Claims:WE CLAIM
1. A pharmaceutical composition administered by inhalation comprising aqueous chlorine solution;
wherein subjecting the individual to 1 to 6 cycles of intermittent inhalation of composition;
wherein, each cycle of intermittent inhalation comprises, inhalation of chlorine of at least 0.2 mg to 6 mg.
2. The pharmaceutical composition as claimed in claim 1, wherein time period of inhalation for each cycle ranges from 5 to 30 minutes.
3. The pharmaceutical composition as claimed in claim 1, wherein individual is subjected to intermittent inhalation of chlorine of at least 1 to 6 cycles in a day for at least 1 to 3 days.
4. The pharmaceutical composition as claimed in claim 1, wherein during a day concentration of chlorine in the inhalation ranges from at least 4 to 16 mg for a day.
5. The pharmaceutical composition as claimed in claim 1, wherein chlorine solution prepared by dissolving an effective amount of chlorine in purified water and optionally adding stabilizing agent.
6. The pharmaceutical composition as claimed in claim 1, wherein concentration ratio of chlorine in water is ranging from 0.02 mg/ml to 0.6 mg/ml.
7. The pharmaceutical composition as claimed in claim 1, wherein subjecting the individual for 15 min inhalation of 1 mg/10 ml of the aqueous chlorine solution.
8. The pharmaceutical composition as claimed in claim 1, wherein composition is administered by inhalation via nose inhaler or mouthpiece as gas, mixture of gas, liquid, mist, spray, aerosol or steam.
9. The pharmaceutical composition as claimed in claim 1, wherein composition is further administered by dilution with medium; wherein dilution medium is selected form group consisting of compressed air, inert carrier gas or oxygen or mixture thereof.
10. The pharmaceutical composition as claimed in claim 1, wherein subjecting an individual to intermittent inhalation of chlorine for the treatment of respiratory viral infection.
11. The pharmaceutical composition as claimed in claim 10, wherein respiratory infection associated with the viruses are selected from a group consisting of coronavirus (COVID), avian virus, swine virus, respiratory syncytial virus (RSV), a rhinovirus, an enterovirus, an influenza A virus, an influenza B virus, a parainfluenza 1 virus, a parainfluenza 2 virus, a parainfluenza 3 virus, a bocavirus, a human metapneumo virus, SARS Virus, and an adenovirus.
12. The pharmaceutical composition as claimed in claim 10, wherein respiratory viral infection associated with COVID.

, Description:THIS APPLICATION IS A PATENT OF ADDITION TO THE INDIAN PATENT APPLICATION NUMBER 202041017016 FILED ON APRIL 21, 2020
FIELD OF THE INVENTION
The present invention relates to an inhalational composition for treatment of respiratory viral infection. More particularly, the invention relates to the pharmaceutical composition of chlorine administered by inhalation for treatment of respiratory viral infection. The present invention also provides a method for the management of respiratory viral infection associated with COVID and viral morbidities by administering lower and effective concentration of chlorine by inhalation.
BACKGROUND OF THE INVENTION
The virus, technically named SARS-CoV-2, is a newly identified virus, but it is the seventh coronavirus known to infect humans. Scientists are trying hard to find drugs to treat COVID-19. According to research there are more than 30 agents including western medicines, natural products and traditional Chinese medicines that may have potential efficacy against COVID-19. However, a number of medicines have been suggested as potential investigational therapies, many of which are now being or will soon be studied in clinical trials, including the solidarity trial co-sponsored by WHO and participating countries. Several drugs such as chloroquine, arbidol, remdesivir, and favipiravir are currently undergoing clinical studies to test their efficacy and safety in the treatment of coronavirus disease 2019 (covid-19). Crucially, enveloped viruses are easier to kill: SARS-CoV-2, the virus responsible for the COVID-19 outbreak, is an enveloped virus and therefore the easiest to kill.
It is discovered that each disinfectant has totally different effects, attacking one or several of the virus’ functions. Even though the outcome is the same, the eradication methods are different. Ozone, ultraviolet irradiation, liquid chlorine, chlorine dioxide, and sodium hypochlorite disinfections are commonly used technologies for disinfection. Using the correct disinfectant is an important part of preventing and reducing the spread of illnesses. Isopropanol or Ethanol (Alcohol), Quaternary Ammonium Compounds, Sodium Hypochlorite (Bleach), Hydrogen Peroxide are some common disinfectants which kills viruses, bacteria as well as fungi. Chlorine is a kind of strong oxidizer, which is one of the most early used disinfection methods. WHO recommended use of chlorine to make inactive COVID-19 for treatment of drinking water and swimming pools.
US6333054 discloses topical hydrogel disinfectant composition comprising active chlorine.
Various non-patent literatures disclosed the Effects of a Low Concentration hypochlorous acid (HOCl) in the treatment of various diseases. Hyun Jik Kim et al (Laryngoscope 118: October 2008) disclose Effects of a Low Concentration hypochlorous acid nasal irrigation solution on bacteria, fungi and viruses. This literature disclosed the virucidal effects of HOCl and used the human influenza a virus to challenge the cells.
The treatment for COVID-19 is symptomatic because there is no specific drug or cure for it. It is essential to develop effective, economical, user friendly method of treating COVID-19.
Thus, the inventors of the present invention have successfully addressed the existing drawbacks and formulated pharmaceutical composition of chlorine for treatment of viral respiratory infection by inhalation.
OBJECTIVE OF THE INVENTION
An object of the present disclosure is to provide a pharmaceutical composition of chlorine for treatment of respiratory viral infection by inhalation.
Another object of the present disclosure is to provide a pharmaceutical composition comprising aqueous solution of chlorine to treat COVID-19.
Another object of the present disclosure is to provide an effective formulation of chlorine for treatment of respiratory viral infection by inhalation.
Another object of the present disclosure is to provide pharmaceutical composition with lower concentration of chlorine for treatment of respiratory viral infection.
Yet another object of the present disclosure is to provide a method for administration of the pharmaceutical composition by inhalation for treatment of respiratory viral infection.
SUMMARY OF THE INVENTION
The present disclosure provides a liquid composition of chlorine for treatment of respiratory viral infection and method thereof.
In one aspect the present invention provides the pharmaceutical composition administered by inhalation, comprising chlorine solution;
wherein subjecting the individual to 1 to 6 cycles of intermittent inhalation of composition;
wherein, each cycle of intermittent inhalation comprises of continuous inhalation of available chlorine of at least 0.2 mg to 6 mg.
According to an embodiment, the present invention provides pharmaceutical composition wherein the concentration of available chlorine is in the range of 0.2 mg to 6 mg.
According to an aspect of some embodiments the present invention provides pharmaceutical composition wherein time period of inhalation cycle ranges from 5 to 30 minutes. Particularly, time period of inhalation cycle ranges from 10 to 25 minutes. More particularly, time period of inhalation cycle ranges from 15 to 20 minutes.
According to an embodiment, the individual is subjected to intermittent inhalation of pharmaceutical composition for atleast 1 to 6 days. Particularly 1 to 4 days. More particularly, 1 to 3 days.
According to another embodiment the present invention provides the pharmaceutical composition wherein individual is subjected to intermittent inhalation for atleast 1 to 6 days and during a day concentration of chlorine in the continuous inhalation ranges from atleast 4 to 7 mg for a day. Particularly, 5 mg to 6 mg for a day; more particularly, 6 mg for a day.
In another aspect the present invention provides the pharmaceutical composition comprising an effective amount of chlorine dissolved in an inactive ingredient and optionally a stabilizing agent.
In another embodiment the present invention provides the composition; wherein said chlorine is chlorine gas or chlorine releasing compound.
In another embodiment, the chlorine releasing compounds are selected from mixture of calcium hypochlorite with the salt of sodium, potassium, ammonia; wherein salt of sodium, potassium, and ammonia are sodium sulphate, potassium sulphate and ammonium sulphate.
Yet according to another embodiment, said inactive ingredient is selected from sterile water, saturated edible oil, saturated neuzoil oil, saline water, glycols and mixture thereof. More particularly, said inactive ingredient is sterile water.
Yet according to another embodiment, the present invention provides the pharmaceutical composition comprising chlorine solution prepared by dissolving an effective amount of chlorine in purified water and optionally adding stabilizing agent.
In another embodiment the present invention provides the composition wherein said stabilizing agent is selected from alkali salt of mineral acids, saline water, sodium sulphate, sodium carbonate, sodium bicarbonate, potassium sulphate, potassium chloride, boric acid and mixture thereof. The concentration of said stabilizing agent is in the range of 0.1 to 10%.
In another embodiment the present invention provides the composition; wherein pH of solution is in the range of 1 to 8.
According to an embodiment, the present invention provides pharmaceutical composition wherein the concentration of chlorine in water is ranging from 0.02mg/ml to 0.6mg/ml; both inclusive. In another embodiment concentration of chlorine in water is ranging from 0.2 mg/10ml to 6 mg/10ml; both inclusive. In another embodiment concentration of chlorine in water is ranging from 0.5 mg/10ml to 1.5 mg/10ml. In another embodiment concentration of chlorine in water is ranging from 1 mg/10ml to 6 mg/10ml.
In another embodiment the pharmaceutical composition as described hereinabove is further administered by diluting with medium. The dilution medium is selected form group consisting of compressed air, inert carrier gas or oxygen or mixture thereof.
The composition described in present invention is further administered to a subject requiring oxygen support for the treatment of respiratory viral infection. More particularly, the pharmaceutical composition of the present invention can also be administered by inhalation to the subject having oxygen support.
In accordance with another aspect the invention provides a process of preparing aqueous chlorine solution for inhalation, comprising of the following steps:
(a) Dissolving chlorine into inactive ingredient;
(b) Adding stabilizing agent to solution of step (a);
(c) Sterilizing by filtration method and
(d) Filling in pre-sterilized vials or added into respule or ampules and sealed.
According to another embodiment, the invention provides a process of preparing aqueous chlorine solution for inhalation, wherein the sterilization of step (c) is carried out through 0.22 µ filter.

According to another embodiment, the invention provides a process of preparing aqueous chlorine solution for inhalation, wherein pre-sterilized vials comprising aqueous chlorine solution, aseptically packed in amber colour glass vial. The invention also provides pre-sterilize ampules or respules.
In accordance with another aspect the invention provided a method of treating a respiratory viral infection which includes administrating aqueous chlorine solution described hereinabove by inhalation.
According to another embodiment, the invention provides method of treating respiratory viral infection by subjecting the individual for 15 min inhalation of 1 mg/10 ml of the aqueous chlorine solution.
In accordance with another aspect the invention provides the method of treating a respiratory viral infection by subjecting an individual to intermittent continuous inhalation of chlorine for predetermined time period.
In accordance with another embodiment, the virus is selected from a group consisting of coronavirus (COVID), avian virus, swine virus, respiratory syncytial virus (RSV), a rhinovirus, an enterovirus, an influenza A virus, an influenza B virus, a parainfluenza 1 virus, a parainfluenza 2 virus, a parainfluenza 3 virus, a bocavirus, a human metapneumovirus, SARS Virus, and an adenovirus.
According to a further embodiment, the virus is coronavirus; more particularly COVID-19.
According to another embodiment, the present invention provides a pharmaceutical composition used for treating or preventing respiratory viral infection associated with an upper respiratory condition. When the infection is associated with an upper respiratory condition or lower respiratory tract infection including lung tissue; the composition of present invention is preferably administered to the upper airway, e.g., as a spray, mist, aerosol or steam. Numerous inhalation delivery systems have been developed and studied to treat respiratory viral infection. All are encompassed in the scope of present invention.
According to another embodiment, the invention provides the pharmaceutical composition administered by inhalation as nose inhaler or mouthpiece as gas, mixture of gas, liquid, mist, spray, aerosol or steam.
According to another embodiment, for the application of the present invention any suitable device may be used to disperse chlorine, but not limited to, spray bottle, aerosol, humidifiers, misters, foggers, vaporizers, atomizers, water sprays, and other devices.
While several embodiments have been described in detail, it will be apparent to those skilled in the art that the disclosed embodiments may be modified. Therefore, the foregoing description is to be considered exemplary rather than limiting.
BRIEF DESCRIPTION OF THE DRAWING
The following figures are illustrative of particular examples for enabling embodiments of devices and methods of the present disclosure, are descriptive of some of the embodiments and are not intended to limit the scope of the disclosure. The figures are not to scale (unless so stated) and are intended for use in conjunction with the explanations in the following detailed description. Wherever applicable, the words and phrases used herein should be understood and interpreted to have a meaning consistent with the understanding of those words and phrases by those skilled in the relevant art. Persons skilled in the art will appreciate that elements in the figures are illustrated for simplicity and clarity and may have not been drawn to scale. For example, the dimensions of some of the elements in the figure may be exaggerated relative to other elements to help to improve understanding of various exemplary embodiments of the present disclosure. Throughout the drawings, it should be noted that like reference numbers are used to depict the same or similar elements, features, and structures.
Figure 1: Illustrates mechanism of action of liquid composition disclosed in the present invention.
Figure 2: Illustrates the (A) LifeViroTreat unit vial,(B) method of application by nebulizer and (C) LifeViroTreat pilot batch for clinical testing.
DESCRIPTION
In the description that follows, a number of terms are used, the following definitions are provided to facilitate understanding of various aspects of the disclosure. Use of examples in the specification, including examples of terms, is for illustrative purposes only and is not intended to limit the scope and meaning of the embodiments of the invention herein. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to,” and the word “comprises” has a corresponding meaning.
The terms and words used in the following description are not limited to the bibliographical meanings, but, are merely used to enable a clear and consistent understanding of the disclosure. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present disclosure are provided for illustration purpose only and not for the purpose of limiting the disclosure as defined by the appended claims and their equivalents.
Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.
The therapeutically effective amount administered to the individual e.g., a mammal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic or prophylactic response in the individual over a reasonable time frame. The dose can be readily determined using methods that are well known in the art. One skilled in the art will recognize that the specific dosage level for any particular individual will depend upon a variety of potentially therapeutically relevant factors.
The term "subject" includes mammals, which can alternatively be used as ‘Individual’ (especially humans) and other animals, such as domestic animals (e.g., household pets including cats and dogs) and non-domestic animals (such as wildlife).
The term "Treating" or "treatment" of a disease includes (1) preventing the disease from occurring in subject that may be predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e. arresting its development, or (3) relieving the disease, i.e. causing regression of the disease.
Viral infections may include for example infections by respiratory viruses, including but not limited to, various types of coronavirus (COVID), avian virus, swine virus, respiratory syncytial virus (RSV), a rhinovirus, an enterovirus, an influenza A virus, an influenza B virus, a parainfluenza 1 virus, a parainfluenza 2 virus, a parainfluenza 3 virus, a bocavirus, a human metapneumovirus, SARS Virus, and an adenovirus.
“Breathing air” is a term used to loosely describe the extent to which air in a local environment (ambient air) is sufficiently to be safe to breathe (Rajagopal Kannan et al, IJISET, Vol. 2 Issue 4, April 2015).
Chlorine group comprises aqueous solution of chlorine, hypochlorite, or hypochlorous acid. Occasionally, chlorine-releasing compounds and their salts are included in this group. Frequently, a concentration of < 1 ppm of available chlorine is sufficient to kill bacteria and viruses, spores. Acute Exposure Guideline Levels (AEGLs) set levels of chemical concentration that pose a defined level of risk to humans. The AEGL values are determined for varying times of exposure, such as ten minutes, thirty minutes, one hour, four hours and eight hours. The AEGL values describe the expected effects of inhalation exposure to certain compounds (airborne concentrations in ppm or mg/m3). Each AEGL is determined by different levels of a compound's toxicological effects, based on the 4 Ds: detection, discomfort, disability and death. There are three levels of AEGL-values: AEGL-1, AEGL-2 and AEGL-3. AEGL-1 is the airborne concentration above which notable discomfort or irritation could be experienced. However, the effects are not disabling and reversible once exposure stops. AEGL-2 is the airborne concentration above which irreversible or other serious, long-lasting adverse health effects or an impaired ability to escape could be experienced. AEGL-3 is the airborne concentration above which life-threatening health effects or death could be experienced.
The present invention is further described by reference to the following examples, which are illustrative only and not limiting of the claimed invention.
(I) Examples
Following Examples 1 to 6 describe the process of preparing pharmaceutical composition (LifeViroTreat)
Example-1: Preparation of Ampules
The chlorine gas (1 mg) is dissolved in the WFI grade water (10 ml) in presence of stabilizers to form a chlorine gas solution at 0-10°C.
Example-2: Preparation of Respule
The chlorine gas (1.5mg) is dissolved in the WFI grade water (10 ml) in presence of stabilizers to form a chlorine gas solution at 0-10°C.
Example-3: Preparation of Respule
The chlorine gas (1gm) is dissolved in the WFI grade water (1000 ml) in presence of stabilizers to form a chlorine gas solution at 0-10°C. Then chlorine gas solution is diluted into respule to make 2 mg dosage form.
Example-4: Preparation of Respule
The chlorine gas (0.5gm) is dissolved in the WFI grade water (1000 ml) in presence of stabilizers to form a chlorine gas solution at 0-10°C. Then chlorine gas solution is added into respule to make 5 mg dosage form.
Example-5: Preparation of Respule
The chlorine gas (1.5gm) is dissolved in the WFI grade water (1000 ml) in presence of stabilizers to form a chlorine gas solution at 0-10°C. Then chlorine gas solution is added into respule to make 8 mg dosage form.
Example-6: Preparation of Vials
Aqueous chlorine solution is prepared by simple solubilization method. An amount of 1000 mg chlorine dissolved in 1000 mL of triple distilled water along with stabilizer and stirred for 15 minutes to get a uniform solution. Aqueous chlorine solution is sterilized by filtration method and passed through 0.22µ filter. The sterilized aqueous chlorine solution filled in pre-sterilized vials and aseptically sealed with closure (Figure 2 A). Filled vials are further packed in primary followed by secondary carton and stored at USP cool temperature.
(II) Stability studies
Stability studies were carried out. The composition described hereinabove was stored at 2 to 8° C in amber color glass container and stable for 1 year.
(III) Safety and toxicological studies
Preclinical studies of Lifevirotreat composition were studied to determine the safety of the composition. The Clinical trials studies of the present invention are carried out by Panexell clinical lab Pvt ltd.
Pre-clinical studies were performed by administering a multiple inhalation doses to rodent rats and non-rodents rabbits (Table 1), followed by observation period of 14 days, 3 female animals per dose level were used in this study as described in the OECD guidelines for testing of chemicals. Animals were kept in animal restrainer and connected with nebulizer. Nebulizer chamber was filled before starting the experiment with aqueous chlorine formulation (1 mg/m3) for 15 minutes (LifeViroTreat, 10 ml glass vial). Animals were exposed to the aqueous chlorine formulation for 15 minutes and for 6 and 12 times in a day. Animals were assessed for their mortality and morbidity. After blood collection animals were sacrificed and subjected to gross necropsy, which included careful examination of the external surface of body, all orifices, cranial, thoracic and abdominal cavities and their contents. Once gross necropsy is done the organs like brain, lung, liver, heart, adrenal gland, kidney, uterus and ovaries were surgically removed, weighed and stored. Lung tissues were studied for further histopathological studies.
Table 1 Experimental detail for preforming safety and toxicological studies for LifeViroTreat Composition
AnimalModel Dose Frequency of dose No. of Animals Sex Route of administration

Rat Air control - 3

Female

Inhalation through Nebulizer

5Times
(4.18mg/kg) LD2
(Every 4 hour for 3 days)
3
LD4
(Every 2 hour for 3 days)
3

10 Times
(8.37mg/kg) HD2
(Every 4 hour for 5 days)

3
HD4
(Every 2 hour for 3 days)
3

Rabbit
Air Control -
3

Female

Inhalation through Nebulizer

5 Times
(2.09mg/kg) LD2
(Every 4 hour for 3 days)
3
LD4
(Every 2 hour for 3 days)
3

10 Times
(4.19mg/kg) HD2
(Every four hour for 5 days)
3
HD4
(Every four hour for 5 days)

3
Moreover, after the exposure period on 14th day biochemical parameters (Table 2 and table 3) and hematological (Table 4 and Table 5) were investigated. Animals were evaluated for anxiety, motor activity and other behavioral changes.
Table 2 Effects of LifeViroTreat composition by inhalation route on some biochemical parameters in the acute toxicity study in rats

PARAMETERS Treatment Groups Reference
Values
Control Group 5 Times
(4.18mg/kg) 10 Times
(8.37mg/kg)
Air Control LD2
(Every 4 hour for 3 days) LD4
(Every 2 hour for 3 days) HD2
(Every 4 hour
For 3 days) HD4
(Every 2 hour for 3 days)
ALB(g/dL) 3.82± 0.27 3.93± 0.21 3.81±0.31 3.67± 0.19 3.42±0.44 3.4– 4.8
ALP(U/L) 217.33±18.15 230.00 ± 50.74 209.33± 14.72 178.33± 33.61 162.67 ± 7.02 62-237
ALT(U/L) 54.86 ± 7.29 45.90±12.64 52.80±4.00 53.50± 4.93 51.00±1.25 18-76
AST(U/L) 95.63 ± 12.47 100.07 ± 15.59 125.47± 20.10 116.67± 12.15 102.97 ± 17.39 74-143
BIL (mg/dL) 0.10± 0.00 0.10± 0.00 0.10±0.00 0.10± 0.00 0.10±0.00 0.05-0.15
CA(mg/dL) 10.38 ± 0.61 10.93±0.67 10.00±1.75 9.23± 1.10 9.17±2.11 9.5-11.5
CHO(mg/dL) 51.10 ± 6.27 59.77±2.67 55.23±6.82 44.83± 5.14 50.87±4.35 37-85
CREJ(mg/dL) 0.36± 0.05 0.32± 0.02 0.30±0.01 0.32± 0.07 0.31±0.02 0.2-0.5
PHOS(mg/dL) 6.54± 1.40 7.57 ± 1.58 7.07 ± 1.72 7.03± 1.59 8.33±1.56 5.58-10.41
TP(g/dL) 6.30± 0.54 6.64 ±0.84 7.04± 0.29 6.81± 0.45 6.67±0.06 5.2-7.1
UREA(mg/dL) 15.60 ± 1.62 17.97± 3.47 20.40± 1.21 19.10 ± 4.20 22.17 ± 2.35 12.3-24.6
GLU(mg/dL) 144.33±15.28 133.00± 10.58 132.33±14.64 120.00± 12.77 142.33± 17.24 70-208
Note: Each value represents the mean± standard deviation (n=3)

Table 3 Effects of LifeViroTreat composition by inhalation route on some biochemical parameters in the acute toxicity study in rabbits

PARAMETERS Treatment Groups Reference Values
Control Group 5 Times
(4.18mg/kg) 10Times
(8.37mg/kg)
Only Air Group I (Every 4 hour for 3 days) Group II (Every 2 hour for 3 days) Group III
(Every 4 hour for 3 days) Group IV (Every 2 hour for 3 days)
ALB (g/dL) 34.03 ± 6.09 3.93± 0.05 3.81±0.31 3.67±0.19 3.42 ±0.44 23-49
ALP(U/L) 204.44±17.64 196.67±102.02 259.33±32.52 278.33± 33.61 229.33± 51.47 44-402
ALT(U/L) 56.15 ± 7.29 45.90±12.64 52.80±4.00 53.50±4.93 51.00± 1.25 34-146
AST (U/L) 22.36 ± 4.63 138.40±18.40* 107.37± 5.41 106.70 ±1.23 103.97± 2.10 7.5-39.7
BIL(mg/dL) 0.10± 0.00 0.10± 0.00 0.10±0.00 0.10±0.00 0.10 ±0.00 0.019-0.2
CA (mg/dL) 10.90 ± 1.99 5.93±0.67 5.00±0.26 5.57± 0.45 5.00 ±0.17 9.22-13.63
CHO (mg/dL) 53.46 ± 5.04 59.77±2.67 55.23±6.82 44.83±5.14 50.87± 4.35 30-65
CREJ(mg/dL) 0.81± 0.22 0.32± 0.02 0.30±0.01 0.32±0.07 0.31 ±0.02 0.5-2.6
PHOS(mg/dL) 5.12± 0.13 6.23± 0.06 5.03±0.40 5.37±0.49 4.67 ±0.49 4-6
TP(g/dL) 60.62 ± 8.64 7.09± 0.21 7.04±0.29 6.81±0.45 6.67 ±0.06 49-79
UREA (mg/dL) 48.04 ± 9.41 34.63±3.61 34.07±4.70 33.17±2.37 33.50± 0.75 25.48-71.41
GLU(mg/dL) 150.67±11.06 136.67±9.28 143.33±13.01# 132.33 ±11.59 140.00 ±11.00 89-150
Note: Each value are presents the mean ± standard deviation (n=3)
ALB2:Albumin,ALP:Alkalinephosphatase, ALT: Alanine Aminotransferase, AST: Aspartate Aminotransferase,BILT2: Total Bilirubin,CA2: Calcium,CHOL:Cholesterol,CREJ:Creatinine,PHOS:Phosphorous,TP:Total protein, UREL: Urea level, GLU: Glucose

Table 4 Effects of LifeViroTreat composition by inhalation route on some hematological markers in the acute toxicity study in rats

PARAMETERS Treatment Groups Reference Values
Control Group 5 Times
(4.18mg/kg) 10 Times
(8.37mg/kg)
Only Air LD2
(Every 4 hour
For 3 days) LD4
(Every 2 hour
For 3 days) HD2
(Every 4 hour
For 3 days) HD4
(Every 2 hour
For 3 days)
WBC
(x10E03cells/µL) 6.62 ± 1.45 5.24± 1.26 7.30 ± 1.15 6.99 ± 1.71 6.14 ± 2.07 1.13-7.49
RBC
(x10E06cells/µL) 7.72 ± 1.12 8.28± 0.40 7.63 ± 0.49 7.56 ± 0.18 7.15 ± 1.24 7.07-9.03
HGB(g/dL) 14.59±1.08 15.13± 1.31 13.13± 1.44 14.23± 2.16 16.00± 1.71 13.7-16.8
HCT (%) 42.00± 3.61 42.17± 3.70 39.03± 0.75 36.07± 0.05 37.90± 2.62 35-52
MCV (fL) 55.89± 4.06 50.83± 2.23 50.43± 2.50 54.33± 4.71 54.67± 5.11 49.90-60.1
MCH(pg) 18.83± 3.55 18.40± 2.16 18.49±1.22 17.90± 2.15 17.80± 1.37 17.8-20.9
MCHC(g/dL) 35.78± 1.77 34.73± 6.79 34.47± 6.91 35.20± 6.63 35.13± 5.37 32.7-37.9
RDW(%) 12.60± 0.64 13.43± 1.11 13.70± 1.15 13.43± 1.96 14.30± 1.50 10.5-14.9
PLT(x10E03 cells/µL) 781.33±105.64 710.33± 86.59 871.00± 24.98 891.67±59.53 796.33± 79.43 680-1200
MPV(fL) 8.01 ± 1.07 9.57± 1.17 9.37 ± 0.61 8.40 ± 2.13 9.57 ± 1.46 6.2-9.8
%NEUT(%) 19.27± 4.33 13.87± 4.78 10.67± 0.64 12.67± 4.69 15.03± 3.51 7.1-33.2
%LYM(%) 77.00±11.19 78.30± 3.32 86.07± 2.79 81.87± 5.42 79.60± 6.91 62.2-90
%MONO(%) 2.69 ± 0.51 4.47± 0.15 2.90 ± 0.72 3.07 ± 0.21 4.77 ± 0.21 0.8-5.9
%EOS(%) 2.42 ± 0.52 1.50± 0.36 1.57 ± 0.29 0.80 ± 0.10 1.57 ± 1.24 0.4-4.5
%BASO(%)
0.16 ± 0.06 0.43± 0.06 0.60± 0.35 0.45 ±00.02 0.43 ± 0.15 0-0.8

Note: Each value represents the mean ± standard deviation(n=3)

Table 5 Effects of LifeViroTreat composition by inhalation route on some hematological markers in the acute toxicity study in rabbits

PARAMETERS Treatment Groups Reference Values
Control Group 5 Times
(4.18mg/kg) 10 Times
(8.37mg/kg)
Only Air LD2
(Every 4 hour
For 3 days) LD4
(Every 2 hour
for3 days) HD2
(Every 4 hour for 3 days) HD4
(Every 2 hour
For 3 days)
WBC(x10E03
cells/µL) 11.64± 1.16 11.13 ±0.80 9.87 ±0.61 11.12± 1.39 11.33 ± 0.88 5.2-16.5
RBC(x10E06
cells/µL) 5.59 ± 0.88 5.18± 0.54 5.79 ±0.69 5.03 ±0.33 4.93 ± 0.21 3.7-7.5
HGB(g/dL) 8.56 ± 0.79 8.47± 1.27 6.07 ±0.57 8.10 ±1.65 8.7 ± 0.15 7.8-15.4
HCT (%) 30.14± 1.98 28.03 ±2.81 29.37± 0.87 27.30± 1.78 26.73 ± 1.05 26.7-47.2
MCV(fL) 53.60± 5.50 54.13 ±0.25 53.57± 1.01 54.30± 0.00 57.60 ± 5.63 55.0-79.6
MCH(pg) 24.93± 4.41 22.50 ±2.40 24.17 ±4092 23.53 ±4.26 23.87 ± 6.80 19.2-29.5
MCHC (g/dL) 27.39± 3.51 28.77± 4.58 25.13 ±4.03 25.77 ±3.20 26.50 ± 3.58 25.52-37
RDW (%) 13.78± 1.25 15.80± 3.46 14.47 ±4.57 15.27± 0.70 13.10 ± 3.72 11.5-16.2
PLT (x10E03
cells/µL) 683.00± 95.77 609.33±59.68 723.33 ±87.51 764.67 ±68.42 746.67 ± 83.27 112-795
MPV (fL) 6.60 ± 0.62 6.13± 0.95 7.37 ±0.38 6.47 ±1.36 6.83 ± 1.32 5.2-9.9
%NEUT(%) 50.16± 8.12 46.63 ±1.00 49.40± 1.32 53.20 ±4.17 50.13 ± 2.87 21-73
%LYM(%) 45.88± 5.45 42.40 ±3.87 39.03± 0.72 44.93 ±4.57 40.53 ± 8.12 9-64
%MONO(%) 9.19 ± 1.59 9.57± 1.50 8.73 ±1.33 7.37 ±1.90 2.60 ± 0.61 1-15
%EOS(%) 0.45 ± 0.25 0.57 ±0.23 0.47 ±0.29 0.27 ±0.25 0.23 ± 0.23 0.0-0.7
%BASO(%) 8.27 ± 0.55 6.73± 2.40 6.33 ±2.52 4.33 ±1.55 6.33 ±2.31 0.5-9
Note: Each value represents the mean ± standard deviation(n=3)

WBC: White blood cells, LY: Lymphocyte, MON: Monocytes, GRAN: Granulocytes, HGB: Hemoglobin, HCT: Hematocrit, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, RDW: Red blood cell distribution width, PLT: Platelets, MPV: Mean platelet volume,

Table 6 Summary of Mortality & Morbidity; Clinical signs and Gross pathology
Day Animal Model Dose
conc Frequency of dose Animal No. Clinical Signs Mortality & Morbidity Day of
Necropsy Gross Pathology

1st Day

Rat Air Control -
1
2
3
NAD

Nil

14th Day

NAD

5 Times
(4.18mg/kg) LD2
(Every 4 hour for 3 days) 1
2
3

NAD

Nil 14th Day

NAD
LD4
Every 2 hour for 3 days 1
2
3

NAD
Nil 14th Day
NAD

10 Times
(8.37mg/kg) HD2
Every four hour for 5 days 1
2
3 All animals were in somnolence condition with decreased motor activity following test item administration on day 0. Nil 14th Day NAD
HD4
(Every 2 hour for 3 days)

1st Day

Rabbit
Air Control

- 1
2
3

NAD
Nil 14th Day
NAD

5 Times
(2.09mg/kg) LD2
Every 4 hour for 3 days

1
2
3

NAD
Nil 14th Day
NAD
LD4
Every 2 hour for 3 days 1
2
3

NAD
Nil 14th Day
NAD

10 Times
(4.19mg/kg) HD2
Every four hour for 5 days 1
2
3 All animals were in somnolence condition with decreased motor activity following test item administration on day 0.

Nil 14th Day

NAD

HD4
Every four hour for 5 days
NAD-No Abnormality Detected;

Table 7 Organ weight of each rat
Group AnimalNo. Organ Weight(gm.)
Liver Lung Heart Kidney Brain Pancreas Spleen Ovaries
Right Left

Air Control 1 12.55 2.21 0.91 0.99 0.95 1.92 1.35 1.42 0.094
2 13.34 1.79 1.08 1.08 1.14 1.87 1.37 1.74 0.087
3 17.36 2.11 1.12 1.28 1.23 2.05 1.15 1.30 0.109
LD2
(Every 4 hour
For 3 days) 1 11.78 2.03 1.01 1.03 1.01 1.93 1.03 1.86 0.078
2 15.64 2.90 1.30 1.16 1.28 1.96 0.97 2.03 0.092
3 17.49 2.64 1.27 1.30 1.24 1.97 0.86 1.34 0.108
LD4
Every 2 hour
For 3 days 1 12.25 1.96 0.90 1.09 1.10 1.88 1.76 1.66 0.096
2 14.83 2.67 1.39 1.15 1.22 2.75 2.42 2.90 0.088
3 17.46 2.63 1.29 1.25 1.22 1.81 1.28 1.07 0.100
HD2
Every four hour for 5days 1 11.61 2.65 0.92 0.96 1.06 1.80 1.16 1.10 0.074
2 11.63 2.03 1.02 1.17 1.04 1.77 1.60 1.14 0.080
3 11.90 2.02 1.00 0.94 0.92 1.98 1.19 1.71 0.078
HD4
(Every 2 hour
For 5 days) 1 12.56 2.03 1.02 0.96 1.11 1.84 1.21 1.69 0.089
2 11.61 2.4 1.14 1.12 1.06 1.81 1.18 1.72 0.075
3 13.9 2.8 0.98 1.14 1.04 2.10 1.36 1.80 0.083

Table 8 Organ weight of each rabbit

Group Animal No. Organ Weight(gm.)
Liver Lung Heart Kidney Brain Pancreas Spleen Ovaries
Right Left

Air Control 1 45.65 9.02 4.91 4.82 4.73 6.48 1.06 0.46 0.094
2 53.34 7.68 4.08 6.30 6.84 5.81 0.95 0.44 0.087
3 47.36 9.52 3.98 5.83 5.33 6.0 1.15 0.49 0.109
LD2
(Every 4 hour
For 3 days) 1 61.78 8.03 4.56 4.35 4.66 5.78 0.83 0.51 0.078
2 55.64 7.10 5.10 5.16 5.28 4.75 0.97 0.48 0.092
3 57.49 9.24 4.87 4.49 4.86 4.97 0.92 0.54 0.108
LD4
Every 2 hour
For 3 days 1 52.25 8.46 3.90 4.97 5.20 5.06 1.01 0.48 0.096
2 64.83 7.89 4.39 6.15 6.22 6.66 0.84 0.50 0.088
3 67.46 7.47 5.29 5.54 5.20 4.78 0.89 0.57 0.100
HD2
Every four hour for 5 days 1 61.61 8.73 6.02 5.89 6.02 5.77 0.90 0.51 0.074
2 51.63 9.09 5,40 5.17 5.90 4.36 0.84 0.44 0.080
3 50.90 7.04 3.90 4.94 4.81 5.02 1.01 0.47 0.078
HD4
(Every 2 hour
For 5 days) 1 52.56 9.02 4.47 5.96 6.11 4.78 0.91 0.49 0.089
2 61.61 7.6 5.14 5.12 5.06 6.18 1.06 0.57 0.075
3 53.9 8.7 4.98 6.14 6.04 5.10 0.98 0.41 0.083

No mortality and morbidity were observed in any of the animals administered with the test item (Table 6). All the values were in normal range as per reference values and no significant variation was observed in any parameter in all treated group as compared to normal control(Air control) Group. In all the steps, animals were in somnolence condition with decreased motor activity. None of the animals exhibited gross lesions related to test item administration. Increase in body weight was observed in all the treated animals at the end of the experiment. Based on the results obtained, it is concluded that, LifeViroTreat at 5 Times (4.18 mg/kg) and 10 Times (8.37 mg/kg) in rat and 5 Times (2.09 mg/kg), 10 Times (4.19 mg/kg) in rabbit was found to be safe.