation, preferably obtained by spray drying . The kit preferably comprises a means for a subj ect to inhale the preparation comprising 2-DG . Preferably, the kit comprises a pressurized meter dose inhaler (pMDIs ) , nebulizer, or a dry powder inhaler ( DPIs ) .

In an embodiment , the application relates to a method for applying a substance to a human body, use of the substance , as well as a method and device for dispensing the substance .

A further embodiment of the present application is to provide an improved method for applying a substance to a human body, a novel use of the substance , as well as an improved method and device for applying the substance .

According to some embodiments , a method of applying a substance or formulation to a human body is provided . The method comprises providing the substance and delivering the substance in the form of aerosol particles or powder particles to the nose or mouth of a person or animal . In particular , these particles can be fine or sub-micron particles , such that , from the nose and mouth, they can also travel along the entire respiratory tract , in particular, to the low respiratory tract of the person . The particles comprise at least one active ingredient , in particular the particles comprise 2-DG .

In some embodiments , the particles can comprise a carrier material carrying the active ingredient . The carrier material can be in particular provided in the form of a matrix in which the active ingredient resides . The carrier material can in particular facilitate the handling and dispensing of the active agent .

The carrier material may comprise a liquid out of the group comprising water, alcohol , liquid glucose , or aqueous solution . The active ingredient or agent , which may be suspended in the liquid, can be thus easily dispensed together with the liquid . The aqueous solution with 2% salt may serve as a preservation medium for the active agent until it is dispensed . In particular, the particles may be provided in a suspension or spray formulation . The liquid carrier material can facilitate quantification or dosage of the active ingredient released together with the liquid .

In some embodiments , the particles comprise at least a lactose and/or a liposome . Lactose or liposome can serve as a carrier for the active ingredient , such that they can be easily transferred or delivered to the human body . In some embodiments , the active agent is encapsulated in a liposome , in order to achieve a sustained release with a long-lasting or retarded effect of the active agent , thus increasing the duration of the desired effect . Particles may also comprise cholesterol , which stabilizes liposomes such that an even greater delay of the activation of the agent or active ingredient can be achieved .

The particles may comprise a mixture of different liposomes .

In particular , a mixture of small liposomes , with an average size of less than 100 nm and large liposomes , with an average size of more than 150 nm . By providing different liposome sizes , a desired time profile of the active agent activity can be achieved .

The method may further comprise propelling the substance by means of at least one propellant comprising CFG ( chlorofluorocarbon) and/or HFA (hydrofluoroalkane ) . The propellant can in particular, facilitate the delivery and dosage of the active ingredients . The amount of the active agents and the dosage may be kept in the range of 5 to 10 millimoles . By limiting the amount of the active agent in the particles , the side effects related with too high dosage of the active ingredients can be avoided . In some embodiments the liposomes have a transition temperature , from solid to liquid, in the range from approximately 35 ° C to 45 ° C, more preferably, between approximately 37 ° C and 40 ° C degrees , and most preferably approximately 37 ° C . Thus , the liposomes may easier dissolve after the substance has been applied to the human body .

In an embodiment , the present application provides a use of a substance as described herein for inhalation . Thereby, the substance is preferably provided in the form of aerosol particles or powder particles , wherein the particles comprise at least one active ingredient or active agent , in particular 2-DG, and wherein the substance is preferably delivered to the mouth or nose of a subj ect ( a "person" ) , and preferably a subj ect in need thereof . In particular , the substance is preferably delivered to the mouth or nose of the person by means of an inhalator which can be operated personally by the user .

In an embodiment , the present application provides a method for dispensing a substance . The method preferably comprises providing the substance in the form of particles or powder, creating an aerosol with the particles suspended in the aerosol , and creating a directed flow of the aerosol such that the suspended particles move essentially along the flow direction of the aerosol , wherein the particles comprise at least one active ingredient , in particular 2-DG . The particles may preferably be provided in the form of compound particles comprising a matrix or carrier material and the active ingredient . The matrix or carrier material can preferably facilitate keeping , handling and dispensing of the active agent in a controlled way . The method preferably further comprises directing the directed flow or j et of the aerosol towards target areas of the human body ( or the body of an animal ) for dispensing the substance . Hence , the substance with the active agent can be purposefully applied to specific areas of the human body .

In an embodiment , the present application provides a device for inhaling a substance in the form of aerosol particles or powder particles . The device preferably comprises a container for receiving and keeping the substance . The device preferably further comprises an actuator for activating the device , the actuator is configured to release a certain amount or dose of the substance kept in the container for conveying through the discharge nozzle of the device , wherein the particles comprise at least one active ingredient , in particular 2 -DG .

Thus , the substance can preferably be dispensed in small doses , in order to keep the concentration of the active agent at a moderate level , in particular, to avoid an overdosage of the active ingredient and the side effects associated with the overdosage .

In an embodiment , the actuator comprises a manual actuator which can be activated or triggered manually . Thus , the user themself can activate the device whenever it is needed .

In an embodiment , the device comprises a dosage valve defining the amount of the released substance , each time when the activator is triggered, and the actuator may be configured to activate the dosage valve . By means of the dosage valve , a precise dosage of the substance , in particular , of the active agent can be achieved . The dosage valve may be an adj ustable dosage valve such that the dosage of the substance can be adj usted prior to dispensing the substance .

In some embodiments , upstream to the discharge nozzle , an air flow channel or chamber for conveying the substance released by the actuator to the discharge nozzle is arranged . The air flow channel may be , in particular, configured to support a turbulent air flow in the air flow channel . The turbulences in the air flow can facilitate entraining the particles released from the container and propel them towards the discharge noz zle of the device . Further , due to the turbulences in the air flow, the phase space occupied by the released particles can be increased such that a broad spatial distribution of the particle j et can be achieved . The broad spatial distribution of the particles may be particularly helpful to avoid local overdoses of the active ingredients at the exposed living tissues .

In some embodiments , the airflow channel is configured such that the air flow in the air flow channel can be created by the user by inhaling the air while keeping the nozzle of the device in a nostril or in the mouth . Such a device does not require any energy supply for providing the air flow .

The actuator can be configured to provide a pressurized air flow in the air flow channel . Release of pressurized air can, in particular, support turbulences which can help to entrain the substance particles located at an outlet of the container when the dosage valve is open .

In some embodiments , the device also comprises a chamber for propellant , and the actuator is configured to release the propellant such that the substance may be conveyed by droplets of propellant along the flow channel. As propellant, CFC (chlorofluorocarbon) and/or HFA (hydrofluoroalkane) may be used.

In some embodiments, the particles may comprise a carrier material or matrix material carrying the active ingredient. The carrier material may be, in particular, provided in the form of a matrix in which the active ingredient resides. The carrier material can preferably facilitate the handling and dispensing of the active agent.

The carrier material may comprise a liquid selected from the group comprising: water, alcohol, liquid glucose, or aqueous solution. The active ingredient or drug can be thus easily dispensed together with the liquid. The aqueous solution with 2% salt may serve as a preservation medium for the active agent until it is dispensed .

In some embodiments, the substance, in particular substance particles, comprise lactose and/or liposome. Lactose or liposome can serve as a carrier for the active ingredient, such that the particles can be easily delivered to the body of the subject.

In some embodiments, the active agent is encapsulated in liposome, preferably in order to achieve a long-lasting or retarded effect in the human body, thus increasing the duration of the desired effect. Particles may also comprise cholesterol, which stabilizes liposomes such that an even greater delay of the activation of the agent or active ingredient can be achieved.

The particles may comprise a mixture of different liposomes. In particular, a mixture of small liposomes, with an average size of less than 100 nm and large liposomes, with an average size of more than 150 nm. By providing different liposome sizes , a desired time profile of the active agent activity can be achieved .

Viral Infections and Diseases

The herein described preparations and forms of 2 -DG may be used in the following embodiments of the first aspect of the invention in relation to viral infections and diseases .

In the first aspect of the application, 2 - DG is provided for use in a medical method to prevent and/or treat a viral disease , in particular Covid-19 , influenza , or Dengue fever . In an embodiment of the application, 2- DG is provided for use in a medical method to prevent and/or to treat Covid-19 , wherein 2 -DG is provided as a preparation in an amount and a formulation that results in an effective tissue concentration that achieves partial or complete inhibition of glycosylation of a SARS- CoV-2 spike protein .

In an embodiment , the application provides 2 - 2-DG for use in a medical method to prevent and/or to treat a viral infection in a subj ect by a virus comprising a spike protein, wherein 2 -DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of the spike protein .

In an embodiment , the application provides a method of preventing and/or treating a viral infection in a subj ect by a virus comprising a spike protein, the method comprising administration of 2 -DG to tissue of the subj ect in an amount and a formulation that results in an effective tissue concentration to partially or completely inhibit glycosylation of the spike protein . In an embodiment, the application further provides a method of preventing and/or treating a viral infection in a subject by a virus comprising a spike protein, the method comprising administration of at least one of: 2-DG, ribavirin, emetine, and NMS-873 to tissue of a subject in an amount and a formulation that results in an effective tissue concentration to partially or completely inhibit glycosylation of the spike protein.

The effective tissue concentration preferably inhibits at least 30%, in particular at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the glycosylation of the spike protein.

The effective tissue concentration of 2-DG is preferably in a range of between approximately 0.1 mM to

25 mM.

In an embodiment, the tissue comprises respiratory tissue. More preferably, the respiratory tissue comprises epithelial cells .

In an embodiment, the virus is enveloped. More preferably, the virus is a Coronavirus. Even more preferably, the Coronavirus is SARS-CoV-2.

In an embodiment, the viral spike protein comprises a SARS-CoV-2 spike protein.

The viral infection is preferably in cells of the airways and respiratory tissue of a subject. More preferably, the viral infection has developed into the viral disease Covid-19. In an embodiment , the virus is enveloped . More preferably, the virus is a Flavi virus . Even more preferably, the Flavi virus is Dengue virus .

In a further embodiment , the viral envelope glycoprotein comprises a Dengue virus envelope glycoprotein E .

The viral infection is preferably in keratinocytes and monocytes of a subj ect . More preferably, the viral infection has developed into the viral disease Dengue fever .

In an embodiment , the virus is enveloped .

More preferably, the virus is a Orthomyxovirus ( e . g . Influenza A, B or C viruses ) . Even more preferably, the Orthomyxovirus is Influenza A virus . Even more preferably, the Influenza A virus is H1N1 virus .

In a further embodiment , the viral spike protein comprises two types of spike proteins of Influenza A virus . More preferably, these spike proteins are cylinder shaped haemagglutinin or mushroom-shaped neuraminidase .

The viral infection is preferably in epithelial cells of the respiratory tract of a subj ect . More preferably, the viral infection has developed into the viral disease Influenza ( commonly called "the flu" ) .

In an embodiment of the application, 2-DG is provided for use in a medical method to prevent and/or to treat a viral disease caused by an enveloped virus comprising a spike protein, wherein 2-DG is provided as a preparation in a liposomal or a proliposomal formulation .

In an embodiment , the application further provides a process for preparing a liposome-encapsulated pharmaceutical composition comprising 2-DG for use in a medical method to prevent and/or to treat a viral infection in a subj ect by a virus comprising a spike protein, wherein 2 -DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of the spike protein .

In a preferred embodiment of the application, 2-DG is provided for use in a medical method to prevent and/or to treat a viral disease comprising a coronavirus , in particular Covid-19 , or influenza, or Dengue fever , wherein 2 -DG is provided as a preparation for administration by inhalation, wherein in particular 2-DG is formulated as a micron or a submicron particle .

In an embodiment , a substance as described herein is preferably for use in the therapy of COVID-19 . The substance preferably comprises non-toxic concentration of the active ingredient for preventing SARS-CoV-2 replication in human lung tissue cells or in human nasal mucosa cells .

The application further provides a method of manufacturing a proliposome- or liposome-encapsulated pharmaceutical composition comprising 2-DG for use in a medical method to prevent and/or to treat a viral infection in a subj ect by a virus comprising a spike protein, wherein 2 -DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of the spike protein .

In an embodiment , the application provides a use of a pharmaceutical composition comprising at least one active ingredient selected from the group comprising : ribavirin, emetine , 2 -DG and NMS-873 , for the treatment of a viral infection in the airways of a subj ect . More preferably the active ingredient comprises 2 -DG and at least one selected from the group comprising ribavirin, emetine , and NMS-873 . In a preferred embodiment , the application provides a use of a pharmaceutical composition comprising 2 -DG for the treatment of a viral infection in the airways of a subj ect .

In an embodiment , the application provides a use of a pharmaceutical composition comprising at least one active ingredient selected from the group comprising : ribavirin, emetine , 2 -DG and NMS-873 , in the manufacture of a medicament for the prevention and/or treatment of a viral infection in the airways of a subj ect . More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising ribavirin, emetine , and NMS-873 . In a preferred embodiment , the application provides a use of a pharmaceutical composition comprising 2 -DG in the manufacture of a medicament for the prevention and/or treatment of a viral infection in the airways of a subj ect .

In an embodiment of the application, an anti- COVID-19 method for applying a substance as described herein to a human body, use of the substance , as well as a method and device for dispensing the substance is provided .

Some active ingredients can serve as translation inhibitors for preventing or suppressing viral replication and/or as agents for suppressing the growth and reproduction of the host cells attacked by viruses . Due to prevention of the viral replication and suppressing the growth and reproduction of the host cells , these active ingredients can serve not only as a medication against viral-infectious diseases but also as a prophylaxis for preventing a viral attack or contagion of the human body .

For example , ribavirin with the molecular formula C ₈ H ₁₂ N ₄ O ₅ is a nucleoside analogue and antiviral agent with an activity against hepatitis C virus . Emetine with the molecular formula C ₂ 9H4 ₀ N ₂ O4 can be isolated from the root of the plant Psychotria Ipecacuanha ( ipecac root ) and other plants with antiemetic and anthelminthic properties and inhibits protein synthesis in eukaryotic cells by irreversibly blocking ribosome movement along the mRNA (messenger Ribonucleic acid) strand and inhibits DNA ( Deoxyribonucleic acid ) replication in the early S phase ( Synthesis Phase ) of the cell cycle . 2 -DG ( 2-deoxy- glucose ) , with the molecular formula C _e H ₁₂ O ₅ , is a glucose molecule which has 2-hydroxyl group replaced by hydrogen . NMS-873 with the molecular formula C ₂ 7H ₂₈ N4O ₃ S ₂ can activate protein response and modulate autophagosome maturation . In some embodiments , the substance is delivered in such a way that the concentration of the active ingredient in the body or organism remains in a non-toxic concentration range .

In the delivery of a substance as described herein to the nose or the mouth of a person, at least a portion of the active ingredients can preferably arrive at inner regions of the mouth and the nose and also deeper in the respiratory tract , in particular the lungs , of the person . These active ingredients can preferably serve as translation inhibitors for preventing or subduing viral replications in human cells . Due to prevention of the viral replication, these active ingredients can also preferably serve as a prophylaxis for preventing pathological development if a person is exposed to a viral infection . In an embodiment , the present application provides a substance for applying to a human or animal body for the treatment of lung tissue cells by inhalation . In particular the substance is preferably provided in the form of aerosol particles or powder particles . The substance preferably comprises at least one active ingredient . More preferably, the active ingredient comprises 2-DG . These active ingredients preferably can serve as translation inhibitors for preventing or subduing viral replication in the human or animal cells and/or as agents for suppressing the growth and reproduction of the host cells attacked by viruses . Due to prevention of the viral replication, these active ingredients can preferably also serve as a prophylaxis for preventing diseases or pathological developments caused by viral infections . The substance can be in particular applied to animals from a group comprising horses , swine , bovine animals as well as hen and/or other poultry .

The substance may be provided for use in the therapy of COVID-19 ( Coronavirus Disease 2019 ) , a respiratory disease caused by SARS-CoV-2 ( severe acute respiratory syndrome coronavirus 2 ) . The substance may, in particular , comprise a non-toxic concentration of the active ingredient , in particular 2-DG for preventing SARS-CoV-2 replication in human lung tissue cells or in human nasal mucosa cells . The prevention of the replication of the SARS-CoV-2 in human lung tissue cells or in human nasal mucosa cells can help to avoid or mitigate the respiratory syndromes of the patients and, help not only in prophylaxis but also in the therapy of the COVID-19 .

The substance may also be provided for use in the therapy of Influenza, a respiratory disease caused by Influenza type A, B or C viruses , in particular H1N1 . The substance may, in particular , comprise a non-toxic concentration of the active ingredient , in particular 2-DG for preventing Influenza virus replication in human respiratory epithelial cells . The prevention of the replication of the Influenza virus in human cells can help to avoid or mitigate the respiratory syndromes of the patients and, help not only in prophylaxis but also in the therapy of the Influenza ( flu) .

The substance may also be provided for use in the therapy of Dengue fever, a disease caused by Dengue virus . The substance may, in particular, comprise a nontoxic concentration of the active ingredient , in particular 2-DG for preventing Dengue virus replication in human keratinocytes and monocytes . The prevention of the replication of the Dengue virus in human keratinocytes and monocytes can help to avoid or mitigate the syndromes of the patients and, help not only in prophylaxis but also in the therapy of the Dengue virus .

In particular , 2-DG molecules , due to the replacement of the 2-hydroxyl group by hydrogen, are characterized by high stability against metabolism. Due to the similarity with glucose molecules , 2 -DG molecules penetrate the cells , undergo phosphorylation and resulting 2-DG- 6-phosphate can remain for some time in the cell . However, in contrast to phosphorylated glucose , phosphorylated 2 -DG-6-phophate does not further participate in glycolysis . Thus , 2-DG-6-phophate can remain in the cell , in particular , in the host cells attacked by a SARS-CoV-2 virus without undergoing glycolysis and, therefore , without producing energy necessary for cellular activities including biogenesis and reproduction of host cells . In other words , 2-DG takes the place of glucose , keeps the host cell busy, but does not produce energy, thus suppressing the growth and reproduction of the cells attacked by the SARS-CoV-2 virus .

Cancer The herein described preparations and forms of 2 -DG may be used in the following embodiments of the second aspect of the invention in relation to cancer .

It is a general obj ect of the application in a second aspect to provide a preparation of 2-DG that is suitable for use in the treatment of cancer .

In a further aspect of the application, 2 -DG is provided for use in a medical method to treat cancer patient , wherein 2-DG is provided as a preparation in a liposomal or a proliposomal formulation .

In a preferred embodiment of the application, 2-DG is provided for use in a medical method to prevent and/or to treat cancer, wherein 2-DG is provided as a preparation for administration by inhalation, wherein in particular 2 -DG is formulated as a micron or a submicron particle .

In an embodiment , a substance as described herein is preferably for use in the therapy of cancer .

The application further provides a method of manufacturing a proliposome- or liposome-encapsulated pharmaceutical composition comprising 2-DG for use in treating cancer, wherein 2 -DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration for use in treating the cancer .

Cancer and lomustine

In an embodiment , the application provides a use of a pharmaceutical composition comprising at least one active ingredient selected from the group comprising : lomustine, 2-DG, NMS-873 and ascorbate, for the treatment of a cancer in a subject.

Also provided is a method of treating a cancer in a subject comprising administering at least one active ingredient selected from the group comprising: lomustine, emetine, 2-DG, NMS-873 and ascorbate.

More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising lomustine, emetine, and NMS-873.

Also provided is a method of treating a cancer in a subject comprising administering 2-DG and at least one active ingredient selected from the group comprising: lomustine, emetine, and NMS-873.

In a preferred embodiment, the application provides a use of a pharmaceutical composition comprising 2-DG for the treatment of cancer in a subject.

Also provided is a method of treating a cancer in a subject comprising administering a pharmaceutical composition comprising 2-DG.

In an embodiment, the application provides a use of a pharmaceutical composition comprising at least one active ingredient selected from the group comprising: lomustine, 2-DG and ascorbate, in the manufacture of a medicament for the prevention and/or treatment of cancer in a subject.

Also provided is a method of preventing or treating a cancer in a subject comprising administering at least one active ingredient selected from the group comprising: lomustine, 2-DG and ascorbate. More preferably the active ingredient comprises 2-DG and at least one selected from the group comprising lomustine and ascorbate .

Also provided is a method of preventing or treating a cancer in a subj ect comprising administering 2-DG and at least one active ingredient selected from the group comprising : lomustine and ascorbate .

In a preferred embodiment , the application provides a use of a pharmaceutical composition comprising 2-DG in the manufacture of a medicament for the prevention and/or treatment of cancer in a subj ect in combination with lomustine or/and ascorbate .

Also provided is a method of preventing or treating a cancer in a subj ect comprising administering a pharmaceutical composition comprising 2-DG .

Inventors studied results of combination index (CI ) achieved on glioma DK-MGhigh and normal BALB 3T3 cells , after adding 2 -DG as a first compound and then combining it with lomustine which gave a lower CI indicating a synergistic effect . Adding lomustine to the cell culture and then combined with 2 -DG gives higher CI values indicating an antagonistic effect . The isobologram analysis suggests there is an antagonism in the action of 2-DG and lomustine in BALB/c 3T3 cells . This supports a rationale of the use of 2-DG in combination with lomustine , but only when 2-DG is used as the first treatment regime compound .

Inventors further identified that co-encapsu- lation 2-DG and sodium ascorbate in 2 : 1 molar ratio decreases formulation IC50 from 0 . 125 micromolar concentration for only liposomal 2-DG to 68 micromolar concentration for 2-DG co-encapsulated with sodium ascorbate . In an embodiment of the application, an anticancer method for applying a substance (wherein, for the purposes of defining the invention, "a substance" may comprise more than one substance ) as described herein to a human body, use of the substance , as well as a method and device for dispensing the substance is provided .

In the delivery of a substance as described herein to the nose or the mouth of a person, at least a portion of the active ingredients can preferably arrive at inner regions of the mouth and the nose and also deeper in the respiratory tract , in particular the lungs , of the person . These active ingredients can preferably assist in the treatment of cancer .

In an embodiment , the present application provides a substance for applying to a human or animal body for the treatment of lung tissue cells by inhalation . In particular the substance is preferably provided in the form of aerosol particles or powder particles . The substance preferably comprises at least one active ingredient . More preferably, the active ingredient comprises 2-DG . These active ingredients preferably can preferably assist in the treatment of cancer . The substance can be in particular applied to animals from a group comprising horses , swine , bovine animals as well as hen and/or other poultry .

The substance may be provided for use in the therapy of cancer . The substance may, in particular , comprise a non-toxic concentration of the active ingredient , in particular 2-DG for use in treating cancer in human lung tissue cells or in human nasal mucosa cells .

In some embodiments , the dose-response relationship of 2-DG on blocking of glycosylation of proteins in cancer cells is observed in the concentration range in particular : for NCI-H1975 adenocarcinoma of the lung the IC50 range from 0 . 5 mM to 5 mM and the ED50 proteins glycosylation range from 0 . 25 mM to 1 mM . These data show that 2-DG is toxic for lung cancer cells and inhibits glycosylation of lung cancer cells proteins .

In an embodiment of the invention, 2-DG is toxic for glioblastoma cells and inhibits glycosylation of glioblastoma cells proteins .

In an embodiment of the invention, 2-DG is toxic for colorectal cancer cells and more particularly, inhibits glycosylation of colorectal cancer cells proteins .

In an embodiment of the invention, 2-DG is toxic for vulva cancer cells and more particularly, inhibits glycosylation of vulva cancer cells proteins .

In an embodiment of the invention, 2 -DG is toxic for non-small-cell lung cancer cells and more particularly, inhibits glycosylation of non-small-cell lung cancer cells proteins .

In an embodiment of the invention, 2-DG is toxic for glioblastoma cells and more particularly, inhibits glycosylation of glioblastoma cells proteins .

In an embodiment of the invention, 2-DG is toxic for breast cancer cells and more particularly, inhibits glycosylation of breast cancer cells proteins .

In an embodiment of the invention, 2-DG is toxic for lymphoblastic leukemia cells and more particularly, inhibits glycosylation of lymphoblastic leukemia cells proteins . In an embodiment of the invention, 2-DG is toxic for fibrosarcoma cells and more particularly, inhibits glycosylation of fibrosarcoma cells proteins .

In an embodiment of the invention, 2-DG is toxic for breast cancer cells and more particularly, inhibits glycosylation of breast cancer cells proteins .

In an embodiment of the invention, 2-DG is toxic for cervix cancer cells and more particularly, inhibits glycosylation of cervix cancer cells proteins .

In an embodiment of the invention, 2-DG is toxic for adenocarcinoma cells and more particularly, inhibits glycosylation of adenocarcinoma cells proteins .

In an embodiment of the application, 2-DG is provided as a preparation to a tumour of a subj ect , a human or an animal , that results in an effective concentration to partially or completely inhibit glycosylation of cancer cells glycoproteins of the tumour .

In an embodiment of the invention, 2-DG targets tumour cells growing under normoxic conditions . This is beneficial since many cancers often rely on glycolysis even under aerobic conditions , the so-called Warburg effect .

A slow-release formulation, obtained by encapsulating 2-DG in liposomes , or co-encapsulating 2-DG with ascorbate ( ascorbic acid or sodium ascorbate ) in liposomes , can maintain a constant or even an increase in the concentration of 2-DG in both tumours and virus-infected cells , and as shown herein, more effective than previous attempts to use 2 -DG in anticancer therapies and allows targeted therapy ( EPR effect ) . The primary mechanism responsible for the preferential accumulation of the liposomal formulation in the tumour is the enhanced permeability and retention ( EPR) effect , which results from the interaction of the biophysical effect of leaky tumour blood vessels and poor tumour lymphatic drainage , allowing liposomes to spontaneously extravasate from blood vessels into tumour tissues .

Alternatively, liposome targeting may comprise use of one or more naturally occurring molecules , for example , folic acid or transferrin, amongst others , which attach to the surface of liposomes and can be used for liposomes targeting . Many tumours overexpress receptors for folic acid and for transferrin which facilitate cancer cells targeting, but also allow efficient blood brain barrier ( BBB ) targeting as transferrin receptors are present on epithelial cells of the brain blood vessels , and transferrin coated particles can be transported through the BBB intact . This can substantially increase drug accumulation within brain tumour tissue .

Natural Preparation

It is a general obj ect of the application in a third aspect to provide a natural preparation of 2-DG with natural minerals for use as a health supplement .

In an embodiment , the application provides a preparation comprises 2 -DG and natural minerals , including at least NaCl ( sodium chloride ) and Selenium, and natural mineral water , the 2 -DG being dissolved in the natural mineral water . The concentrations of NaCl in the natural preparation are preferably between approximately 0.5% to 1.5% solution of NaCl, more preferably 0.009 mg/ml (0.9% solution) .

The concentrations of Selenium will preferably provide a subject taking the natural preparation with at least 30% of their daily selenium requirement following the prescribed daily dosage of natural preparation.

In an embodiment, the preparation preferably comprises other natural minerals including one or more of those selected from the group comprising: sulfur, calcium, potassium, oxygen, and other natural ingredients under 1 g/kg including those that may be present in the natural mineral water.

Concentrations of sulfur in the preparation are preferably between approximately 5 g/kg to 30 g/kg, and more preferably approximately 12 g/kg.

Concentrations of calcium in the preparation are preferably between approximately 2 g/kg to 6 g/kg, and more preferably approximately 4 g/kg.

Concentrations of potassium in the preparation are preferably between approximately 2 g/kg to 6 g/kg, and more preferably approximately 3.5 g/kg.

Concentrations of oxygen in the preparation are preferably between approximately 0.05 g/kg to 0.014 g/kg, and more preferably approximately 0.012 g/kg.

In some embodiments of the application, per 1 ml dose of natural preparation, the natural preparation may also comprise any one or more of the following minerals : - Amounts of bicarbonates in the natural preparation may be between approximately 0.2 mg to 0.5 mg, and more preferably approximately 0.35 mg;

- Amounts of calcium in the natural preparation may be between approximately 0.05 mg to 0.2 mg, and more preferably approximately 0.1 mg;

- Amounts of magnesium in the natural preparation may be between approximately 0.015 mg to 0.045 mg, and more preferably approximately 0.03 mg;

- Amounts of sulphates in the natural preparation may be between approximately 0.005 mg to 0.02 mg, and more preferably approximately 0.01 mg;

- Amounts of chlorides in the natural preparation may be between approximately 0.005 mg to 0.02 mg, and more preferably approximately 0.01 mg;

- Amounts of silica in the natural preparation may be between approximately 0.01 mg to 0.02 mg, and more preferably approximately 0.015 mg;

- Amounts of potassium in the natural preparation may be between approximately 0.005 mg to 0.02 mg, and more preferably approximately 0.01 mg; and

- other ingredients under 0.01 mg include sodium, nitrates, fluorides, and selenium.

In an embodiment of the application, 2-DG is at a concentration of between approximately 0.05 mg and 0.5 mg, and more preferably approximately 0.15 mg or as much as 10 mg per 1 ml dosage of natural preparation.

Some parts of the embodiments have similar parts. The similar parts may have same names or similar part numbers. The description of one part applies by reference to another similar part, where appropriate, thereby reducing repetition of text without limiting the disclosure . Brief Description of the Figures

The application will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes reference to the annexed drawings, wherein:

Fig. 1 Structural comparison of glucose and 2-deoxy-D- glucose (2-DG) .

Fig. 2 Analysis of Spike protein glycosylation in human bronchial epithelial cells infected with SARS- CoV-2 in the presence and absence of 2-DG.

Fig. 3 Analysis of Spike protein glycosylation in normal renal epithelial cells infected with SARS- CoV-2 in the presence of 2-DG with dose-response relationship.

Fig. 4 Determination of IC50 values for 2-DG in human bronchial epithelial cells (short exposure) .

Fig. 5 Determination of IC50 values for 2-DG in human bronchial epithelial cells (long exposure) .

Fig. 6 Evaluation of antiviral activity of 2-DG on blocking the infection and replication of SARS- CoV-2 in primary bronchial epithelial cells .

Fig. 7 Evaluation of antiviral activity of 2-DG on blocking the infection and replication of SARS- CoV-2 in Vero E6 cell line.

Fig. 8 Evaluation of antiviral activity of 2-DG on blocking the multiplication of SARS-CoV-2 virus 8 hours after infection (post-treatment) in Vero E6 cell line.

Fig. 9 Analysis of Spike protein glycosylation in lysates obtained directly from normal renal epithelial cells infected with SARS-CoV-2 virus, in the presence and absence of 2-DG and with doseresponse relationship.

Fig. 10 Uptake measurement of 2-DG in lung tissue lysates of mice treated with inhalation with 2-DG.

Fig. 11 Analysis of ACE2 protein expression in lysates obtained directly from normal renal epithelial cells, in the presence and absence of 2-DG and with dose-response relationship. "Ctrl" means that cells were not exposed to 2-DG.

Fig. 12 Evaluation of antiviral activity of 2-DG on blocking the multiplication of H1N1 (type of Influenza A virus) after infection in MDCK cell line .

Fig. 13 Schematic drawing of a compound particle according to an exemplary embodiment. Diagram of a particle formed by spray drying a solution containing 2-DG. The black points are 2-DG contained in the particle structure.

Fig. 14 Size distribution chart of the particles containing 20% by weight of the 2-DG produced in example 10.

Fig. 15 Circularity chart of the particles containing 20% by weight of the 2-DG produced in example 10. Fig . 16 Schematic drawing of a compound particle according to another exemplary embodiment . Diagram of a unilamellar liposome containing 300 mM of 2 -DG solution within water space prepared in the examples 20-24 .

Fig . 17 Schematic view of a device for inhaling a substance according to an exemplary embodiment .

Fig . 18 Flow chart of a method of dispensing a substance according to an embodiment .

Fig . 19 Determination of ED50 value of 2-DG measured as inhibition of proper glycosylation of viral hemagglutinin HA protein .

Fig . 20 Determination of IC50 values for 2 -DG in human neoplastic glioblastoma DK-MG cells .

Fig . 21 Plot of percentage of viable cells depending on the concentration of 2 -DG and lomustine combined with constant concentration of lomustine and 2- DG, respectively, used in DKMG cells .

Fig . 22 Determination of ED50 value in glioblastoma and lung cancer cell lines by means of inhibition of protein glycosylation .

Fig . 23 Release of an encapsulated 2 -DG in HSPC/DSPE-PEG 2000 95 : 5 m/m liposomes in 0 , 9% NaCl in H ₂ O, measured in units of hours and days .

Detailed Description of the Application The following provides general remarks regarding the embodiments , examples , and aspects of the application .

Further description is provided as follows with respect to the first aspect of the invention to provide a preparation of 2 -DG that is suitable for treatment of a viral disease caused by an enveloped viruses , in particular by SARS-CoV-2 .

In this text , the term viral disease caused by an enveloped virus comprising a spike protein refers to a viral disease which is caused by infection of a host with an enveloped virus comprising a spike protein as defined above . Spike proteins are visible structures in electron microscopes on the surface of such enveloped viruses . Exemplary enveloped viruses comprising a spike protein include Arenaviruses , Bunyaviruses , Coronaviruses ( e . g . Sars-Cov-2 virus ) , Filoviruses Hepadnaviruses , Herpesviruses , Orthomyxoviruses ( e . g . Influenza A, B or C viruses ) , Paramyxoviruses , Poxviruses , Retroviruses and Togaviruses . As an example , attachment of influenza A viruses by their spikes to sialic acid on the cell surface is a critical step in the initiation of infection . The receptor-binding site of the viral hemagglutinin glycoprotein binds to sialic acids expressed by cell surface glycoproteins and/or glycolipids to mediate virus entry .

Exemplary enveloped viruses comprising other than spike glycosylated enveloped proteins include Fla- vi viruses ( e . g . Dengue virus ) . More preferably, the viral glycoproteins include glycoprotein E which is responsible for host cell entry by Dengue virus .

In this text , the term glycosylation refers to the attachment of sugar moieties to proteins . It is a post-translational modification . Glycosylation is critical for a wide range of biological processes , including cell attachment to the extra-cellular matrix and protein-ligand interactions in the cell . This post-trans- lational modification is characterized by various glyco- sidic linkages , including N- , O- and C-linked glycosylation, glypiation (GPI anchor attachment ) , sialylation and phosphoglycosylation . Sialic acids , terminal , negatively charged monosaccharides , are commonly a part of glycoproteins , glycolipids or gangliosides , where they decorate the end of sugar chains at the surface of cells or soluble proteins . As an example , Orthomyxoviruses can use si- alylated structures for binding to their target host cell , thus sialic acids plays an important role in several human viral infections .

In this text , the term viral envelope refers to the outer structure that encloses the nucleocapsids of some viruses .

In this text , the term spike protein refers to a glycoprotein that protrudes from the envelope of some viruses ( such as e . g . a Coronavirus , Orthomyxoviruses , Herpesviruses ) and facilitates entry of the virion into a host cell by binding to a receptor on the surface of a host cell followed by fusion of the viral and host cell membranes .

In this text , the term "other enveloped glycosylated proteins" refers to a glycoprotein E that protrudes from the envelope of some viruses ( such as e . g . Flaviviruses ) and facilitates entry of the virion into a host cell by binding to a receptor on the surface of a host cell , in particular a DC-SIGN, a C-type lectin receptor present on the surface of monocyte-derived cells , followed by fusion of the viral and host cell membranes . In this text , the term effective tissue concentration of 2-DG relates to the amount of 2-DG that is available in an infected or unaffected tissue that is targeted for treatment by 2-DG such as e . g . a tissue of the lower respiratory tract , the heart or the liver , monocytes or keratinocytes .

In the context of cancer cells , the term "effective tissue concentration" is used, but in other contexts , such as in "effective tissue concentration to partially or completely inhibit glycosylation of receptors or other glycoproteins on human or animal cells" , it is allowable to use the same term "effective tissue concentration" in two ways . One definition is for the treatment of cancer , while the other definition is for treatment of viruses .

The application relates to 2 -deoxyglucose ( otherwise known as 2 -deoxy-D-glucose or 2 -Deoxy-D-xyl- hexose ; hereinafter referred to as 2-DG) with the molecular formula C6H12O5 for use in the treatment and prevention of viral infections , particularly viral infections caused by SARS-CoV-2 or other viruses particularly Influenza or Dengue viruses , whose proteins require glycosylation to fold properly . 2-DG is a synthetic glucose analog where the C2 hydroxyl group is replaced with hydrogen ( Figure 1 ) . 2-DG was provided by Sigma-Aldrich; catalog number D8375 ) .

In the case of the SARS-CoV-2 , the application found that that blocking or even hindering spike glycosylation is an effective way to inhibit infection because glycosylation of the spike protein is a requirement for infection .

The glycosylation dependent interaction between spike and ACE2 receptor has been described . In the case of ACE2 , the application found that 2 -DG also decreases the expression of ACE2 on the surface of epithelial cells . Thus , it suggested the dual role of 2 -DG by blocking as well as preventing from infection . Moreover, the SARS-CoV-2 virus multiplication in cells causes the rapid increase in energy demand of infected cells .

Blocking the glycosylation of the spike protein or other glycoproteins of the virus that mediate in human cell entry may have a positive effect also in the second stage of the disease , when viral remnants would not be able to play such a negative role due to the inactivation of a protein such as spike . For example , properly glycosylated and folded spike protein can penetrate into various cells and tissues , thereby leading to unfavorable effects . Blocking spike glycosylation therefore also plays a positive role in the second stage of the disease .

The application is based on the use of 2-DG in the treatment of viruses , and particularly Covid-19 and the prevention of SARS-CoV-2 virus infection of eukaryotic cells . The mechanism of action of 2 -DG in this case is mainly based on the influence on the metabolism of infected cells , and in particular on blocking glycolysis and glycosylation of the SARS-CoV-2 virus spike protein . Spike glycosylation plays a fundamental role in infecting cells because the interaction between spike and its maj or ACE-2 receptor depends on glycosylation .

It is shown herein that 2 -DG by preventing the spread of SARS-CoV-2 infection - by preventing virus multiplication in eukaryotic cells - has both a preventive effect in blocking the infection as observed when first exposing cells to 2-DG and subsequently to SARS- CoV-2 , as well as a therapeutic effect as observed when first exposing cells to SARS-CoV-2 and subsequently to 2 - DG or when exposing cells simultaneously to both . It is also shown herein that 2-DG completely inhibits the standard/regular glycosylation of the spike protein that is observed in the absence of 2-DG . Blocking these processes are effective at concentrations that are much lower than concentrations which cause toxic effects in normal cells .

The application is also based on the use of 2-DG in the treatment of other viral diseases , in particular Influenza caused by Influenza A, B or C viruses , in particular H1N1 , or in the treatment of Dengue fever caused by Dengue virus , and the prevention of Influenza A, B or C or Dengue virus infection of eukaryotic cells . The mechanism of action of 2 -DG in these cases is mainly based on blocking glycosylation of the Influenza hemagglutinin ( HA) spike proteins as well as blocking glycosylation of glycoprotein E , an enveloped protein from Dengue virus . Spikes or other viral enveloped proteins glycosylation plays a fundamental role in infecting cells because the interaction between these proteins and their maj or receptors ( e . g . DC-SIGN for Dengue virus or sialic acid on the cell membrane glycans of the upper respiratory tract for Influenza virus ) depends on glycosylation ( in case of Influenza infection also sialylation as a particular type of glycosylation on the cell membrane of the upper respiratory tract ) .

To evaluate the toxicity of 2-DG to normal cells as well as to assess the inhibition of viral multiplication the human bronchial epithelial cells ( HBEpiC ) line and Vero6 cell line were obtained . 2-DG was supplied by Sigma-Aldrich ( catalog number D8375 ) . To evaluate the inhibition of Influenza viral multiplication, the most widely used cell line for the isolation and propagation of human influenza viruses ( expressing high levels of human influenza virus receptors ) - MDCK cell line was used . In this aspect , the human bronchial epithelial cells ( hereinafter referred to as HBEpiC ) and primary human bronchial epithelial cells ( hereinafter referred to as PBEC ) are the same cells but provided from different vendors ( from Innoprot ( Cat no . P10557 ) and Promocell (C- 12640 ) , respectively) . Thus , the phrases HBEpiC and PBEC are used interchangeably within text .

Thus , 2 -DG exhibits many advantages making it a promising pharmaceutically active ingredient for use in a medical method to prevent and/or to treat a viral disease , in particular Covid-19 , including advantages such as :

- 2 -DG is non-toxic or minimally toxic or harmless against normal (uninfected ) cells at concentrations which generally exhibit a therapeutic effect on infected cells ;

- 2-DG exhibits only a slight toxicity compared to other drugs used against SARS-CoV-2 ;

- 2 -DG reduces SARS-CoV-2 virus multiplication;

- 2 -DG reduces multiplication of SARS-CoV-2 virus in cells infected with this virus before exposure to 2 -DG;

- 2 -DG reduces or blocks glycosylation of viral proteins , especially spike proteins ;

- 2-DG reduces or blocks glycosylation in eukaryotic cells infected by Influenza, in particular blocking glycosylation of ACE2 proteins ;

- 2 -DG reduces or blocks glycolysis in eukaryotic cells infected by SARS-CoV-2 .

2-DG exhibits many advantages making it a promising pharmaceutically active ingredient for use in a medical method to prevent and/or to treat a viral disease , in particular Influenza , including advantages such as :

- 2 -DG is non-toxic or minimally toxic or harmless against normal (uninfected ) cells at concentrations which generally exhibit a therapeutic effect on infected cells ;

- 2 -DG reduces Influenza virus multiplication;

- 2 -DG reduces multiplication of Influenza virus in cells infected with this virus before exposure to 2 -DG;

- 2 -DG reduces or blocks glycosylation of viral proteins , especially Influenza spike proteins including hemagglutinin;

- 2 -DG reduces or blocks glycosylation in eukaryotic cells infected by Influenza, in particular blocking sialylation of membrane glycans .

2 -DG reduces or blocks glycolysis in eukaryotic cells infected by Influenza .

2-DG exhibits many advantages making it a promising pharmaceutically active ingredient for use in a medical method to prevent and/or to treat a viral disease , in particular Dengue fever , including advantages such as :

- 2 -DG is non-toxic or minimally toxic or harmless against normal (uninfected ) cells at concentrations which generally exhibit a therapeutic effect on infected cells ;

- 2 -DG reduces Dengue virus multiplication;

- 2 -DG reduces multiplication of Dengue virus in cells infected with this virus before exposure to 2- DG;

- 2 -DG reduces or blocks glycosylation of viral proteins , especially Dengue enveloped glycoprotein;

- 2 -DG reduces or blocks viral entry by blocking interactions between DC-SIGN - the best characterized molecule among the candidate protein receptors for Dengue viral entry and glycosylated E proteins of the virus .

Liposomes are among the best drug delivery systems . They are most often non-toxic, biocompatible and can be made up of the same components as the cells of the human body . Liposomes can be used intravenously, intraperitoneally, orally and pulmonally .

There are some limitations to the use of liposomes , however . In the case of long storage of liposome preparations , leakage of the encapsulated substances , crystallization or their gradual hydrolysis in water is observed . To avoid these problems , it is possible to use so-called proliposomes . These can be both lyophilized liposomes as well as lyophilized lipids ( or organic solutions of lipids with API ) which form liposomes in an aqueous solution due to the natural features of phospholipids related to their structure .

A further aspect of the application relates to a proliposome preparation containing the active substance 2-deoxyglucose ( 2-DG) .

As described above and below, 2-deoxyglucose ( otherwise known as 2 -deoxy-D-glucose or 2 -Deoxy-D-xyl- hexose ; herein referred to as 2-DG) , with the molecular formula C6H12O5 , is a synthetic glucose analog where the C2 hydroxyl group is replaced with hydrogen . 2-DG was provided by Sigma-Aldrich; catalog number D8375 ) .

2-DG is a simple reducing sugar which is very difficult to maintain in the form of very small particles because of their relatively high hygroscopicity and low glass transition temperature resulting of stacking together sticky particles . One of the solutions to the problem of both 2-DG spray drying of glucose and obtaining delayed release of this substance in the lungs is the possibility of using proliposomes .

The preparation of proliposomes

In the case of 2-DG solution spray drying mixed with solution of other excipients and phospholipids such as naturally occurring dipalmitoyl phosphatidylcholine ( DPPC ) in the lungs , it is possible to obtain particles wherein 2 -DG and excipients are mixed with phospholipids to form particles of small size .

The hydration of such particles causes the organization of phospholipids into the lipid bilayer and the partial entrapment of the substances that formed the particle in the water space of the liposomes . Leucine is an excipient that significantly improves the aerological properties of the particles obtained by spray drying . In the case of drying from an organic-water solution, its diffusion to the surface of the particles is observed . This allows for the surrounding of a more hydrophilic 2- DG inside the particles and for obtaining another phospholipid layer above or below the leucine layer , depending on the drying method and the solvents used .

After hydration, the obtained particles form liposomes which partially encapsulate 2-DG ( Figure 16 ) . 2-DG encapsulation efficiency depends on the shell composition and phospholipid content and mixture of solvents used . In some embodiments , the active agent is encapsulated in proliposomes in which the amount of active ingredient ranges from 1 to 80% . Each particle formed as a result of spray drying gives one liposome , the size of which depends on the amount of phospholipid and the fluidity of the lipid bilayer of the liposomes . Stiffer membranes produce smaller liposomes and more fluid membranes produce larger liposomes . This effect is due to difference in osmotic pressure after the substance is encapsulated which then causes the causes the liposomes to swell . Thereby, liposomes with more fluid membranes increase in size , whereas liposomes with stiff membranes break apart .

In general to produce proliposomes by spraydrying process excipients such as mannitol , trehalose , amino acids ( glycine , leucine ) and phospholipids forming the lipid bilayer ) can be used . Preferably, the formulation of proliposomes contains mannitol in the amount of 0 to 60 % by weight of the preparation, glycine or leucine amino acid in the amount of 0 to 80 % by weight of the preparation and trehalose in the amount of 0 to 60 % by weight of the preparation . Additionally, cholesterol and negatively or positively charged phospholipids can be used, using them in small amounts as liposome bilayer stabilizers . Examples of negatively charged phospholipid species include natural phosphatidylglycerols , dimir- istoyl phosphatidylglycerol , dipalmitoylphosphatidylglycerol , and other phospholipids . In some embodiments , the nicotinic acid amide was used in proliposome formulation to facilitate phospholipids dissolving . Molten nicotinic acid amide dissolves phospholipids and many poorly soluble substances .

The ratio of phospholipids to active substance and excipients determines the amount of substances in the internal water space of liposomes . More often the more phospholipids , the higher the entrapment efficiency of the active ingredient and the proliposome-forming excipient . But this parameter is also related to the nature of the excipients and other factors . Preferably a suitable ratio between the phospholipids and the excipient and active substance should be from 1 to 10 to 1 to 1 . The parameter determining the rate of release of the active substance from liposomes is the phase transition temperature of the membrane of the liposomes used in the formulation . In the case of liposomes composed of DPPC alone , the release rate of the active substance can be very slow . By changing the composition of the lipid bilayer by using increasing amounts of DMPC , it is possible to obtain a bilayer with ever greater permeability to active substances , and thus it is possible to control the leakage of this substance over a wide time range . DPPC is a lipid whose phase transition temperature is around 41 . 5 ° C . Below temperature , this lipid does not form a lipid bilayer, therefore an addition ( 5 to 30 molar percent ) of another phospholipid ( DMPC ) with lower phase transition temperature is important . Preferably a suitable ratio between DPPC and DMPC should be from 10 to 1 to 10 to 2 . The use of a formulation containing DPPC and DMPC in such a weight ratio that causes a phase transition at the temperature of the human body causes a rapid release of the entire content of liposomes within several minutes . By increasing the proportion of DPPC to DMPC by a few percent by weight can extend the release time of the substance to several hours . It is therefore possible to consciously control the release time of the encapsulated 2 -DG by varying the ratio of

DMPC to DPPC and by changing the lamellarity of liposomes .

Another approach to proliposomes production is the use of API and lipids (phospholipids or phospholipid and sterols ) solution dissolved in the water/or- ganic cosolvents system being able to dissolve both API and lipids . This solution when nebulized forms liposomes upon contact with water . The API is the partially encapsulated and the encapsulation efficiency depends on API concentration, API lipids ratio and its concentration within water/organic cosolvent system . The pharmaceutically approved solvents , miscible with water which dissolves lipids are propylene glycol , glycerol and ethyl alcohol and can be used alone or in desired combination . The addition of water does not produce lipids precipitation until certain water concentration is achieved . In many cases the water soluble substance can be dissolved in such a solution to form API lipid solution . This solution if mixed with water forms multilamellar , oligolamel- lar or unilamellar liposomes , depending on the lipid concentration, organic excipients type or temperature . The solution can be nebulized by commercially available systems to form very fine solution droplets containing 2 -DG phospholipids or phospholipids and sterols which form different types of liposomes with varying 2-DG encapsulation efficiency .

Alternatively, 2-DG proliposomes can be prepared by dehydration-rehydration method followed by extrusion method in order to achieve large unilamellar liposomes . Non encapsulated 2 -DG in liposomes can remain in suspension and does not need to be separated . The encapsulation efficiency varies from 25-70% depending on the method used, liposomes composition and size . The resulting suspension is then mixed with water/ethanol solution of excipients such like leucine and trehalose and then spray-dried . Preferably, formulation of proliposomes contains trehalose in the amount of 0 to 60% by weight of the preparation . Resulting proliposomes particles have the size of 1-3 pm depending on the final mixture composition, concentration and spray-drying conditions . The non-encapsulated 2-DG exerts its local activity immediately while the encapsulated is slowly released and constitutes a depot that gradually replenishes the metabolism of free 2 -DG . Liposomal 2-DG for intravenous application

2-DG can be also used for intravenous administration . In order to extend the circulation time a proper type of liposomes must be used . Liposomes of a size close to 100 nm with 2-DG may be encapsulated within the internal space of liposomes by one of the available passive methods of drug encapsulation . In some embodiments , the liposomes used for intravenous delivery have sizes from 30 nm to 200 nm . 2-DG can be co-encapsulated with sodium ascorbate or ascorbic acid in order to achieve a synergistic effect . The weight combination between 2 -DG and ascorbate can be modulated and adj usted to the cancer type . Both 2DG and ascorbate increases free radical levels in the cancer cells and act synergistically . 2-DG can also be also used for pulmonary application . In some embodiments , the liposomes used to pulmonary delivery have sizes ranging from 50 nm to 2 pm .

The phospholipids which can be used include : hydrogenated soya or egg PC, DPPC, DSPC, negatively charged lipid : DPPG and DSPG . Also , natural egg sphingomyelin can be used as a base for liposomal composition . Positively charged lipids can be DOTAP, DOTMA, DDAB , DC- chol and others .

The liposomes may contain cholesterol . In most commercially available formulations , a preferable range of 30 to 50 molar percent in the relation to other lipids is be applied . In case of 2-DG liposomes cholesterol free liposomes are preferable or low liposomal composition can be further enriched in polymer modified lipids in order to extend their circulation time . As a polymer polyethylene glycol , polyvinyl alcohol or polyglycerol may be used . For targeting purpose , the pegylated lipids can be modified by attaching targeting molecules at the end of PEG chains . The molecules used as targeting moieties can be for examples : folic acid, transferrin, and others . The targeting molecules can be attached to PEG molecules using several commonly used reactions . Once these molecules are on the surface of liposomes and preferably are attached to the PEG molecules that are longer than used for liposomes PEGylation . For example , for liposomes PEGylation the PEG 2000 is commonly used whereas for targeting moieties PEG 3200 , 3400 or PEG 5000 is used . The 2-DG will be encapsulated by one of existing method such like dehydration-rehydration method, polyol dilution method or the passive equilibration method which utilize the 30% ethanol in liposomes suspension . More preferably, the 2-DG will be encapsulated by one of existing method such like dehydration-rehydration method, polyol dilution method or the passive equilibration method which utilize the 30% ethanol in liposomes suspension . That is , the efficiency of encapsulating 2-DG in liposomes can be higher than 30% and can preferably be up to 70% . One method to achieve this is to use tert-butanol and ethanol ( 2 : 1 ) co-solvent system to dissolve lipids and then adding water solution of the 2-DG, to produce 70% 2-DG encapsulation . The size of the liposomes may be controlled by liposomes extrusion, high pressure homogenization or a similar technique . Non encapsulated 2 -DG may be removed by size exclusion chromatography or dialysis .

Figure 1 . Structural comparison of glucose and 2-deoxy-D-glucose .

Figure 2 . Analysis of Spike protein glycosylation in human bronchial epithelial cells ( HBEpiC ) showing that 2-DG inhibits glycosylation of spike protein from SARS-CoV-2 virus . "Ctrl" means that cells were not exposed to 2 -DG . Each monomer of Spike protein is estimated to be 180 kDa in size . Figure 3. Analysis of Spike protein glycosylation in normal renal epithelial cells (Vero-E6) showing that 2-DG inhibits glycosylation of spike protein from SARS-CoV-2 virus . "Ctrl" means that cells were not exposed to 2-DG. The dose-response relationship for concentrations from 0.7 mM to 15 mM is clearly visible. Each monomer of Spike protein is estimated to be 180 kDa in size .

Figure 4. Determination of IC50 values for 2- deoxy-D-glucose in human bronchial epithelial cells (short exposure) revealed lack of cytotoxic effects on epithelial cells after 15 minutes to 6 hours exposure to 2-DG. An IC50 value cannot be determined due to lack of toxicity.

Figure 5. Determination of IC50 values for 2- deoxy-D-glucose in human bronchial epithelial cells (long exposure) revealed very high IC50 value after exposing human epithelial cells to 2-DG.

Figure 6. Evaluation of antiviral activity of 2-DG on blocking the infection and replication of SARS- CoV-2 in primary bronchial epithelial cells (PBEC) . 2-DG induces a dose dependent decrease in SARS-CoV-2 virus production on primary human bronchial epithelial cells, with effect equivalent to the positive control remdesivir above 0.78 mM. Effect of increase concentrations of 2-de- oxy-D-glucose on SARS-CoV-2 replication in primary human bronchial epithelial cells (HBEpiC) . Limit of detection (LOD) and viral titers without compound (T-) or with remdesivir (T+, 6 pM - 3 times the EC90) are indicated by dotted lines. Tested compound was used at 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 mM. Viral titers were determined by TCID50 method on Vero-E6 cells and calculated by the Spearman and Karber algorithm. Figure 7. Evaluation of antiviral activity of 2-DG on blocking the infection and replication of SARS- CoV-2 in Vero E6 cell line. 2-DG induces a strong decrease in the production of infectious SARS-CoV-2 particles. Effect is impressive on TCID50 calculation but less pronounced on qPCR experiments . Effect of increase concentrations of 2-deoxy-D-glucose on SARS-CoV-2 replication in Vero E6 cells. Limit of detection (LOD) and viral titers without compound (T-) or with remdesivir (T+, 6 pM - 3 times the EC50) are indicated by dotted lines. Tested compound was used at 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 mM. A) Viral titers were determined by TCID50 method on Vero-E6 cells and calculated by the Spearman and Karber algorithm. B) Copy numbers of gene E of SARS- CoV-2 were determined by TaqMan One Step RT-qPCR with E Sarbeco primers and probe (Charite, Corman et al Eurosurveillance; PMID: 31992387 ) and following instructions of the Qiagen QuantiNova Probe RT-PCR Kit. IC50 and IC90, calculated from nonlinear regressions, are indicated below .

Figure 8. Effect of increase concentrations of 2-Deoxy-D-glucose on SARS-COV-2 replication in Vero E6 cells. Viral titers without compound ("[0 pM]") or with 6 pM of remdesivir ("RMD") are indicated by dotted lines. Tested compound was used at 0.19, 0.56, 1.67, 5, 15, and 45 mM. 2-DG was added at 0 h.p.i for the "treatment" group and at 8 h.p.i for the "post-treatment" group. Viral titers were determined by the TCID50 method on Vero- E6 cells and calculated by the Spearman & Karber algorithm. Non-linear curve fitting is indicated by full lines for A) "treatment" (blue) and "post-treatment" (red) groups, B) "treatment" group only and C) "posttreatment" group only.

Figure 9. Analysis of Spike protein glycosylation in Vero-E6 cells showing that 2-DG inhibits glycosylation of Spike protein of SARS-CoV-2 virus isolated directly from cells infected with this virus. Line "33" and line "57" represent lysates from cells that were not exposed to 2-DG.

Figure 10. Uptake measurement of 2-DG in lung tissue lysates of mice treated with inhalation with 2-DG showing the that 2-DG was accumulated in the lungs of mice in this animal model study. Temporal changes of OD for all the samples included in the analysis are visible.

Figure 11. Analysis of ACE2 protein expression in lysates obtained directly from normal renal epithelial cells, in the presence and absence of 2-DG and with dose-response relationship. "Ctrl" means that cells were not exposed to 2-DG. The dose-response relationship for concentrations from 0.7 mM to 15 mM is visible.

Figure 12. Evaluation of antiviral activity of 2-DG on blocking the infection and replication of H1N1 Influenza A type virus in MDCK cell line. 2-DG induces a strong decrease in the production of infectious H1N1 viral particles. Effect is impressive on TCID50 calculation Effect of increase concentrations of 2-deoxy-D-glucose on H1N1 replication in MDCK cells . Tested compound was used at 0.02, 0.06, 0.19 0.56, 1.67,5,15 and 45 mM. IC50 and IC90, calculated from nonlinear regressions, are indicated below.

Examples

Example 1. Analysis of Spike protein glycosylation in human bronchial epithelial cells

Human Bronchial Epithelial Cells (HBEpiC) were obtained from Innoprot (Cat no. P10557, batch no. 7475) and cultured in Bronchial Epithelial Cell Medium (Innoprot, Cat no. P60151) supplemented with 0.2% gentamicin (Gibco, Life Technologies, USA, Cat. no. 15710- 049) under standard cell culture conditions (5% CO ₂ , 16% O ₂ , 37°C) . HBEpiC at passage 2, were plated in Bronchial Epithelial Cell Medium at 297.0 thousand cells per well of a 6-well plate and left for 48 hours in an incubator. The plates were coated with collagen (Collagen I-cell surface coating kit, Innoprot, Cat no. P8188) prior to cell seeding. The cells were transduced with lentiviral vectors encoding the SARS-CoV-2 Spike Protein or Spike SI domain. After 24h post transduction, 25 mM or 15 mM of 2- deoxy-D-glucose (Sigma-Aldrich; Cat no. D8375) was added to the cells for next 24h. The cells were lysed for 30 min at 4°C in cell lysis buffer (50 m Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM Sodium Orthovanadate , 10 pM p- glycerophosphate , 5 pM Sodium Pyrophosphate and 0.5% Triton X-100) freshly supplemented with Proteases and Phosphatases Inhibitor Cocktail. Lysates were clarified at 7,000 x g for 6 min at 4°C. The samples were mixed with Laemmli with p-mercaptoethanol , heated at 98 °C for 3 minutes and 25 pg (as determined by BCA assay (Thermo Scientific, Cat. no. 23225) ) of the proteins were applied to the gel for Western Blot analysis. Bands were visualized using Opti-4CN Substrate Kit (Bio-Rad, Cat. no. 1708235) .

Antibodies that were used in this study included :

- anti-SARS-CoV-2 Spike Glycoprotein SI antibody (Abeam, Cat. no. ab275759) , dilution 1:500;

- anti-Actin Antibody, clone C4 (Sigma-Aldrich, Cat. no. MAB1501) , dilution 1:4000;

- anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-2357) , dilution 1:4000;

- anti-mouse IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-516102) , dilution 1:4000. Results and conclusions : The experiment shows that 2-DG inhibits glycosylation of spike protein from SARS-CoV-2 virus (Figure 2) . The effect of dose and timing of dose (the dose-response relationship as well as the time-response relationship) of 2-DG on blocking of spike glycosylation is observed in the concentration range of 10 pM (0.01 mM) to 10 mM. In Figure 2 at a concentration of 15 mM total inhibition was observed.

Example 2. Analysis of the glycosylation of Spike protein in normal renal epithelial cells

Renal epithelial cells (VERO, clone E6) were obtained from ATCC (Cat no. CRL-1586, batch no. 70034994) and cultured in Eagle's Minimum Essential Medium (ATCC, Cat no. 30-2003) supplemented with 0.2% gentamicin (Gibco, Life Technologies, USA, Cat. no. 15710-049) , 1% Penicillin with Streptomycin (Biowest, Cat. no. L0022- 100) and 10% fetal bovine serum (Biowest, Cat. no. S181H- 500) under standard cell culture conditions (5% CO2 , 16% 02, 37°C) . Vero E6 cells at passage 4, were plated in EMEM at 297.0 thousand cells per well of a 6-well plate and left for 24 hours in an incubator. The cells were transduced with lentiviral vectors encoding the SARS-CoV- 2 Spike Protein. After 24h post transduction, 0.7 mM, 1.5 mM, 5 mM and 15 mM of 2-deoxy-D-glucose (Sigma-Aldrich; Cat no. D8375) was added to the cells for next 24h. The cells after incubation with 2-deoxy-D-glucose (or without this compound as in control group) were lysed for 30 min at 4°C in cell lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM Sodium Orthovanadate, 10 pM p-glyc- erophosphate , 5 pM Sodium Pyrophosphate and 0.5% Triton X-100) freshly supplemented with Proteases and Phosphatases Inhibitor Cocktail. Lysates were clarified at 7,000 x g for 6 min at 4°C. The samples were mixed with Laemmli with p-mercaptoethanol, heated at 98°C for 3 minutes and 25 pg (as determined by BCA assay (Thermo Scientific, Cat. no. 23225) ) of the proteins were applied to the gel for Western Blot analysis. Bands were visualized using Opti-4CN Substrate Kit (Bio-Rad, Cat. no. 1708235) .

Antibodies used in the study:

- anti-SARS-CoV-2 Spike Glycoprotein SI antibody (Abeam, Cat. no. ab275759) , dilution 1:500

- anti-Actin antibody, clone C4 (Sigma-Aldrich, Cat. no. MAB1501) , dilution 1:4000

- anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-2357) , dilution 1:4000

- anti-mouse IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-516102) , dilution 1:4000

Results and conclusions : The experiment shows that 2-DG inhibits glycosylation of spike protein from SARS-CoV-2 virus (Figure 3) . The effect of dose and timing of dose (the dose-response relationship as well as the time-response relationship) of 2-DG on blocking of spike glycosylation is observed in the concentration range of 10 pM (0.01 mM) to 10 mM. In Figure 3 the doseresponse relationship is clearly visible (selected concentrations for this blot ranges from 0.7 mM to 15 mM) .

Example 3. Determination of IC50 values for 2-deoxy-D-glucose in human bronchial epithelial cells (short exposure)

Human Bronchial Epithelial Cells (HBEpiC) were obtained from Innoprot (Cat no. P10557, batch no. 7475) and cultured in Bronchial Epithelial Cell Medium (Innoprot, Cat no. P60151) supplemented with 0.2% gentamicin (Gibco, Life Technologies, USA, Cat. no. 15710- 049) under standard cell culture conditions (5% CO2, 16% O ₂ , 37°C) . HBEpiC at passage 2, were plated in Bronchial Epithelial Cell Medium at 10.0 thousand cells per well of a 96-well plate and left for 48 hours in an incubator. The plates were coated with collagen (Collagen I-cell surface coating kit, Innoprot, Cat no. P8188) prior to cell seeding. 2-deoxy-D-glucose (Sigma-Aldrich; Cat no. D8375) was added to the cells for 15 min, 30 min, Ih, 2h and 6h.

Cells were exposed to the compound at the following concentrations of 2-deoxy-D-glucose in medium as a solvent: 200 mM; 100 mM; 50 mM; 25 mM; 12.5 mM; 6.25 mM.

In order to assess cell viability, medium containing 2-deoxy-D-glucose was removed, the fresh medium was added to the cells and CellTiter 96® AQueous Solution Assay (Promega) was used in accordance with the manufacturer's instructions, 20 pl of reagent was added per 100 pl of cell culture medium and cells were incubated at 5% CO2, 16% 02, 37°C. Absorbance was measured at 490 nm. The results obtained 1 hour after addition of the CellTiter reagent were analyzed in GraphPadPrism 5.01. Data normalization and a nonlinear regression model were applied in order to determine the IC50.

Results and conclusions : The experiment shows lack of cytotoxic effects on epithelial cells after 15 minutes to 6 hours exposure to 2-DG. 2-deoxy-D-glucose at a concentration of even 200 mM within this range of incubation times did not cause cell death in a sufficient amount allowing for a IC50 calculation (Figure 4) .

Example 4. Determination of IC50 values for 2-deoxy-D-glucose in human bronchial epithelial cells (long exposure)

Human Bronchial Epithelial Cells (HBEpiC) were obtained from Innoprot (Cat no. P10557, batch no. 7475) and cultured in Bronchial Epithelial Cell Medium (Innoprot, Cat no. P60151) supplemented with 0.2% gentamicin (Gibco, Life Technologies, USA, Cat. no. 15710- 049) under standard cell culture conditions (5% CO ₂ , 16% O ₂ , 37°C) . HBEpiC at passage 6, were plated in Bronchial Epithelial Cell Medium at 9.0 thousand cells per well of a 96-well plate and left for 48 hours in an incubator. The plates were coated with collagen (Collagen I-cell surface coating kit, Innoprot, Cat no. P8188) prior to cell seeding. 2-deoxy-D-glucose (Sigma-Aldrich; Cat no. D8375) was added to the cells for 48h.

Cells were exposed to compounds at the following concentrations of 2-deoxy-D-glucose in medium as a solvent: 100 mM; 50 mM; 25 mM; 12.5 mM; 6.25 mM; 3.13 mM.

In order to assess cell viability, CellTiter 96® AQueous Solution Assay (Promega) was used in accordance with the manufacturer's instructions, 20 pl of reagent was added per 100 pl of cell culture medium and cells were incubated at 5% CO2 , 16% 02, 37°C. Absorbance was measured at 490nm. The results obtained 4 hours after addition of the CellTiter reagent were analyzed in GraphPadPrism 5.01. Data normalization and a nonlinear regression model were applied in order to determine the IC50.

Results and conclusions : The experiment shows a very high IC50 value after exposing human epithelial cells to 2-DG (Figure 5) .

Example 5. Evaluation of 2-DG antiviral activity in primary bronchial epithelial cells (PBEC) infected by SARS-CoV-2 virus .

Work plan:

1. Reception and amplification of PBEC cells.

2. Seeding of PBEC and calibration of the infection with SARS-CoV-2. 3. If calibration is successful, treatment with compound of interest in triplicate.

4. Infection with SARS-CoV-2 at one MOI (multiplicity of infection) .

5. Recovery of viral particles in the supernatant after incubation and quantification using TCID50 (tissue culture infectious dose 50%) and RT-qPCR on Vero E6 cells. Data analysis.

Calibration protocol :

Day 1. Human PBEC cells from Promocell (C- 12640) in passage 3 were seeded in 48 wells plate. Growth conditions: Airway epithelial cell growth medium (Promocell C-21060) .

Day 2. Infection with SARS-CoV-2 ( Ih with either 1, 2 or 3xPBS wash) at MOI IO ^-1 .

Day 3. Recovery of virus particle and measurement of the production by RT-qPCR on triplicate pool.

Screening protocol:

Day 1. PBEC human cells from Promocell (C- 12640) were seeding in 48 wells plate in growth conditions included Airway epithelial cell growth medium (Promocell C-21060) .

Day 2. Pre-treatment of PBEC cells in 48 wells plate with 2-DG prior to SARS-CoV-2 infection (2h) , and treatment with 2-DG for 48 h. SARS-CoV-2 was added at MOI 10-1 (MOI 0.1) and removed after 2 hours. Then, the cell culture was washed with PBS (3x0.5 mL) and 300pL of medium with 2-DG from 50 mM to 0.39 mM, or medium with or without remdesivir for control, were added.

Day 4. Viral titer was assessed by the TCID50 (Median Tissue Culture Infectious Dose) method on Vero-E6 cells and calculated by the Spearman & Karber algorithm.

Infection process was conducted according to below presented scheme: T=-2h or t=0 Treatment with compound from 50mM to 0.39 mM

T=0h Infection with SARS-CoV-2 at MOI IO ^-1 (MOI

0.1) with 2-DG 50 mM to 0.39 mM

T=2h Virus removal and PBS wash (3x0.5 mL) .

Add 300 pL medium with 2-DG from 50 mM to 0.39 mM

[48h incubation]

T=50h Recover 150 pL of supernatant and TCID50 processing

- RT-qPCR targeting SARS-CoV-2 E gene

[4 day incubation]

Endpoint TCID50 reading and calculation.

Results and conclusions : It was demonstrated that 2-DG influences on SARS-CoV-2 propagation in human epithelial cells (Figure 6) . In the conditions with 2h Pretreatment (according to above listed schedule) 2-DG induces a decrease in the production of infectious SARS- CoV-2 particles starting at 0.78 mM. In Treatment conditions only SARS-CoV-2 virus production was stronger (around 2,51ogl0 (TCID50) ) . In these conditions 2-DG induces a massive decrease in the production of infectious SARS-CoV-2 particles starting at 0.78 mM. At conditions above 0.78 mM, 2-DG effect is equivalent to the positive control remdesivir.

To conclude, 2-DG induces a dose dependent decrease in SARS-CoV-2 virus production on primary human bronchial epithelial cells, with effect equivalent to the positive control remdesivir above 0.78 mM. No toxicity of 2-DG was observed even during the 48h incubation. 2-DG concentration required to inhibit SARS-CoV-2 replication in human epithelial cells was at least 50 times lower than IC50 concentration. Example 6. Evaluation of antiviral activity of 2-DG on blocking the infection and replication of SARS-CoV-2 in Vero E6 cell line.

Work plan:

1 . Seeding of cells and treatment with compound of interest in triplicate.

2 . Infection with SARS-CoV-2 at one MOI (multiplicity of infection) .

3 . Recovery of viral particles in the supernatant after incubation and quantification using TCID50 (tissue culture infectious dose 50%) and RT-qPCR. Data analysis .

Screening protocol:

Day 1: Vero E6 cells in passage 41 were seeding in 96 wells plate in growth conditions including medium DMEM with high glucose (Dutscher L0104-500, lot MS008A) .

Day 2: 2-DG was diluted at IM in medium and added to a final concentration of 50 mM, then diluted/2 in triplicate until 0.39 mM.

In parallel: toxicity assessment on Vero cells treated with 2-DG at the same concentrations, fixed at 24 h post treatment and tested with a cytotoxicity algorithm (number of cells, nuclear morphology) in two media (DMEM high glucose and F12 low glucose) .

Infection process was conducted according to below presented scheme:

T=-3h or t=0 Treatment with compound from 50mM to 0.39 mM

T=0h Infection with SARS-CoV-2 at MOI 10~ ³ (MOI

0.001) with 2-DG 50 mM to 0.39 mM

T=lh Virus removal and PBS wash (2x1 mL) . Add

1 mL medium with 2-DG from 50 mM to 0.39 mM [24h incubation]

T=25h Recover 500 pL of supernatant and TCID50 processing

- RT-qPCR targeting SARS-CoV-2 E gene

[4 day incubation]

Endpoint TCID50 reading and calculation.

Results and conclusions: 2-DG induces a strong decrease in the production of infectious SARS-CoV- 2 particles in Vero E6 cells (Figure 7) . Effect is impressive on TCID50 calculation but less pronounced on qPCR experiments. Effect of increase concentrations of 2- deoxy-D-glucose on SARS-CoV-2 replication in Vero E6 cells is clearly visible.

Example 7. Evaluation of antiviral activity of 2-DG on blocking the multiplication of SARS-CoV-2 in Vero E6 cell line 8 hours after infection (post-treatment) in Vero E6 cell line

Work plan:

1. Seeding of cells and treatment with compound of interest in triplicate.

2. Infection with SARS-CoV-2 at one MOI (multiplicity of infection) .

3. Recovery of viral particles in the supernatant after incubation and quantification using TCID50 (tissue culture infectious dose 50%) . Data analysis.

Screening protocol:

Day 1: Vero E6 cells were seeding in 96 wells plate in growth conditions including medium DMEM with high glucose (Dutscher L0104-500, lot MS008A) .

Day 2: 2-DG was diluted at IM in medium and added to a final concentration of 45 mM, then diluted/3 in triplicate until 0.19 mM. In parallel: toxicity assessment on Vero cells treated with 2-DG at the same concentrations, fixed at 24 h post treatment and tested with a cytotoxicity algorithm (number of cells, nuclear morphology) in two media (DMEM high glucose and F12 low glucose) .

Infection with SARS-CoV-2 at MOI equal 10-3 was conducted at T=0h and 2-DG was added at 0 h.p.i for the "treatment" group and at 8 h.p.i for the "post-treat- ment" group.

Results and conclusions: 2-DG induces a strong decrease in the production of infectious SARS-CoV- 2 particles in Vero E6 cells when added 8 hours after cells infection (Figure 8) . Effect is impressive on TCID50 calculation. Effect of increase concentrations of 2-deoxy-D-glucose on SARS-CoV-2 replication in Vero E6 cells is clearly visible. Results showed that 2-DG can be used not only in preventing but also in treatment of COVID-19 by suppressing viral replication.

Example 8. Analysis of the glycosylation of Spike protein in renal epithelial cells

The analysis was performed on the samples

(see table 1 below) obtained within experiment described in example 6.

Table 1.

The samples were mixed with Laemmli with p- mercaptoethanol, heated at 60°C for 5 minutes and 20 g (as determined by BCA assay (Thermo Scientific, Cat. no. 23225) ) of the proteins were applied to the gel for Western Blot analysis. Bands were visualized using Opti-4CN Substrate Kit (Bio-Rad, Cat. no. 1708235) .

Antibodies that were used in the study:

- anti-SARS-CoV-2 Spike Glycoprotein SI antibody (Abeam, Cat. no. ab275759) , dilution 1:500

- anti-Actin antibody, clone C4 (Sigma-Aldrich, Cat. no. MAB1501) , dilution 1:4000

- anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-2357) , dilution 1:4000

- anti-mouse IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-516102) , dilution 1:4000 Results and conclusions : The experiment shows that 2-DG inhibits glycosylation of spike protein in lysates of SARS-CoV-2 virus infected Vero-E6 cells (Figure 9) . In Figure 9 the dose-response relationship is clearly visible (selected concentrations for this blot ranges from 0.19 mM to 45 mM) .

Example 9. 2-deoxy-D-glucose (2-DG) uptake measurement in lung tissue lysates of mice treated with 2-DG for different time periods

Procedure

The analysis involved six lung tissue sections of mice (both male and female) treated by inhalation with 2-DG at a concentration of 30 mM thrice a day for 24 hours, 7 days and two weeks. The tissues were weighed, suspended in 10 mM Tris-HCl lysis buffer (pH 8.0) and then subjected to homogenization with the use of homogenizer (MPW-302, Precision Mechanics) . Each analyzed sample is described in table 2 below:

Table 2.

As a negative control, two normal cell lines were used - BALB 3T3/c and human dermal fibroblasts.

The 2DG6P accumulation in cells was determined with the use of 2-Deoxyglucose (2-DG) Uptake Measurement Kit (Cat. No. CSR-OKP-PMG-KOITE; Cosmo Bio Co., LTD. , Tokyo, Japan) according to the manufacturer's protocol .

1. Cell and tissue lysates were heat treated at 80°C for 15 min and then centrifuged at 4°C, 15 000 x g for 20 min.

2. The supernatants were transferred to new tubes and used as unknown samples for measurement method (point 5 ) .

3. The 2DG6P standards were prepared by serial dilution of 1 mM 2DG6P in lx Sample Diluent Buffer in the following ranges: 0; 0.3125; 0.625; 1.25; 2.5 and 5 pM (Figure 10, samples B2-G2) . 4. 60 L of Reagent Mix A (including NAD and low glucose-6-phosphate dehydrogenase (G6PDH) ) were added to each well of 96-ell plate.

5. Then 2DG6P standard and unknown samples (20 pL) were added to each well and incubated for over 19 hours at room temperature.

6. 5 pL of Solution B (acid solution) were added to each well and incubated at 37°C for 1 h.

7. 5 pL of Solution C (acid neutralizing solution) were added to each well and incubated at RT for 10 min.

8. 30 pL of Reaction Mix D (including NADH and high G6PDH) were added to each well and incubated at 37°C for 1 h.

9. 5 pL of Solution E (alkali solution) were added to each well and incubated at 70°C for 1 h and then chilled on ice for 5 min.

10.5 pL of Solution F (alkali neutralizing solution) were added to each well and incubated at RT for 15 min.

11.70 pL of Enzyme Cycling Solution (including glutathione disulfide (GSS) and glucose 6-phosphate (G6P) , high G6PDH and glutathione reductase (GR) ) were added to each well.

12. The optical density (OD) was read at 420 nm in every 2.5 min over a period of 30 min using a microplate reader preheated to 30°C.

The 96-well plate template was prepared as below and included: 2DG6P standards (B2-G2, samples (B3- G3 ) , BALB 3T3 (B4) as well as fibroblasts (C4) .

Results and conclusions :

Temporal changes of OD for all the samples included in the analysis (Figure 10) show that 2-DG was accumulated in the lungs of mice in animal model study due to the inhalation. Obtained data shows a plateau effect in 2-DG accumulation.

Example. 10. Analysis of ACE2 protein expression in renal epithelial cells

Renal epithelial cells (VERO, clone E6) were obtained from ATCC (Cat no. CRL-1586, batch no. 70034994) and cultured in Eagle's Minimum Essential Medium (ATCC, Cat no. 30-2003) supplemented with 0.2% gentamicin (Gibco, Life Technologies, USA, Cat. no. 15710-049) , 1% Penicillin with Streptomycin (Biowest, Cat. no. L0022- 100) and 10% fetal bovine serum (Biowest, Cat. no. S181H- 500) under standard cell culture conditions (5% CO2 , 16% 02, 37°C) . VERO cells at passage 4, were plated in EMEM at 297.0 thousand cells per well of a 6-well plate and left for 24 hours in an incubator. The cells were transduced with lentiviral vectors encoding the SARS-CoV-2 Spike Protein. After 24h post transduction 2-deoxy-D-glu- cose (Sigma-Aldrich; Cat no. D8375) was added to the cells for next 24h. The cells after incubation with 2-de- oxy-D-glucose (or without this compound as in control group) were lysed for 30 min at 4°C in cell lysis buffer (50 mM Tris-HCl pH 7.5 , 1 mM EDTA, 1 mM EGTA, 1 mM Sodium Orthovanadate, 10 pM p-glycerophosphate , 5 pM Sodium Pyrophosphate and 0.5% Triton X-100) freshly supplemented with Proteases and Phosphatases Inhibitor Cocktail. Lysates were clarified at 7,000 * g for 6 min at 4°C. The samples were mixed with Laemmli with p-mercaptoethanol, heated at 98 °C for 3 minutes and 25 g (as determined by BCA assay (Thermo Scientific, Cat. no. 23225) ) of the proteins were applied to the gel for Western Blot analysis. Bands were visualized using Opti-4CN Substrate Kit (Bio-Rad, Cat. no. 1708235) and Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Cat. no. RPN2232 ) .

Antibodies used in the study:

- anti-ACE2 Antibody (Cell Signaling Technology, Cat no. 4355) , dilution 1:100

- anti-Actin Antibody, clone C4 (Sigma-Aldrich, Cat. no. MAB1501) , dilution 1:4000

- anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-2357) , dilution 1:4000

- anti-mouse IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-516102) , dilution 1:4000

Results and conclusions: The experiment shows that 2-DG inhibits expression of ACE2 protein on human renal epithelial cells (Figure 11) . The effect of dose and timing of dose (the dose-response relationship as well as the time-response relationship) of 2-DG on blocking of ACE2 expression is observed in the concentration range of 10 pM (0.01 mM) to 10 mM. In Figure 11 the dose-response relationship is clearly visible (selected concentrations for this blot ranges from 0.7 mM to 15 mM) . Visualization using the Amersham ECL Prime Western Blotting Detection Reagent. „Ctrl" means that cells were not exposed to 2-DG.

This was a significant result identifying that 2-DG can act on host cells in blocking glycosylation of certain receptors including receptors such as ACE2 which viruses use to bind to a host cell for cell infection. Thus, virus interaction with host cells can be reduced thereby reducing the capability of viruses to infect host cells and use them to replicate within a host. This can decrease the rate of viral replication within the cells of a host thereby providing a more effect innate immune response by the host against the viral infection.

Example 11. Evaluation of antiviral activity of 2-DG on blocking the infection and replication of H1N1 Influenza A type virus in MDCK cell line.

Work plan:

1. Seeding of cells and treatment with compound of interest in triplicate.

2. Infection with H1N1 at one MOI (multiplicity of infection) .

3. Recovery of viral particles in the supernatant after incubation and quantification using TCID50 (tissue culture infectious dose 50%) and RT-qPCR. Data analysis .

Screening protocol: Day 1: MDCK cells were seeding in 96 wells plate in growth conditions including medium Eagle's Minimum Essential Medium (EMEM) .

Day 2: 2-DG was diluted at IM in medium and added to a final concentration of 45 mM, then diluted/2 in triplicate until 0.02 mM.

Infection process was conducted according to below presented scheme:

T=-2h or t=0 Treatment with compound from 45 mM to 0.02 mM

T=0h Infection with H1N1 at MOI 10~ ² (MOI 0.01) with 2-DG 40 mM to 0.02 mM

T=2h Virus removal and PBS wash (2x1 mL) . Add

1 mL medium with 2-DG from 45 mM to 0.02 mM [24h incubation]

T=25h Recover 500 pL of supernatant and TCID50 processing Endpoint TCID50 reading and calculation .

Results and conclusions : 2-DG induces a strong decrease in the production of infectious H1N1 particles in MDCK cells ( Figure 12 ) . Effect is impressive on TCID50 . Effect of increase concentrations of 2-deoxy-D- glucose on H1N1 replication in MDCK cells is clearly visible .

In the further aspect of the application, a formulation comprising a 2 -DG ( as an active ingredient ) encapsulated in a dry particles composed from different excipients releasing 2-DG in the respiratory tract or forming liposomes having extended and consciously controlled the release time of the substance to several hours . By varying the ratio of DMPC to DPPC or changing lipid composition by addition varying amount of cholesterol to liposomal bilayer composed from natural soy or sunflower lecithin of the pharmaceutical purity .

To prepare inhalable particles containing 2-DG a spray drying procedure can be applied ( Figure 13 ) . The particles of the size lower than 5 pm can according to the application penetrate lower parts of respiratory track . The production of fine particles composed solely from 2 -DG is very difficult because 2-DG has a low melting temperature and is hygroscopic . As excipients used for 2-DG particles preparation by spray-drying method mannitol , trehalose or amino acids ( leucine , glycine ) can be used . The spray-drying method of producing very small particles involves preparing a solution of 2 -DG in water with various ingredients such as mannitol or trehalose and amino acids such as leucine or glycine . A solution containing from 0 . 1 to 5 % solids in the solution may then be spray dried . Amino acids such as leucine or glycine form a hydrophobic shell surrounding particles containing 2 -DG and mannitol . This allows to solve the problem of sticking of particles prepared from the 2 -DG alone .

Example 12 .

200 mg of 2-DG, 300 mg of leucine and 500 mg of mannitol were dissolved in 100 mL of deionized water . The resulting solution was spray-dried at a temperature of 150 degrees Celsius using a Mini Spray-dryer Buchi 290 device . The particles obtained have a size of about 4 , 6 micrometers and a very high roundness ( Figure 13 , Figure 14 , Figure 15 ) .

Example 13 .

200 mg of 2-DG, 300 mg of leucine and 500 mg of trehalose were dissolved in 100 mL of deionized water . The resulting solution was spray-dried at a temperature of 150 degrees Celsius using a Mini Spray-dryer Buchi 290 device . The particles obtained have a size of about 5 micrometers and a very high roundness .

Example 14 .

200 mg of 2-DG, 800 mg of trehalose and 100 mg of leucin were dissolved in 100 mL of deionized water . The resulting solution was spray-dried at a temperature of 100 degrees Celsius using a Mini Spray-dryer Buchi 290 device . The particles obtained have a size of about 4 micrometers and a very high roundness .

Example 15 .

200 mg of 2-DG, 800 mg of mannitol and 100 mg of leucin were dissolved in 100 mL of deionized water . The resulting solution was spray-dried at a temperature of 100 degrees Celsius using a Mini Spray-dryer Buchi 290 device . The particles obtained have a size of about 4 micrometers and a very high roundness .

Example 16 . 200 mg of 2-DG, 200 mg of DPPC, DMPC and DPPG 85:10:5 mol/mol ratio, 400 mg of leucine and 200 mg of mannitol were dissolved in 100 mL of 60% ethanol at 50°C. The mixture was spray dried in a Mini Buchi machine and the obtained proliposome particles had size of about 4pm. The particles were mixed with 37 °C physiological saline to obtain liposomes with an average size of 2 pm. At time t = 0, it was determined that about 60% of the active substance was released, the rest was slowly released within 18 hours.

Example 17

200 mg of 2-DG, 300 mg of DPPC and DMPC (70:30 mol/mol) , 400 mg of leucine and 100 mg of trehalose were dissolved in 100 mL of 60% ethanol at 50°C. The mixture was spray dried in a Mini Buchi machine and the obtained proliposome particles had size of about 4,5 pm. Proliposomes particles were mixed with 37°C physiological saline to obtain liposomes with an average size of 2,7 pm. At time t = 0, it was determined that about 50% of the active substance was released, the remaining amount was released over the next 8 hours .

Example 18

200 mg of 2-DG, 300 mg of DPPC and DMPC (70:30 mol/mol) , 300 mg of glycine and 200 mg of trehalose were dissolved in 100 mL of 60% ethanol at 50°C. The mixture was spray dried in a Mini Buchi machine and the obtained proliposome particles were mixed with 37 °C physiological saline to obtain liposomes with an average size of 3 pm. At time t = 0, it was determined that about 45% of the active substance was released, the remaining amount was released over the next 8 hours .

Example 19

200 mg of 2-DG, 300 mg of SPC/Chol (70:30 mol/mol) , 300 mg of urea and 200 mg of leucin were dissolved in 100 mL of 50% tert-butanol at 50°C. The mixture was spray dried in a Mini Buchi machine and the obtained proliposome particles were mixed with 37 °C physiological saline to obtain liposomes with an average size of 1,2. At time t = 0, it was determined that about 65% of the active substance was released, the remaining amount was released over the next 8 4 hours .

Example 20

900 mg of DPPC, DMPC and DPPG 85:10:5 mol/mol ratio were dissolved in 3 mL of ethanol at 50°C and then 10 mL of water with 0.5 g of 2-DG dissolved were purred in to achieve oligolamellar liposomes . The suspension was then extruded 6 times through 200 nm polycarbonate filter in a thermobarrel extruder set at 55° . The resulted unilamellar liposomes were then incubated 15 min at 70°C to increase 2-DG encapsulation. The liposomal suspension was next cooled down to 20 °C and diluted with water solution containing trehalose and leucine. Final lipid concentration was 20 mg/mL, 5% trehalose and 1% leucine concentration .

After spray-drying the particle size was 4.5 pm, the 41% of the encapsulated 2-DG remained encapsulated after reconstitution of the particles in 0.9% NaCl at 37°C. Liposomes size remained essentially unchanged and was 186 nm.

Example 21

200 mg of DPPC, DMPC and DPPG 85:10:5 mol/mol ratio were dissolved in 4 mL of ethanol/propylene glycol mixture (1:1) at 50 °C. This solution was then mixed with 1 mL of the solution containing 50 mg 2-DG and clear solution was achieved. This solution forms liposomes when mixed with 150 mM NaCl solution at 37°C. The encapsulation efficiency of the 2-DG is near 72%.

Example 22 1 g of lipids of the composition HSPC/ DSPG/DSPE-PEG 2000 (75:20:5, mol/mol) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen. The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of 600 mM solution of the 2- DG at 64°C. The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . The resulting liposomes were then frozen and freeze-dried. Liposomal powder was then rehydrated by addition of 10 ml distilled water at 64 °C. The oligolamellar liposomes were next extruded through 80 nm polycarbonate filter on thermobarrel extruder. The nonencapsulated 2-DG was removed by dialysis method. The encapsulation efficiency of the 2-DG was 26%, the liposomes size was 110 nm (Figure 16) .

Example 23

1g of lipids of the composition SM/DSPE-PEG 2000 (95:5, mol/mol) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen. The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of 300 mM solution of the 2-DG at 70°C. The multilamellar liposomes were then extruded 4 times through 400 nm and then 8 times through 80 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . To the liposomal suspension ethanol was slowly pipetted to achieve 30% concentration. The liposomal suspension was next incubated to 75°C for 10 min. The encapsulation efficiency of the 2-DG was 27%, the liposomes size was 105 nm.

Example 24

1 g of lipids of the composition HSPC/DSPE- PEG 2000 (95:5, mol/mol) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen. The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of 300 mM solution of the 2-DG at 64 °C. The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes. The resulting liposomes were then frozen and freeze-dried. Liposomal powder was then rehydrated by addition of 10 ml distilled water at 64°C. The oligo- lamellar liposomes were next extruded through 80 nm polycarbonate filter on thermobarrel extruder. The non-encap- sulated 2-DG was removed by dialysis method. The encapsulation efficiency of the 2-DG was 21%, the liposomes size was 107 nm.

Example 25

1 g of lipids of the composition DPPC/DSPE- PEG 2000 (95:5, mol/mol) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen. The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of 300 mM solution of the 2-DG at 55°C. The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . The resulting liposomes were then frozen and freeze-dried. Liposomal powder was then rehydrated by addition of 10 ml distilled water at 64°C. The oligolamel- lar liposomes were next extruded through 80 nm polycarbonate filter on thermobarrel extruder. The non-encapsu- lated 2-DG was removed by dialysis method. The encapsulation efficiency of the 2-DG was 19%, the liposomes size was 103 nm.

Example 26

1 g of lipids of the composition HSPC/ DDAB/DSPE-PEG 2000 (75:20:5, mol/mol) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen. The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of 300 mM solution of the 2- DG at 64 ° C . The multilamellar liposomes were then extruded through 400 and then 80 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . The non-encapsulated 2-DG was removed by dialysis method . The encapsulation efficiency of the 2-DG was 13% , the liposomes size was 103 nm .

Example 27

1 g of lipids of the composition HSPC/DSPE- PEG 2000 ( 95 : 5 , mol/mol ) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen . The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of solution of 200 mM 2-DG and 100 mM sodium ascorbate at 64 ° C . The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . The resulting liposomes were then frozen and freeze-dried . Liposomal powder was then rehydrated by addition of 20 mL distilled water at 64 ° C . The oligolamellar liposomes were next extruded through 80 nm polycarbonate filter on thermobarrel extruder . The non-encapsulated 2-DG and sodium ascorbate were removed by dialysis method . The encapsulation efficiency of the 2-DG was 27% , the liposomes size was 109 nm.

Example 28

1 g of lipids of the composition HSPC/DSPG/DSPE-PEG 2000 ( 75 : 20 : 5 , mol/mol ) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen . The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of solution of 200 mM 2-DG and 100 mM sodium ascorbate at 64 ° C . The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . The resulting liposomes were then frozen and freeze-dried . Liposomal powder was then rehydrated by addition of 20 mL distilled water at 64 °C. The oligolamellar liposomes were next extruded through 80 nm polycarbonate filter on thermobarrel extruder. The non-encapsulated 2-DG and sodium ascorbate were removed by dialysis method. The encapsulation efficiency of the 2-DG was 16%, the liposomes size was 102 nm.

Example 29

1 g of lipids of the composition HSPC/ DDAB/DSPE-PEG 2000 (75:20:5, mol/mol) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen. The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of solution of 200 mM 2-DG and 100 mM sodium ascorbate at 64 °C. The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . The resulting liposomes were then frozen and freeze-dried. Liposomal powder was then rehydrated by addition of 20 mL distilled water at 64°C. The oligolamellar liposomes were next extruded through 80 nm polycarbonate filter on thermobarrel extruder. The non-encapsulated 2-DG and sodium ascorbate were removed by dialysis method. The encapsulation efficiency of the 2-DG was 17%, the liposomes size was 96 nm.

Example 30

1 g of lipids of the composition HSPC/DSPE- PEG 2000/DSPE-PEG3200-FA (95:4.7:0.3) , mol/mol) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen. The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of solution of 300 mM 2-DG and at 64 °C. The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes. The resulting liposomes were then frozen and freeze-dried. Liposomal powder was then rehydrated by addition of 20 mL distilled water at 64°C. The oligolamellar liposomes were next extruded through 80 nm polycarbonate filter on thermobarrel extruder . The non-encapsulated 2-DG was removed by dialysis method . The encapsulation efficiency of the 2-DG was 24% , the liposomes size was one of 102 or 86 nm.

Example 31

1g of lipids of the composition SM/DSPE-PEG 2000 ( 95 : 5 , mol/mol ) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen . The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of 200 mM 2-DG and 100 mM sodium ascorbate solution at 64 ° C . The multilamellar liposomes were then extruded 4 times through 400 and then 8 times through 80 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . To the liposomal suspension ethanol was slowly pipetted to achieve 30% concentration . The liposomal suspension was next incubated to 75 ° C for 10 min . The non-encapsu- lated 2 -DG and sodium ascorbate were removed by dialysis method . The encapsulation efficiency of the 2-DG was 32% , the liposomes size was 117 nm.

Example 32

1 g of lipids of the composition HSPC/DSPE- PEG 2000 ( 85 : 2 , 5 , mol/mol ) was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen . The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of 300 mM solution of the 2-DG at 64 ° C . The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . The non-encapsulated 2 -DG was removed by dialysis method . The resulting liposomes were next incubated at 37 ° C with mixed micelles composed from DSPE-PEG 2000 and DSPE-PEG 3400-Transferrin for Ih . The final molar ration DSPE-PEG 2000 and DSPE-PEG 3400-Transferrin was 1 : 1000 . The encapsulation efficiency of the 2 -DG was 23% , the liposomes size was 101 nm.

Example 33

1 g of lipids of the composition POPC/DSPE- PEG 2000 ( 95 : 5 , mol/mol ) and 30 mg of lomustine was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen . The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of 300 mM solution of the cucrose at R . T . The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . The non-incorporated lomustine was removed during extrusion as white deposit on the filter . The incorporation efficiency of the lomustine was 78 % , the liposomes size was 98 nm.

Example 34

1 g of lipids of the composition POPC/DOTAP/DSPE-PEG 2000 ( 75 : 20 : 5 , mol/mol ) and 30 mg of lomustine was dissolved in 10 mL of cyclohexane and frozen in liquid nitrogen . The resulting ice was subsequently freeze-dried and dry lipid cake was suspended in 20 mL of 300 mM solution of the cucrose at R . T . The multilamellar liposomes were then extruded through 400 and then 100 nm polycarbonate filter on the thermobarrel extruder in order to achieve large unilamellar liposomes . The non-incorporated lomustine was removed during extrusion as white deposit on the filter The incorporation efficiency of the lomustine was 89% , the liposomes size was 95 nm.

Fig . 17 shows a schematic view of a device for inhaling a substance according to an embodiment . The device 1 comprises a discharge nozzle 2 and a container 4 for receiving and keeping the substance 5 as well as an actuator 6 for activating the device 1 . The actuator 6 is configured to release a certain amount or dose of the substance 5 which is kept in the container 4 for transferring the substance 5 through the dis- charge noz zle 2 of the device 1 . In the embodiment of Figure 17 , the device 1 comprises an air flow channel 7 or chamber for conveying the substance 5 released by the actuator 6 to the discharge nozzle 2 . The substance 5 in the container 4 , shown in Figure 17 is a powder comprising a carrier material and an active ingredient out of a group comprising in particular 2 -DG .

In some embodiments , the container 4 is configured to keep a substance in liquid form . The substance 5 may, in particular, comprise a liquid out of the group comprising water , alcohol , liquid glucose , or aqueous solution, in which the active agent is contained .

In some embodiments , the substance 5 in the container 4 is in the form of powder of particles with lactose and/or liposome . Lactose or liposome can serve as a carrier for the active ingredient , such that they can be easily transferred or delivered to the human body . In some embodiments , the active agent is encapsulated in liposomes , in order to achieve a longer or delayed effect in the human body, thus increasing the duration of the therapeutic or prophylactic effect . Particles may also comprise cholesterol which stabilizes liposomes such that an even stronger delay of the agent can be achieved . The particles may comprise a mixture of different liposome . In particular, a mixture of small liposomes , with an average size of less than 100 nm and large liposomes , with an average size of more than 150 nm . By providing different liposome sizes , a desired time profile of the active agent activity can be achieved .

The Device 1 may comprise a chamber or reservoir for a propellant , such as CFG ( chlorofluorocarbon) and/or HFA (hydrofluoroalkane) , for propelling the substance along the flow channel towards the nozzle. The propellant can in particular, facilitate the delivery and dosage of the active ingredients .

In the embodiment of Figure 17, the device 1 comprises a dosage valve 8 arranged at an outlet of the container 4 and the actuator 6 may be functionally connected to the dosage valve 8 and be configured to activate the dosage valve 8. The dosage valve may be configured to release a define an amount of the substance 5, which is to be released each time when the activator 6 is activated. By means of the dosage valve 8, a precise dosage of the substance 5, in particular, of the active ingredient or agent comprised in the substance 5 can be achieved. The dosage valve 8 may be an adjustable dosage valve such that, prior to dispensing the substance, the dosage of the substance can be adjusted.

The dosage valve 8 can be, in particular, configured to keep the amount of the active agents in the released portion of the substance 5 and the dosage in the range of 5 to 10 millimoles. By limiting the amount of the active agent in the particles, side effects related with too high dosage can be avoided. In some embodiments, the liposomes have a transition temperature, from solid to liquid, in the range from 35°C to 45°C, more specifically, between 37°C and 40°C degrees about 37°C. Thus, the liposomes may easier dissolve after the substance has been applied to the human body.

The air flow channel 7 may be configured to support turbulences in the air flow. The turbulences in the air flow can facilitate entraining the particles released from the container and propel them towards the discharge nozzle 2 of the device 1. Further, due to the turbulences in the air flow, the phase space occupied by the particles released from the container 4 can be increased such that a broad distribution of the resulting particle j et can be achieved . The broad distribution of the particles may be particularly helpful to avoid local overdoses of the active ingredients at the human tissues exposed to the substance .

In some embodiments , the airflow channel 7 is configured such that the air flow in the air flow channel can be created by the user by inhaling the air while keeping the discharge nozzle 2 of the device in a nostril or in the mouth . Such a device does not require any additional source of energy for providing the air flow .

The actuator 6 can be configured to provide a pressurized air flow in the air flow channel . The pressurized air released by the actuator can, in particular, support turbulences which can help to entrain the substance particles located at the outlet of the container 4 when the dosage valve 8 is open .

Figure 13 shows a compound particle according to an embodiment .

The particle 10 comprises a carrier material 11 with an active ingredient 12 , wherein the active ingredient 12 is encapsulated or integrated in the carrier material 11 . In this embodiment , the particle is formed by spray drying and the active ingredient 12 comprises 2 - DG . In some embodiments the carrier material may comprise trehalose and/or phospholipids . The active ingredient may comprise one or more different types of agents out of the group comprising ribavirin, emetine , 2-DG and NMS-873 . These active ingredients can serve as translation inhibitors for preventing or suppressing viral replication and/or as agents for suppressing the growth and reproduction of the host cells attacked by viruses . Due to prevention of the viral replication and suppressing the growth and reproduction of the host cells, these active ingredients can serve not only as a medication against viral-infectious diseases but also as a prophylaxis for preventing a viral attack or contagion of the human body. In some embodiments, the carrier material may comprise a mixture of different liposomes showing different stability and transition temperature characteristics.

Figure 16 shows a compound particle according to an embodiment. The particle 10, similar to the particle of Figure 13, is a compound particle comprising a carrier material 11 (liquid) with an active ingredient 12. However, in this embodiment, the carrier material 11 and the active ingredient 12 is surrounded by lipid bylayer 13. The active ingredient 12 may comprise one or more different types of agents and in particular comprises 2-DG.

The carrier material 11, in this embodiment, is a liquid. The carrier material may comprise a liquid out of the group comprising water, liquid glucose or aqueous solution. In some embodiments, the carrier material comprises an aqueous solution of one or more salt. In some embodiments, the salt in the aqueous solution is NaCl . The salt concentration in the aqueous solution may vary in the range from 1% to 5%, in particular, 1.5% to 2.5%, or from 1.8% to 2.2%. In some embodiment, the aqueous solution comprises an additive for regulating the pH value of the aqueous solution. By regulating the pH value in the aqueous solution, the stability of the compound particle, in particular, the shell 13 of the compound particle, can be adjusted such that a desired sustained release of the active agent in the human body can be achieved . The shell 13 incapsulating the carrier material 11 and the active agent 12 may comprise lipid bylayer composed of phospholipids , PEGylated phospholipids , sterols and surfactants . In some embodiments , the shell 13 may comprise a mixture of different phospholipids showing different stability and transition temperature characteristics .

The size of the particles of Figure 13 may vary from in a range 0 . 5 - 5 pm and the size of the particles of Figure 16 may vary from in a range 50 nm - 1000 nm.-Such fine particles 10 can be easily applying to a target region of the human body, in particular, by spreading them as aerosol particles or powder particles .

Further , liposomes with different properties can be used . Thus , by choosing different size and/or properties of liposomes in the shell of the compound particles , a desired time profile for releasing the active agent in the human body can be achieved . The liposome encapsulation can help to reduce toxic reactions or irritations , when the substance is applied to the human body . In particular, the during inhalation, the liposome shell covers the carrier material with the active agent , such that the active agent can get to deeper zones of the respiratory tract without getting in direct contact with the tissues , before reaching the target location . Thus , unnecessary exposure of the human tissue to the active agent , in particular in the regions unaffected by the virus , can be avoided .

Figure 18 shows a flow chart of a method of dispensing a substance according to an embodiment . According to the method 100 , in step 110 , a substance in the form of aerosol particles or powder particles is provided . The provided particles may be kept , in particular , in a container of a device for inhaling the substance . Further , in step 120 , an aerosol is created, the particles being suspended in the aerosol . The aerosol can be created, in particular, in an air flow channel or chamber of the device for inhaling the substance .

Further , in step 130 , a directed flow of the aerosol is created such that the suspended particles move essentially along the flow direction of the aerosol . The particles may comprise at least one active ingredient or active agent out of the group comprising ribavirin, emetine , 2 -DG and NMS-873 . The method further comprises directing 140 the directed flow or j et of the aerosol towards target areas of the human body for dispensing the substance . In some embodiments , the target areas may comprise a throat or nasal area , pharynx or nasal mucosa as well as lung tissue of humans . By discharging the substance with the active ingredients , viral activity can be affected .

By discharging translation inhibitors in the target areas , viral replications in the human cells can be prevented or suppressed . Furthermore , by suppressing the viral replication in the early stage , later pathological consequences can be avoided as well . Thus , the method can also serve as a prophylaxis for avoiding viral diseases .

Even though, the mechanisms are not completely understood, 2 - DG molecules , as compared to glucose , are characterized by stability against cellular metabolism, in particular , in cancerous or virally compromised cells . Because of the similarity with glucose molecules , cells may regard 2-DG molecules as glucose molecules and capture them. Within the cells , 2-DG molecules can even undergo phosphorylation . The resulting 2 - DG-6- phosphate , however, in contrast to phosphorylated glucose , does not further participate in glycolysis . Thus , 2-DG- 6-phophate can remain in a host cell , in particular, in a host cell attacked by a SARS-CoV-2 virus without undergoing glycolysis and, hence , without producing energy which is necessary for cellular activities including biogenesis and reproduction of host cells . The viruses do not have their own metabolism. Instead, the viruses can penetrate into healthy cells or host cells and modify their DNA such that these cells start to produce more viruses and to reproduce themselves as well , resulting in a multiplicity of infected cells which can produce and disseminate more viruses . Similar to cancerous cells , the host cells also require vast amount of energy, in particular, for growth and production of further viruses .

Some viruses , in particular the SARS2 viruses ( from the Corona family) , are transmitted via the respiratory tract . They nest in the frontal sinuses and lungs of the infected patients .

Therefore , by applying the growth and multi- plication-inhibiting 2-DG directly to the airways ( right where the infecting host cells reside ) , are helpful both for prophylaxis and therapy of the diseases caused by the viruses . Further , by providing the active agent , in particular 2 -DG, in the form of very finely ground powder or particles which may be inhaled by means of an inhaler ( Formulation 1 ) such that the growth and reproduction of the cells in the respiratory tract , infected by SARS-CoV- 2 can be suppressed . By adding additional substances , in particular matrix material , to the powder or using a liquid ( suspension) form ( SprayFormulation2 ) the side effects can be reduced and/or absorption rate in the organism can be control the absorption rate can be improved . In particular, some therapeutic substances , in particular, 2-DG, is the short half-life or dwelling time in the organism. In some cases , the active ingredients , e . g . 2- DG, are not detectable in the blood after approximately 48 to 53 minutes after the administration . There are indications that the active substances can quickly decay or enter into reaction with other substances and still remain active . In the body, 2-DG can be detected by a number of alternating defense and excretion mechanisms and can thus trigger reactions which convert or change it very quickly into other substances . By enclosing the active ingredients , in particular 2-DG, in liposomes , or structures with lipids , before applying them through the respiratory tract , the time profile of their activity in the organism can be influenced, in particular while fighting SARS-CoV-2 virus . The liposomes can be mixture of different liposomes configured such that they circulate for several hours without the substance encapsulated or integrated in the liposome becoming detected by the defense mechanisms of the organism. In some embodiments , the liposome is configured such that an essentially constant concentration of the active ingredient , in particular 2-DG, in the blood is maintained during many hours , in some embodiments , even more than 100 hours .

The substance or formulation with liposomes , in particular, liposome encapsulation, also reduces mechanical irritations in the respiratory tract and reactions , such as coughing , sneezing, rash, toxic consequences , allergies etc . The liposomes can also facilitate to reach the target locations , such as lungs , which is crucially important for precise targeting of the active ingredients . Further, liposome pH values can be programmed or adj usted according to specific application, in particular, in accordance with the properties of the target tissue . The adj ustment of the pH value can also influence the sustained release , activity and mobility of the formulation . Liposomes can, in particular, influence the rate of release of the active agent , in particular 2 - DG, after administration . Thus, the frequency of usage of the inhaler for administration of the substance can be reduced. Furthermore, due to the local delivery of 2-DG in small quantities, the overall load of the medication in the organism, can be reduced, resulting in reduction of possible side effects. Further, a combination of active ingredients can be applied locally and/or non-locally. In particular, 2-DG can be combined with one or more further active agents, such that a synergetic effect in fighting the viral infection is achieved.

Example 35. Analysis of Influenza A virus hemagglutinin (HA) protein glycosylation in MDCK cells

Determination of ED50 value

The analysis was performed on the samples of MDCK cells infected with H1N1 (type of Influenza A virus) . The samples were mixed with Laemmli with p- mercaptoethanol, heated at 96°C for 4minutes and 25 pg (as determined by BCA assay (Thermo Scientific, Cat. no. 23225) ) of the proteins were applied to the gel for Western Blot analysis. Bands were visualized using Opti- 4CN Substrate Kit (Bio-Rad, Cat. no. 1708235) .

Antibodies used in the study:

- anti-inf luenza A virus H1N1 HA (Hemagglutinin) antibody produced in rabbit (GeneTex, Cat. GTX127357) , dilution 1:500

- anti-Actin antibody, clone C4 (Sigma- Aldrich, Cat. no. MAB1501) , dilution 1:4000

- anti-mouse IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-516102) , dilution 1:4000

-anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-2963) , dilution 1:4000 Densitometry was conducted using Imaged. The ED50 value was determined.

Results and conclusions : Analysis of Influenza A virus hemagglutinin ( HA) protein glycosylation demonstrated that the ED50 value of 2-DG measured as inhibition of virus hemagglutinin ( HA) protein proper glycosylation is equal to 186 pM ( Figure 19 ) .

Figure 19 . Determination of ED50 value of 2- DG measured as inhibition of proper glycosylation of Influenza A virus hemagglutinin HA protein showed that ED50 is equal to 186 pM .

Further description is provided as follows with respect to the second aspect of the invention to provide a preparation of 2 -DG that is suitable for use in the treatment of cancer .

Targeting cancer protein folding is a new promising strategy to fight cancer . Protein glycosylation is required to make this folding accurate . In rapidly dividing cells as neoplastic cells , lack of proper glycosylation will lead to immediate inhibition of proliferation . 2 -DG can be considered as a small chemical compound inhibiting protein glycosylation . Our analysis showed that it can be more promising than influence on glycolysis .

In a further aspect of the application, 2 -DG is provided for use in a medical method to treat cancer patient , in particular with non-small-cell lung cancer, breast cancer, colorectal cancer as well as fibrosarcoma , glioblastoma and lymphoblastic leukemia .

In this aspect of application, 2-DG is provided as a preparation to tumour of a subj ect , that results in an effective concentration to partially or completely inhibit glycosylation of the cancer cells glycoproteins . Example 36. Determination of IC50 values for 2-deoxy-D-glucose in human neoplastic glioblastoma DK-MG cells .

Determination of IC50 values

Glioblastoma cells (DK-MG) were plated at a density of 5000 cells per well of a 96-well plate and left for 24 hours in an incubator in order to attach to the plate surface. After this time, the RPMI (Biowest, Cat No. L0500) medium was changed to a serum-free medium. The next day, the medium was changed again (to such also without the addition of serum) and 2-deoxy-D-glucose was added in quadruplicate at the following concentrations: 20; 10; 5; 2,5; 1,25 mM. After 1, 2, and 6 hours of incubation with compounds, absorbance was measured at 490 nm using CellTiter 96 aqueous one solution cell proliferation assay (Promega) according to the manufacturer's instructions. Water (solvent control for 2-DG) was added to part of the cells.

IC50 value was determined with GraphPad Prism. Wells with water were used as zero concentration of the tested compounds .

Results and conclusions :

It was demonstrated that 2-DG influences on proliferation of human neoplastic glioblastoma cells (Figure 20) . In the conditions with Ih to 6 h treatment 2-DG induces a decrease in these cells number starting at 1 mM to 5 mM.

Figure 20. Determination of IC50 values for 2-deoxy-D-glucose in human neoplastic glioblastoma DK-MG cells revealed cytotoxic effects on these cells after 1 hour to 6 hours exposure to 2-DG. An IC50 value was determined in range from 1.0 to 5 mM. Exemplary results show A) IC50 for 2-DG [mM] after 1 h of incubation equals 2.241; B) IC50 for 2-DG [mM] after 2 h of incubation equals 1.943; C) IC50 for 2-DG [mM] after 6 h of incubation equals 1.59.

Example 37. Isobologram analysis of 2-deoxy- D-glucose (2-DG) and lomustine combinations after incubation for 6 days in DK-MG.

BALB 3T3 cells and DK-MGhigh glioblastoma cells with high expression of EGFRvIII were cultured in DMEM (Biowest, Cat no. L0102-500) and RPMI 1640 culture medium (Biowest, Cat no. L0500-500) respectively, supplemented with 0.2% gentamicin (Gibco, Life Technologies, USA, Cat. no. 15710-049) , 1% Penicillin with Streptomycin (Biowest, Cat no. L0022-100) and 10% fetal bovine serum (Biowest, Cat no. S181H-500) under standard conditions (5% CO2, 16% 02, 37°C) . Before cytotoxicity assay, DK-MGhigh cells were seeded in duplicate in a 96-well plate at densities of 5,000 cells per well. Subsequently, the cells were exposed to the tested compounds - 2-DG and lomustine at a wide range of concentrations and incubated for 72 hours.

Table 3. Concentrations of compounds used individually for the first 3 days in isobologram.

Both cell lines were cultured within a of range of concentrations of lomustine starting from 20pM, and a range of concentrations of 2-DG starting from 5mM. Compounds were also used separately.

After the first 3 days, combinations of 2-DG and lomustine were added to the cell culture according to the Table 4. Therefore, wide ranges of concentrations were maintained but constant concentrations of lomustine (20pM) or 2-DG (0.5mM) were added.

Table 4. Compound concentrations in combinations.

To summarise:

Day 1-3 : Cells were incubated with lomustine at different concentrations (20; 10; 5; 2.5; 1.25; 0.625; 0.3125; 0.15625 pM) or 2-DG at different concentrations (5; 2.5; 1.25; 0.625; 0.3125; 0.1563; 0.0781 mM) .

Day 3-6: Cells were still incubated with lomustine at different concentrations (20; 10; 5; 2.5; 1.25; 0.625; 0.3125; 0.15625 pM) or 2-DG at different concentrations (5; 2.5; 1.25; 0.625; 0.3125; 0.1563; 0.0781 mM) but the constant concentrations of lomustine (20pM) or 2-DG (0.5mM) were added. 2-DG at concentration of 0 . 5mM was added to the range of concentrations of lomustine and lomustine at concentration of 20pM was added to the range of concentrations of 2-DG .

Finally, after 6 days of incubation with 2-DG and lomustine ( 3 days with single compound and 3 days with combinations ) , the MTS assay was performed in order to assess cell viability .

Cells were then incubated for 3 hours with the CellTiter 96® AQueous One Solution ( Promega , Cat No . G3581 ) and then the absorbance was measured using a microplate reader ( X=490nm) . Subsequently, IC50 , IC30 and IC70 values were calculated by GraphPad Prism 5 . 01 software , where data normalization and a nonlinear regression model were applied .

Figure 21 . Plot of percentage of viable cells depending on the concentration of 2 -DG and lomustine combined with constant concentration of lomustine and 2- DG, respectively, used in DKMG cells .

Table 5 . The inhibitory concentration values were determined based on the MTS assay performed after 6 days of exposure to combinations of lomustine and 2-DG combination on DKMG cells .

Table 6 . The inhibitory concentration values were determined based on the MTS assay performed after 6 days of exposure to combinations of lomustine and 2-DG combination on BALB 3T3 cells.

Conclusions

The outcome of isobologram analyses suggest that the combination index (CI) values depend on combination type. Adding 2-DG as a first compound and then combining it with lomustine gives lower CI which indicated synergistic effect. Adding lomustine to the cell culture and then combined with 2-DG gives higher CI values indicating antagonistic effect.

Example 38. Determination of ED50 values for 2-deoxy-D-glucose in human neoplastic glioblastoma DK-MG cells and lung cancer NCI-H1975 cells by means of protein glycosylation inhibition measurement in Western blotting analysis .

Determination of ED50 value

Human non-small cell lung cancer cell line (H1975) and human glioma cell line (DK-MG) , were cultured in RPMI (Biowest, Cat. no. L0500-500) supplemented with 0.2% gentamicin (Gibco, Life Technologies, USA, Cat. no. 15710-049) , 1% Penicillin with Streptomycin (Biowest, Cat. no. L0022-100) and fetal bovine serum (Biowest, Cat. no. S181H-500) under standard cell culture conditions (5% CO2, 16% 02, 37°C) . The cells were plated in RPMI medium with FBS at 297.0 thousand cells per well of a 6-well plate and left for 24 hours in an incubator. 2-deoxy-D- glucose (Sigma-Aldrich; Cat no. D8375) was added to the cells for 24h. Cells were lysed for 30 min at 4°C in cell lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM Sodium Orthovanadate , 10 pM p- glycerophosphate , 5 pM Sodium Pyrophosphate and 0.5% Triton X-100) freshly supplemented with Proteases and Phosphatases Inhibitor Cocktail. Lysates were clarified at 7,000 x g for 6 min at 4°C. The samples were mixed with Laemmli with p-mercaptoethanol, heated at 98 °C for 3 minutes and 25 pg (as determined by BCA assay (Thermo Scientific, Cat. no. 23225) ) of the proteins were applied to the gel for Western Blot analysis. Bands were visualized using Opti-4CN Substrate Kit (Bio-Rad, Cat. no. 1708235) .

Antibodies used in the study:

- anti- EGFR antibody (Santa Cruz Biotechnology, Cat. no. Sc-373746) , dilution 1:500

- anti-Actin antibody, clone C4 (Sigma- Aldrich, Cat. no. MAB1501) , dilution 1:4000

- anti-mouse IgG-HRP (Santa Cruz Biotechnology, Cat. no. sc-516102) , dilution 1:4000

Results and conclusions :

It was demonstrated that the median effective dose (ED50) of 2-DG on human neoplastic DK-MG glioblastoma cells ranges from 0.1 mM to 1 mM) . The median effective dose (ED50) of 2-DG on human NCI-H1975 lung cancer cells ranges from 0.25 mM to 1 mM (exemplary results on Figure 22) .

Figure 22. Analysis of 2-DG on protein glycosylation in DK-MG glioblastoma and NCI-H1975 lung cancer cell lines. The median effective dose (ED50) of 2- DG on EGFR in human neoplastic DK-MG glioblastoma cells ranges from 0.1 mM to 1 mM. The median effective dose (ED50) of 2-DG on EGFR in human NCI-H1975 lung cancer cells ranges from 0.25 mM to 1 mM. The expected effect is visible as an improper band corresponding to improperly folding protein due to glycosylation inhibition. "Ctrl" means that cells were not exposed to 2-DG.

Other cancer cell lines were tested in vitro and a summary of the results of exposing them to 2-DG is as follows: In some embodiments, the dose-response relationship of 2-DG on blocking of glycosylation of proteins in cancer cells is observed in the concentration range in particular: for NCI-H1975 adenocarcinoma of the lung the IC50 range from 0.5 mM to 5 mM and the ED50 proteins glycosylation range from 0.25 mM to 1 mM. These data show that 2-DG is toxic for lung cancer cells and inhibits glycosylation of lung cancer cells proteins.

For DK-MG glioblastoma cells the IC50 range from 1.0 mM to 5 mM and ED50 proteins glycosylation range from 0.1 mM t 1 mM. These data show that 2-DG is toxic for glioblastoma cells and inhibits glycosylation of glioblastoma cells proteins.

For SW48 Adenocarcinoma (Colorectal; Dukes' type C, grade IV) the IC50 range from 0.5 mM to 5 mM and the ED50 proteins glycosylation range from 0.1 mM to 1 mM. These data show that 2-DG is toxic for colorectal cancer cells and inhibits glycosylation of colorectal cancer cells proteins .

For SW962 carcinoma (derived from metastatic site: lymph node; cancer primary position was vulva) the IC50 range from 0.5 mM to 5 mM and ED50 proteins glycosylation range from 0.25 mM to 1.5 mM. These data show that 2-DG is toxic for vulva cancer cells and inhibits glycosylation of vulva cancer cells proteins .

For NCI-H460 non-small-cell lung cancer the IC50 range from 0.25 mM to 2.5 mM and the ED50 proteins glycosylation range from 0.1 mM to 2.25 mM. These data show that 2-DG is toxic for non-small-cell lung cancer cells and inhibits glycosylation of non-small-cell lung cancer cells proteins . For U87-MG glioblastoma the IC50 range from 0.75 to 5 mM and the ED 50 proteins glycosylation range from 0.5 mM to 0.5 mM. These data show that 2-DG is toxic for glioblastoma cells and inhibits glycosylation of glioblastoma cells proteins.

For MCF7 breast cancer the IC50 range from 0.5 mM to 5 mM and the ED50 proteins glycosylation range from 0.1 mM to 1 mM. These data show that 2-DG is toxic for breast cancer cells and inhibits glycosylation of breast cancer cells proteins .

For RPMI 8402 T-cell acute lymphoblastic leukemia the IC50 range from 0.75 mM to 5 mM and the ED50 proteins glycosylation 0.5 mM to 5 mM. These data show that 2-DG is toxic for lymphoblastic leukemia cells and inhibits glycosylation of lymphoblastic leukemia cells proteins .

For HT 1080 fibrosarcoma the IC50 range from 0.25 mM to 5 mM and the ED50 proteins glycosylation range from 0.25 mM to 1 mM. These data show that 2-DG is toxic for fibrosarcoma cells and inhibits glycosylation of fibrosarcoma cells proteins.

For MDA-MB-468 breast cancer the IC50 range from 0.5 mM to 5 mM and the ED50 proteins glycosylation range from 0.75 mM-1.5 mM. These data show that 2-DG is toxic for breast cancer cells and inhibits glycosylation of breast cancer cells proteins.

For HeLa epithelioid cervix carcinoma the IC50 range from 0.75 mM to 5 mM and the ED50 proteins glycosylation range from 1 mM to 2.5 mM. These data show that 2-DG is toxic for cervix cancer cells and inhibits glycosylation of cervix cancer cells proteins. For SW1417 human colon adenocarcinoma the IC50 range from 0.5 mM to 5 mM and the ED50 proteins glycosylation range from 0.25 mM to 1 mM. These data show that 2-DG is toxic for adenocarcinoma cells and inhibits glycosylation of adenocarcinoma cells proteins.

Example 39. Performance of 2DG release study in 0.9% NaCl

Prepared liposomes were used in the study. To 300 pl of the liposome suspension 200 pl of 0.9% NaCl solution was added. The whole mixture was mixed and applied to a minicolumn filled with Sephadex G50 fine gel. The whole liposomes sample was eluted with 600 pl of NaCl solution.

Liposomes were diluted to 1000 pl and incubated at 37 °C. From the mixture, 110 pl samples were collected 3x each and 100 pl were separated from the released substance at time t=0, and after 1, 3, 6, 12, 24 and 36 hours. 2DG content was determined in the collected samples by comparing the determined amounts with the unseparated sample (100% 2DG) .

The leakage results are shown in Figure 23.

Figure 23 shows the release of an encapsulated 2-DG variant in 0,9% NaCl in H2O, measured in units of days or weeks. This graph shows that the liposomal encapsulation retards the release of 2-DG.

Further description is provided as follows with respect to the third aspect of the invention to provide a natural preparation of 2-DG with natural minerals for use as a health supplement. This is a natural and not a synthesized formulation comprising only natural ingredients . The natural preparation comprises 2-DG in a form and a concentration as described herein in addition to at least NaCl and Selenium. The minerals and 2-DG are in natural mineral water to form the natural preparation. Preferably the 2-DG is dissolved in the natural mineral water .

Concentrations of NaCl in the natural preparation are preferably between approximately 0.009 mg/ml (0.9% solution) , though the natural preparation may have between approximately 0.5% to 1.5% solution of NaCl.

Concentrations of Selenium in the natural preparation will preferably provide the subject taking the natural preparation with at least 30% of their daily selenium requirement following the prescribed daily dosage of natural preparation. Where each dosage is to be administered orally to a subject three times a day, each dosage of natural preparation will comprise between approximately 5% to 15%, and more preferably approximately 10% of the subject's daily selenium requirement. The daily selenium intake for an adult human is approximately 55 micrograms (0.7 micromolar) /day. Therefore 10% (per dose of natural preparation) will be approximately 5.5 micrograms selenium per dose (i.e. in 1 ml of natural mineral water solution of the natural preparation) . Therefore, selenium can be in a 1 ml dose of the natural preparation at between approximately 4 pg/ml to 8 pg/ml, and more preferably approximately at 5.5 pg/ml.

The natural preparation may also comprise other natural minerals including one or more of those selected from the group comprising: sulfur, calcium, potassium, oxygen, and other natural ingredients under 1 g/kg including those that may be present in the natural mineral water. Concentrations of sulfur in the natural preparation may be between approximately 5 g/kg to 30 g/kg, and more preferably approximately 12 g/kg. Concentrations of calcium in the natural preparation may be between approximately 2 g/kg to 6 g/kg, and more preferably approximately 4 g/kg. Concentrations of potassium in the natural preparation may be between approximately 2 g/kg to 6 g/kg, and more preferably approximately 3.5 g/kg. Concentrations of dissolved oxygen in the watery natural preparation may be between approximately 0.05 g/kg to 0.014 g/kg, and more preferably approximately 0.012 g/kg.

Per 1 ml dose of natural preparation, the natural preparation may also comprise any one or more of the following minerals:

- Amounts of bicarbonates in the natural preparation may be between approximately 0.2 mg to 0.5 mg, and more preferably approximately 0.35 mg.

- Amounts of calcium in the natural preparation may be between approximately 0.05 mg to 0.2 mg, and more preferably approximately 0.1 mg.

- Amounts of magnesium in the natural preparation may be between approximately 0.015 mg to 0.045 mg, and more preferably approximately 0.03 mg.

- Amounts of sulphates in the natural preparation may be between approximately 0.005 mg to 0.02 mg, and more preferably approximately 0.01 mg.

- Amounts of chlorides in the natural preparation may be between approximately 0.005 mg to 0.02 mg, and more preferably approximately 0.01 mg.

- Amounts of silica in the natural preparation may be between approximately 0.01 mg to 0.02 mg, and more preferably approximately 0.015 mg.

- Amounts of potassium in the natural preparation may be between approximately 0.005 mg to 0.02 mg, and more preferably approximately 0.01 mg. - other ingredients under 0.01 mg include sodium, nitrates, fluorides, and selenium.

Glucose or the 2-DG already present can act as a preservative for the natural preparation.

2-DG is at a concentration of between approximately 0.05 mg and 0.5 mg, and more preferably approximately 0.15 mg or as much as 10 mg per 1 ml dosage of natural preparation.

The formulation for the natural preparation is preferably taken by a subject using a nebulizer. It is preferred that the oral dose is taken by the subject three times a day.

For use in delivery by a nebulizer, approximately 1 ml of formulation is nebulized to create aerosol .

Approximately 50% of particles of 2-DG had a size smaller than 5 micrometers. This is important as particle sizes larger than 5 micrometers will not make it through the subject's lungs to the lower regions of the lungs. Less than 3 micrometers is more preferred.

Therefore, use of an oral formulation of 2-DG was shown to be beneficial for subjects tested on. When used with a nebulizer, 45 of 50 patients with respiratory type flu symptoms reported an improvement of their wellbeing (measured subjectively) . The natural preparation is for supporting the health and increasing the immunity of the subject's body. Reference symbols and numerals

1 Device

2 discharge noz zle

3 particles

4 container

5 substance

6 actuator

7 flow channel

8 dosage valve

10 particle

11 carrier material

12 active ingredient

13 shell

100 method

110 method step

120 method step

130 method step

140 method step

Below some of the above and further embodiments , in particular of the aspects of the application, are further defined in the following list of embodiment items .

Item 1 . 2 -Deoxy-D-Glucose ( 2-DG) for use in a medical method in a subj ect , wherein 2-DG is provided as a micron or a submicron particle in a preparation, wherein in particular said micron or submicron particle is a mechanically micronized particle or is a micronized particle obtained by spray drying, or 2-DG is provided as a preparation in a liposomal or a proliposomal formulation, in particular obtained by spray drying .

Item 2 . 2 -DG according to item 1 , wherein the preparation comprises an amount of 2-DG in a range of between approximately 1% and 75% w/w of the total weight of the preparation, in particular an amount of 2-DG in a range with lower limit of approximately 10% or 20% or 30% and an upper limit of approximately 35% or 45% to 55% w/w of the total weight of the preparation, in particular between approximately 10% and 40% w/w or between approximately 15 % w/w and 30% w/w of the total weight of the preparation .

Item 3 . 2 -DG according to item 1 or item 2 , wherein the preparation comprises one or more further excipients selected from the group of excipients comprising :

- an amino acid, in particular leucine or glycine , in particular in an amount of 0% w/w up to approximately 80% w/w of the total weight of the preparation, more particular in an amount of approximately 10% w/w up to approximately 80% w/w, more particular in an amount of approximately 10% w/w up to 50% w/w, more particular in an amount of approximately 10% w/w up to 30% w/w;

- trehalose in an amount of 0% w/w up to approximately 60 % w/w of the total weight of the preparation, more particular in an amount of approximately 5% w/w up to 30% w/w;

- mannitol , 0% w/w up to 60% w/w of the total weight of the preparation, more particular in an amount of 5 % w/w up to 30% w/w;

-propylene glycol or/and, glycerol , ethyl alcohol in the concentration range from 10 to 80% of the total weight of the liquid preparation;

- one or more further phospholipid, in particular a natural or a semi-synthetic phospholipid, one or more further negatively or a positively charged phospholipid, in particular in an amount of approximately 1% up to 10% of the molar % of the phospholipid fraction, more particular in an amount of approximately 5 molar % up to 10 molar % , wherein in particular the one or more further phospholipid is in particular selected from the group comprising phosphatidylglycerol , dimiristoyl phosphatidylglycerol , dipalmitoylphosphatidylglycerol , hydrogenated soybean phosphatidylcholine ( HSPC ) , soybean phosphatidylcholine ( SPG ) and wherein optionally the phospholipids comprises DPPE or DSPE with covalently attached hydrophilic polymer , in particular a PEG or polyglycerol in a molar ratio of 0 to approximately 10 molar % of the total lipid fraction, more particular in an amount of approximately 5 molar % of total lipid fraction;

- sterol , in particular cholesterol in an amount of 0 molar % up to approximately 55 molar % of the total the lipid fraction, more particular in an amount of approximately 30 molar % up to 45 molar % ;

- antioxidant , in particular ascorbyl palmitate (AP ) in an amount of 0 molar % to 10 molar % of total lipid fraction

- nicotinic acid amide , in an amount of approximately 10% w/w up to 80% w/w of the total weight of the preparation, more particular in an amount of approximately 20 to 60% w/w of the preparation; and

- urea in an amount of approximately 20% w/w up to 80% w/w of the total weight of the preparation, more particular in an amount of approximately 40 to 60% w/w of the preparation .

Item 4 . 2 -DG according to any one of the preceding items , wherein the preparation further comprises an excipient comprising a lipid fraction comprising or consisting of a phospholipid fraction in an amount of approximately 5% to 80% w/w, in particular approximately 15% to 50% w/w of the total weight of the preparation .

Item 5. 2-DG according to any one of the preceding items, wherein:

- liposome sizes range from approximately 30 nm to 200 nm in particular for intravenous delivery;

- liposome sizes range from approximately 50 nm to 5 pm, in particular for pulmonary delivery;

- unilamellar liposomes sizes range from approximately 30 to 120 nm, in particular for intravenous delivery.

Item 6. 2-DG according to any one of the preceding items, wherein the amount of 2-DG in the liposomal or proliposomal formulation is in a range of approximately 1 mg or 10 mg to 1500 mg, in particular approximately 50 mg to 500 mg, in particular, approximately 100-200 mg per unit dosage.

Item 7. 2-DG according to any one of the preceding items, wherein 2-DG is provided as a preparation for administration by inhalation, wherein the preparation comprises particles for inhalation with a diameter of approximately 10 pm or less, in particular less than approximately 5 pm, 3 pm, 1 pm, 0.3 pm or 0.1 pm, more particular particles with a diameter in a range with a lower limit between approximately 0.1 pm and 1 pm and with an upper limit between approximately 0.5 and 5 pm.

Item 8. 2-DG according to any one of the preceding items, for use in a substance for inhalation, wherein the substance is provided in the form of aerosol particles or powder particles, and wherein the particles comprise the 2-DG.

Item 9. 2-DG according to any one of the preceding items, for use in a device for inhaling a substance in the form of aerosol particles or powder particles, the device comprising: - a discharge nozzle for dispensing the substance in the form of aerosol particles or powder particles ,

- a container for receiving and keeping the substance , and

- an actuator for activating the device , the actuator being configured to release a certain amount or dose of the substance kept in the container for conveying the substance through the discharge nozzle of the device , wherein the particles comprise the 2-DG .

Item 10 . 2-DG according to any one of the preceding items , further comprising at least one of ribavirin, emetine , or NMS-873 .

Item 11 . 2-DG according to any one of the preceding items , for slow release in a subj ect .

Item 12 . 2-DG according to item 11 , wherein the slow release is provided such that the concentration of the 2-DG remains constant or even increases over time .

Item 13 . 2-DG according to item 11 or item 12 , wherein the slow release is provided over a time period that is measured in units of hours or days .

Item 14 . 2-DG according to any one of the preceding items , wherein the amount of 2 -DG in the liposomal or proliposomal formulation is in a range of approximately 1 mg or 10 mg to 1500 mg, in particular approximately 50 mg to 500 mg, in particular , approximately 100-200 mg per unit dosage .

Item 15 . 2-DG according to any one of the preceding items , for use in a substance for intravenous application into a body of a subj ect , wherein the substance is provided in the form of a liquid wherein the liquid comprises the 2-DG

Item 16 . 2-DG according to any one of the preceding items , wherein the subj ect comprises an animal or a human .

Item 17 . 2-DG according to any one of items 1 to 16 , for use in a medical method to prevent and/or to treat a viral infection in a subj ect by a virus comprising a spike protein and/or to treat cancer or other enveloped glycosylated protein, wherein 2 -DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of the spike protein or other enveloped glycosylated protein .

Item 18 . 2-DG for use in a medical method according to item 17 , wherein the effective tissue concentration inhibits at least 30% , in particular at least 50% , 70% , 80% 90% , 95% or 99% of the glycosylation the spike protein or other enveloped glycosylated protein .

Item 19 . 2-DG for use in a medical method according to item 17 or item 18 , wherein the virus comprises one or more of a Coronavirus , Influenza A, B, or C , or Dengue virus .

Item 20 . 2-DG for use in a medical method according to any one of items 17 to 19 , wherein the viral spike protein comprises a SARS-CoV-2 spike protein or an Influenza hemagglutinin spike protein or the viral glycosylated protein comprises an enveloped glycoprotein E from Dengue virus .

Item 21 . 2-DG for use in a medical method according to any one of items 17 to 20 , wherein the viral infection has developed into the viral disease comprising one or more of Covid-19 , Influenza, or Dengue fever .

Item 22 . 2-DG according to any one of items 1 to 16 , for use in a medical method to treat and/or prevent cancer in a subj ect , wherein 2-DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of cancer cell proteins of cancer cells in the subj ect . Item 23 . 2-DG according to any one of items 17 to 22 , wherein the effective tissue concentration of 2-DG is in a range of between approximately 0 . 1 mM to 25 mM, and up to 100 mM .

Item 24 . 2-DG according to any one of items 17 to 22 , wherein the tissue comprises respiratory tissue , monocytes or keratinocyte cells .

Item 25 . process for preparing a proliposome- or liposome-encapsulated pharmaceutical composition comprising 2-DG for use in a medical method to prevent and/or to treat a viral infection in a subj ect by a virus comprising a spike protein or other enveloped glycosylated proteins , wherein 2 -DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of the spike protein or other enveloped glycosylated proteins .

Item 26 . process for preparing a proliposome- or liposome-encapsulated pharmaceutical composition comprising 2-DG for use in a medical method to treat and/or to prevent cancer in a subj ect , wherein 2-DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of cancer cell proteins of cancer cells in the subj ect .

Item 27 . A method of manufacturing a proliposome- or liposome-encapsulated pharmaceutical composition comprising 2-DG for use in a medical method to prevent and/or to treat a viral infection in a subj ect by a virus comprising a spike protein or other enveloped glycosylated proteins , wherein 2 -DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of the spike protein or other enveloped glycosylated proteins . Item 28 . method of manufacturing a proliposome- or liposome-encapsulated pharmaceutical composition comprising 2-DG for use in a medical method to treat and/or to prevent cancer in a subj ect , wherein 2-DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of cancer cell proteins of cancer cells in the subj ect .

Item 29 . method of manufacturing a proliposome- or liposome-encapsulated pharmaceutical composition comprising 2-DG for slow release in a subj ect , wherein 2-DG is provided as a preparation in an amount and a formulation to tissue of a subj ect that results in an effective tissue concentration to partially or completely inhibit glycosylation of a spike protein or other enveloped glycosylated protein of a virus in the subj ect , or inhibit glycosylation of cancer cell proteins of cancer cells in the subj ect .

Item 30 . A method for applying 2 -DG according to any one of items 1 to 16 , for slow release in a sub j ect .

Item 31 . A method for inhibition of glycosylation of receptors or other glycoproteins on normal cells or cancer cells of a subj ect , or a virus that has infected the subj ect , the method comprising introducing 2-DG according to any one of items 1 to 16 into the subj ect .

Item 32 . A method according to item 31 , wherein the inhibition of glycosylation comprises preventing binding of a glycoprotein on a virus to a receptor on the surface of a host cell and thereby preventing fusion of viral and host cell membranes .

Item 33 . A method according to item 32 , wherein the glycoprotein on the virus comprises a spike protein and the receptor comprises an ACE2 receptor . Item 34. A method according to item 31 or item 32, for treating viral diseases, in particular Influenza caused by Influenza A, B or C viruses, in particular H1N1, or in the treatment of Dengue fever caused by Dengue virus, and the prevention of Influenza A, B or C or Dengue virus infection of eukaryotic cells.

Item 35. Preparation for use in a medical method, particularly to prevent a viral infection in a subject, wherein the preparation comprises 2-DG and natural minerals, including at least NaCl and Selenium, and natural mineral water, the 2-DG being dissolved in the natural mineral water.

Item 36. Preparation according to item 35, wherein concentrations of NaCl in the natural preparation are between approximately 0.5% to 1.5% solution of NaCl, in particular 0.009 mg/ml (0.9% solution) .

Item 37. Preparation according to item 35 or item 36, wherein concentrations of Selenium will provide a subject taking the natural preparation with at least 30% of their daily selenium requirement following the prescribed daily dosage of natural preparation.

Item 38. Method of dispensing a substance, the method comprising:

- providing the substance in the form of aerosol particles, creating an aerosol with the particles suspended in the aerosol, creating a directed flow of aerosol such that the suspended particles move essentially along the flow direction of the aerosol, wherein the particles comprise at least one active ingredient or active agent out of the group comprising ribavirin, emetine, 2-DG and NMS-873, and directing the directed flow or jet of the aerosol towards target areas of the human body for dispensing the substance.

Item 39. Use of a pharmaceutical composition comprising at least one active ingredient selected from the group comprising: lomustine, emetine, 2-DG, NMS-873 and ascorbate, for the treatment of a cancer in a sub j ect .

Item 40. Use according to item 39, wherein the active ingredient comprises 2-DG and at least one selected from the group comprising: lomustine, emetine, and NMS-873.

Item 41. Use of a pharmaceutical composition comprising at least one active ingredient selected from the group comprising: lomustine, 2-DG and ascorbate, in the manufacture of a medicament for the prevention and/or treatment of cancer in a subject.

Item 42. Use according to item 41, wherein the active ingredient comprises 2-DG and at least one selected from the group comprising lomustine and ascorbate .

Item 43. Use of a pharmaceutical composition comprising 2-DG in the manufacture of a medicament for the prevention and/or treatment of cancer in a subject in combination with lomustine or/and ascorbate.