FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. Title of the Invention
DRY POWDER INHALATION (DPI) FORMULATION
2. Applicant(s)
Name Nationality Address
Sava Research Centre, 17/6 Block D1,
SAVA HEALTHCARE LTD Indian MIDC Chinchwad, Pune-411019,
Maharashtra, India
3. Preamble to the description
The following specification particularly describes the invention and the manner in which it is to be performed

FIELD
The present disclosure relates to a dry powder inhalation (DPI) formulation.
DEFINITIONS
As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which they are used indicates otherwise.
D10 refers to the portion of particles with diameters below the specified value is 10%.
D50 refers to the portion of particles with diameters smaller and larger than a specified value are 50%. Also known as the median diameter.
D90 refers to the portion of particles with diameters below the specified value is 90%.
MMAD (mass median aerodynamic diameter) refers to the diameter at which 50% of the particles of an aerosol by mass are larger and 50% are smaller.
FPF (Fine-particle fraction) refers to a fraction wherein the fine particle dose is divided by the total emitted dose.
Vero cells refer to mammalian cell lines derived from the kidney of an African green monkey extensively used in virology studies and other applications.
First pass metabolism refers to a phenomenon in which a drug gets metabolized at a specific location in the body that results in a reduced concentration of the active drug upon reaching its site of action or the systemic circulation.
Binding free energy refers to the sum of all the intermolecular interactions that is present between the ligand and the target.

BACKGROUND
The background information hereinbelow relates to the present disclosure but is not necessarily prior art.
Ivermectin is an FDA-approved broad spectrum anti-parasitic drug found to be active against various parasite infestations including head lice, scabies, river blindness, strongyloidiasis, trichuriasis, ascariasis, heartworm and lymphatic filariasis. It also shows anti-viral activity against a broad range of viruses like Dengue, West Nile, Yellow fever and Zika. The drug inhibits the viral replication by inhibiting the human immunodeficiency virus (HIV-1) via the integrase enzyme, through inhibition of nonstructural protein 5, a polymerase for viral RNA synthesis and regulator for immune signaling of Dengue virus, and via DNA polymerase inhibition for herpesvirus, Yellow fever virus, Dengue virus and West Nile virus via nonstructural protein 3 (a DNA helicase enzyme).
Considering the COVID pandemic, absence of any approved medication for effective management and treatment, Ivermectin was preferred based on the available safety and efficacy details. Conventionally available oral formulations for COVID-19 requires a high oral dosage leading to poor compliance of the patients. Further, the oral administration of Ivermectin is also associated with adverse reactions that include severe episodes of confusion, ataxia, seizures, coma and hypotension including nausea, vomiting, diarrhea, an asymptomatic increase of blood uric acid and transaminases, a decrease in the neutrophil counts, and increased levels of liver enzymes such as ALT and AST.
There is, therefore, felt a need to develop a formulation that overcomes the above-mentioned drawbacks.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.

It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
An object of the present disclosure is to provide a dry powder inhalation (DPI) formulation.
Another object of the present disclosure is to provide a dry powder inhalation formulation of Ivermectin.
An object of the present disclosure is to provide a dry powder inhalation formulation that avoids the first-pass metabolism.
Another object of the present disclosure is to provide a dry powder inhalation formulation that exerts equivalent/enhanced efficacy at a reduced dose.
Still another object of the present disclosure is to provide a simple process for the preparation of a dry powder inhalation formulation.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure relates to a dry powder inhalation (DPI) formulation and a process for its preparation.
In an aspect, the dry powder inhalation formulation comprises a micronized Ivermectin having a particle size in the range of 0.1 µm to 5 µm; a first lactose having a particle size in the range of 10 um to 200 urn; a second lactose having a particle size in the range of 1 µm to 150 µm, and optionally at least one excipient. The mass ratio of the first lactose to the second lactose is in the range of 5:1 to 15:1.
In another aspect, the process for the preparation of dry powder inhalation formulation comprises blending of a first lactose and a second lactose to obtain a lactose mixture. The lactose mixture was sifted through 80 mesh to obtain a

homogeneous lactose mixture. The homogeneous lactose mixture is divided into two portions i.e. a first portion (~75%) and a second portion (~25%). At least one additive is mixed with the first portion of the homogenous lactose mixture and blended to obtain a first resultant mixture. Separately, a predetermined amount of Ivermectin is mixed with the second portion of the homogenous lactose mixture to obtain a mixture and the mixture is sifted through 80 mesh to obtain a second resultant mixture. The first resultant mixture and the second resultant mixture are blended at a speed in the range of 10 rpm to 30 rpm for a time period in the range of 10 minutes to 60 minutes to obtain the dry powder inhalation (DPI) formulation. The so obtained dry powder inhalation (DPI) formulation is loaded into size 3 HPMC capsules by using a capsule filler.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
The present disclosure will now be described with the help of the accompanying drawing, in which:
Figure 1A illustrates a graph depicting cumulative (% undersize) particle size distribution of the dry powder inhalation (DPI) formulation of Ivermectin 0.375 mg in accordance with the present disclosure;
Figure 1B illustrates a graph depicting drug deposition of the dry powder inhalation (DPI) formulation of Ivermectin 0.375 mg in accordance with the present disclosure;
Figure 1C illustrates a graph depicting cumulative (% undersize) particle size distribution of the dry powder inhalation (DPI) formulation of Ivermectin 1.7 mg in accordance with the present disclosure;
Figure 1D illustrates a graph depicting drug deposition of the dry powder inhalation (DPI) formulation of Ivermectin 1.7 mg in accordance with the present disclosure;

Figure 2A illustrates a molecular docking of Ivermectin B1a binding with hexokinase I along with 3D model of the interactions and the 2D interaction patterns with H-bond interaction;
Figure 2B illustrates a molecular docking of Ivermectin B1b binding with hexokinase I along with 3D model of the interactions and the 2D interaction patterns without H-bond interaction;
Figure 2C illustrates a molecular docking of Ivermectin B1a binding with hexokinase II (PDB: 2NZT) along with 3D model of the interactions and the 2D interaction patterns with H-bond interaction;
Figure 2D illustrates a molecular docking of Ivermectin B1b binding with hexokinase II (PDB: 2NZT) along with 3D model of the interactions and the 2D interaction patterns with H-bond interaction;
Figure 2E illustrates a molecular docking of 2-Deoxy-D-Glucose (2DG) binding with hexokinase I (PDB ID: 1CZA) along with 3D model of the interactions and the 2D interaction patterns with H-bond interaction;
Figure 2F illustrates a molecular docking of 2-Deoxy-D-Glucose (2DG) binding with hexokinase II (PDB: 2NZT) along with 3D model of the interactions and the 2D interaction patterns with H-bond interaction;
Figure 3 illustrates a graphical representation of percentage hexokinase inhibition activity of Ivermectin against control and 2-deoxy-2 glucose (2DG);
Figure 4 illustrates a molecular docking of mitogen-activated protein kinase 1 binding with Ivermectin shows 3D model of the interactions and the 2D interaction patterns and H-bond interaction and ligand interaction with distance and without distance;
Figure 4A illustrates a molecular docking of cellular tumor antigen P53 binding with Ivermectin shows 3D model of the interactions and the 2D interaction

patterns and H-bond interaction and ligand interaction with distance and without distance; and
Figure 4B illustrates a molecular docking of RAC-alpha serine/threonine-protein kinase binding with Ivermectin shows 3D model of the interactions and the 2D interaction patterns and H-bond interaction and ligand interaction with distance and without distance.
DETAILED DESCRIPTION
Embodiments, of the present disclosure, will now be described with reference to the accompanying drawing.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details, are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure is not to be construed as necessarily

requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
Considering the COVID pandemic, absence of any approved medication for effective management and treatment, Ivermectin was preferred based on the available safety and efficacy details. Conventionally available oral formulations for COVID-19 requires a high oral dosage leading to poor compliance of the patients. Further, the oral administration of Ivermectin is also associated with adverse reactions that include mild to moderate diarrhea, an asymptomatic increase of blood uric acid and transaminases, a decrease in the neutrophil counts, and increased levels of liver enzymes such as ALT and AST.
The present disclosure provided a dry powder inhalation (DPI) formulation and a process for its preparation.
In an aspect of the present disclosure, the dry powder inhalation (DPI) formulation comprises:
• a micronized Ivermectin having a particle size in the range of 0.1 µm to 5 µm;
• a first lactose having a particle size in the range of 10 µm to 200 µm;
• a second lactose having a particle size in the range of 1 µm to 150 µm; and
• optionally at least one excipient.
In an embodiment, the micronized Ivermectin is in an amount in the range of 1 mass% to 15 mass% with respect to the total mass of the formulation.
In an embodiment, the first lactose is in an amount in the range of 70 mass% to 90 mass% with respect to the total mass of the formulation.
In an embodiment, the mean particle size (d10) of the first lactose is in the range of 10 µm to 50µm.

In an embodiment, the mean particle size (d50) of the first lactose is in the range of 51 µm to 120 µm.
In an embodiment, the mean particle size (d90) of the first lactose is in the range of 121 µm to 200 µm.
In an embodiment, the second lactose is in an amount in the range of 5 mass% to 15 mass% with respect to the total mass of the formulation.
In an embodiment, the mean particle size (d10) of the second lactose is in the range of 1 µm to 30 µm.
In an embodiment, the mean particle size (d50) of the second lactose is in the range of 31 µm to 90 µm.
In an embodiment, the mean particle size (d90) of the second lactose is in the range of 91 µm to 150 µm.
In an embodiment, the mass ratio of the first lactose to the second lactose is in the range of 5:1 to 15:1. In the exemplary embodiment of the present disclosure, the mass ratio of first lactose to second lactose is 9.1:1.
In an embodiment, the MMAD of the dry powder inhalation is in the range of 1.5 µm to 2.5 µm.
In an embodiment, the excipient is in an amount in the range of 1 mass% to 5 mass% with respect to the total mass of the formulation.
In an embodiment, the excipient is selected from citric acid monohydrate, magnesium stearate.

In another aspect of the present disclosure, there is provided a process for the preparation of the dry powder inhalation formulation.
The process is described in detail.
In a first step, a first lactose and a second lactose are blended to obtain the lactose mixture. The lactose mixture is sifted through 80 mesh in a controlled temperature and humidity conditions to obtain a homogeneous lactose mixture. The homogeneous lactose mixture is divided into two portions i.e. a first portion (~75%) and a second portion (~25%).
In a second step, additive is mixed with the first portion of the homogenous lactose mixture and blended to obtain a first resultant mixture.
In a third step, separately, Ivermectin is mixed with the second portion of the homogenous lactose mixture to obtain a mixture, the mixture is sifted through 80 mesh size to obtain the second resultant mixture.
In a fourth step, the first resultant mixture and the second resultant mixture are blended at a speed of 10 rpm to 30 rpm for a time period in the range of 10 minutes to 60 minutes to obtain the dry powder inhalation (DPI) formulation.
The so obtained dry powder inhalation (DPI) formulation is loaded into size 3 HPMC (Hydroxypropyl methylcellulose) capsules using a capsule filler.
The present disclosure provides the dry powder inhalation formulation that avoids the first-pass metabolism. The formulation of the present disclosure is therapeutically effective at a reduced dose and also observed increased patient compliance. Further, the dry powder inhalation formulation of the present disclosure has been evaluated for its toxicity on rats. It is observed that the No Observed Effect Level (NOEL) of Ivermectin is >0.27 mg a.i./kg b.wt. when administered through inhalation for up to 2 weeks.

The dose of Ivermectin as a DPI is almost 7 fold less than the oral dose; hence the use of Ivermectin as a DPI is beneficial to avoid such allergic reactions and side effects observed with oral treatment.
In accordance with the embodiment of the present disclosure, the formulation of the present disclosure is used for in the treatment of cancer, bacterial infection, viral infection, fungal infection and autoimmune diseases.
In a preferred embodiment of the present disclosure, the formulation of the present disclosure is used for in the treatment of lung cancer, COVID-19 infection and rheumatoid arthritis.
The foregoing description of the embodiments has been provided for purposes of illustration and is not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment, but, are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure.
The present disclosure is further described in light of the following experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure. The following experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to industrial scale.

EXPERIMENTAL DETAILS
Example 1: Preparation of DPI formulation Dry mixing:
21 mg of a first lactose (respitose SV003) and 2.3 mg of a second lactose (respitose ML006) were mixed using double lined polybags and sifted using 80 mesh size to obtain a homogeneous lactose mixture. The homogeneous lactose mixture was divided into two portions i.e. a first portion (~75%) and a second portion (~25%). 0.25 mg of citric acid monohydrate was added to the first portion of the homogenous lactose mixture and blended to obtain a first resultant mixture. Separately, 1.7 mg of Ivermectin (particle size of NMT 5.0 microns) was mixed with the second portion of the homogenous lactose mixture to obtain a mixture and the mixture was sifted through 80 mesh to obtain a second resultant mixture. The first resultant mixture and the second resultant mixture are transferred to a double cone blender and blended at a speed of 30 rpm for a time period in the range of 45 minutes to obtain the dry powder inhalation (DPI) formulation of Ivermectin (Formulation 1). The so obtained dry powder inhalation (DPI) formulation of Ivermectin was loaded into size 3 HPMC capsules using a capsule filler.

Example 2:
The dry powder inhalation formulations were prepared in the similar manner as disclosed in Example 1, by varying the concentration of ingredients according to the formulations as illustrated in Table 1.
Table 1:

Ingredient (mg) Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7
Ivermectin (micronized) 1.7 1.7 1.7 1.7 1.7 1.7
First lactose (respitose SV003) 20.5 21 2.3 -- 23.05 2.3
Second lactose (respitose ML006) 2.3 2.3 20.75 23.05 -- 20.75
Citric acid monohydrate 0.50 -- 0.25 0.25 0.25 0.25
Magnesium stearate -- -- -- -- -- 0.08
Total (mg) 25.00 25.00 25.00 25.00 25.00 25.00

Example 3: Ivermectin - Stability Study
The stability conditions of Ivermectin are illustrated below in Tables 2-3.
Table 2:

Stability Result
Product: Ivermectin Powder for Inhalation 1.7 mg
Batch Number : 1147-01
Batch Size : 3000 capsules
Date In : 10/7/2020 Tests
Description Water Content Fine Particle Deposition Assay Related Substance
Limit *** T0 T1 T2 Total % Max.
Imp. (NMT
1 %) Total Imp. (NMT 5.0 %)
Pack 1: HDPE bottle Initial *** 4.89 T0: 12.3 T1: 40.2 T2: 32.4 Total 84.9 92.20% 0.71% 3.75%

1M/40/75 *** 4.95 T0: 13.8 T1:46.5 T2:39.4 Not performed 96.6% 0.691% 3.619%

3M/40/75 *** 5.29 T0: 9.9 T1:41.3 T2:36.7 Total 87.9 93.7% 0.721% 3.546%

3M/30/75 *** 5.28 T0: 9.3 T1: 38.9 T2: 41.4 Total 89.6 98.1% 0.717% 3.532%

3M/25/60 *** 5.31 T0: 9.2 T1: 38.8 T2: 41.4 Total 89.4 99.5% 0.724% 3.550%

6M/40/75 *** 4.98 T0: 10.8 T1: 50.9 T2: 23 Total 84.7 93.0% 0.69% 3.74%

6M/30/75 *** 4.98 T0: 12.3 T1: 40.2 T2: 32.5 Total 85.0 92.40% 0.71% 3.71%

6M/25/60 *** 4.99 T0: 12.3 T1: 40.1 T2: 32.6 Total 89.4 95.50% 0.71% 3.74%
*** Description: Wh ite to off white powder filled i n size '3' ca psule with green colo ured body a nd cap.

Table 3:

Stability Result
Product Name : Ivermectin Powder for Inhalation 0.375 mg
Batch Number 1177-04
Batch Size
Date In 1 :2-01-20 Tests
Description Water Content Fine Particle Deposition (by NGI / T1T2) Unit dose delivered (DUSA) Assay NGI
(Fine Particle
Fraction) Related Substance
Pack Limit *** NMT 7.0 Device Induction
port
+Mouthpie Pre Seperator Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Stage 6 Stage 7 MOC Total 65-135% 90-110% NLT 25% % Max. Imp. (NMT Total Imp. (NMT
PP Rolex bottle size "3"
HPMC red / trans capsules 1M 40/75 *** 5.25 6.1 8.8 38.9 4.3 5.3 7.50% 12.00% 7.70% 2.70% 1.10% 0.30% 94.70% Min:104.5 % Max:108.5 % Avg: 106.1 % % RSD: 1.30 93.6% 38.56% 0.791% 4.864%

1M 30/75 *** 5.2 Not Performed 97.3% Not 0.792% 4.801%

1M 25/60 *** 5.44 Not Performed 94.1% Not 0.765% 4.632%

1M 2-8°C *** 5.24 4.80% 6.90% 36.00% 4.80% 5.60% 8.30% 14.10% 10.10% 4.00% 1.30% 0.30% 96.20% Min:91.7 % Max:115.5 % Avg: 105.3 %
% RSD: 8.04 95.2% 44.91% 0.795% 4.896%

3M 2-8°C *** 5.04 Not Performed 94.9% Not 0.752% 4.598%

3M 40/75 *** 5.11 5.30% 9.30% 41.90% 5.10% 5.60% 7.20% 10.70% 6.40% 2.40% 1.10% 0.50% 95.50% Min:94.9 % Max:102.7 % Avg: 98.5 % % RSD: 3.11 96.5% 34.60% 0.756% 4.747%

3M 30/75 *** 5.16 5.60% 8.50% 38.70% 4.50% 5.60% 7.50% 12.80% 8.80% 3.50% 1.30% 0.50% 97.30% Min:93.9 % Max:111.7 % Avg: 99.7 % % RSD: 5.38 96.3% 40.85% 0.741% 4.549%

3M 25/60 *** 5.23 5.30% 8.50% 38.10% 4.50% 5.30% 7.50% 12.80% 8.80% 3.50% 1.30% 0.50% 96.10% Min:94.1 % Max:109.1 % Avg: 99.5 % % RSD: 4.73 97.6% 40.87% 0.753% 4.590%

T0 T1 T2 Total NA NA

6M 2-8°C *** 5.08 11 60.7 27.4 99.1
93.9
0.72% 4.25%

6M 40/75 *** 5.2 11.7 57.9 17.5 87.1
84.3
0.732 4.664

6M 30/75 *** 5.18 11 60 27.2 98.9
94.9
0.741 4.498

6M 25/60 *** 5.16 11.1 60.8 27.3 99.2
93.3
0.71% 4.23%

Inference: The dry powder inhalation (DPI) formulation of Ivermectin in capsules of the present disclosure was subjected to 3 and 6 months accelerated stability testing at 25°C/60% RH and 40°C/75% RH respectively. It is evident from tables 2-3 that Ivermectin (API) is stable for a longer period of time, at 25°C/60% RH, and 40°C/75% RH conditions as there was no change in the Ivermectin content in dry powder inhalation (DPI) formulations of Ivermectin 0.375 mg and Ivermectin 1.7 mg.
The so obtained dry powder inhalation formulation was filled into a size 3 HPMC capsule using a capsule filler.

Example 4: In-vitro Lung Deposition Study
The Aerodynamic Particle Size Distribution (APSD), delivered dose, are the critical quality attributes for the in vitro characterization of orally inhaled drug products (OINDPs).
The APSD of an aerosol determines the portion of DPI particles that deposit in the body especially in the lower respiratory tract. The particles in the range of 1 to 5 microns reach the lower respiratory tract are considered effective, particles with larger than 5 microns will remain in the upper respiratory tract and are likely to impact the oropharynx and be swallowed, particles smaller than 1 micron will be cleared by lungs clearance mechanism.
Dry powder inhalation (DPI) formulation of Ivermectin 0.375 mg was evaluated for Aerodynamic Particle Size Distribution (APSD). The APSD from each dose of the dry powder inhalation formulation of the present disclosure was evaluated using Next Generation Impactor (NGI). The instrument was loaded with content of the 0.375 mg capsule of the dry powder formulation of Ivermectin 0.325mg, the flow rate through the instrument during testing was regulated at 0.74 L/minute. The procedure was repeated for Ivermectin 1.7 mg. The dry powder inhalation formulation collected in the filter was analyzed for Ivermectin content in lungs, fine particle fraction, MMAD, and geometric standard deviation (GSD), results are provided below in Table 4. The cumulative (% undersize) particle size distribution, drug deposition for Ivermectin 0.375mg and Ivermectin 1.7 mg were illustrated in Fig. 1A, 1B, 1C, and 1D respectively.
TABLE 4:

DPI formulation Ivermectin content in lungs FPF (%) MMAD
(µm) GSD
Ivermectin 0.375 mg 0.171mg 45 2 2.5
Ivermectin 1.7 mg 1.012 mg 49 2.1 2

It is evident that irrespective of Ivermectin dose, nearly 58.680% reaches the lungs.
Example 5: In-vivo toxicity studies of the Dry Powder Inhalation formulation of the present disclosure
The toxicity of the dry powder inhalation formulation of the present disclosure was evaluated in Wistar rats to determine the No Observed Effect Level (NOEL). The potential toxicity of the formulation of the present disclosure, when administered via inhalation route for a period of 2 consecutive weeks (7 consecutive days per week), was evaluated. The study animals (24) (6 rats/sex/group) were divided into two groups, control (GI) administered with only air, intervention (GIII) was administered Ivermectin at a dose of 0.27 mg/kg body weight once daily for 14 days.
The Ivermectin was loaded in the rotating brush generator and dust was generated. The Ivermectin DPI was loaded, in a cylindrical powder reservoir of the aerosol generator system, positioned below the cylindrical brush. The airflow rate through the dust generator was maintained approximately at 39 litres per minute for GI and GIII respectively. Rats belonging to GI were exposed for 15 minutes and rats belonging to GIII were exposed for 11 minutes.
The mean delivered doses were 0.27 mg a.i./kg b. wt. for Ivermectin (mean measured value). The mass median aerodynamic diameter (MMAD) of Ivermectin aerosols was found within a range of 3.15 and 3.33 µm with a geometric standard deviation (GSD) within a range of 1.61 and 1.65.
The animals (rats) were observed daily for clinical signs, morbidity, and mortality. The body weight is recorded at regular intervals on day 1 (before treatment), day 4, day 8, and day 15. The feed consumption was calculated on day 4, day 8, and day 15.
After completion of the treatment period, the rats were euthanized using thiopentone sodium overdose, followed by pathological examination. The

pathological examination includes external abnormalities, followed by euthanization, organs were removed intact, examined, and weighed. The organ weights of the left lung, liver, kidneys, spleen, adrenals, heart, testes, epididymides, ovaries, uterus with cervix, thymus, and brain were recorded. Further, the broncho-alveolar lavage (BAL) of the right lung was analyzed.
During the study, it was observed that all the animals well-tolerated the dose and there is no mortality or any clinical signs of toxicity were observed. There was no statistical difference in the body weight, percent body weight change, and feed consumption in the treated groups when compared with that of the control group. The mass median aerodynamic diameter (MMAD) of the dry powder inhalation formulation in the form of aerosols was found within a range of 3.15 and 3.33 µm with a geometric standard deviation (GSD) within a range of 1.61 and 1.65.
In GIII rats were treated with dry powder inhalation formulation of the present disclosure, showed 42.032 to 99.457 ng/mL of Ivermectin in plasma for male rats and 62.481 to 125.265 ng/mL of Ivermectin in plasma for female rats and 55.888 to 180.562 ng/gram of Ivermectin in lungs for male rats and 104.666 to 180562.280 ng/gram of Ivermectin in lungs for female rats.
The No Observed Effect Level (NOEL) of Ivermectin was >0.27 mg a.i./kg b. wt., when administered through inhalation up to 2 weeks in Wistar rats under the procedure and conditions followed in this study.
The detailed observations for each of the parameters are provided below
i. Signs of Toxicity
Rats were observed twice each day in the duration of the experimental period for morbidity and mortality (before and after exposure on treatment days, and morning and evening on non-treatment days). Observations include evaluation of skin and fur, eyes, mucous membranes, respiratory and circulatory effects, autonomic and central nervous system effects, somatomotor activity and behaviour pattern, and observation of tremor, convulsions, salivation, diarrhoea, lethargy, sleep,

and coma. In addition, rats were observed for the detailed clinical signs once daily along with the above observations. Rats were also observed for clinical signs at the end of exposure and one-hour post exposure.
ii. Body Weight
The individual body weight for each rat was recorded on the day of randomization (prior to treatment), 1 (beginning of the treatment), and on days 4, 7, and at 15 (study termination).
The details are illustrated below in Tables 5 and 6, and Tables 7 and 8.
Table 5: Body Weight (g) of Individual Rat (Group GI)

Rat No. Sex On Days

1 4 8 15
1 M 299.0 313.0 325.4 348.5
2 M 279.3 287.6 295.3 311.6
3 M 273.3 281.3 285.4 300.5
4 M 272.1 280.9 285.9 312.9
5 M 264.1 269.3 276.9 290.4
6 M 262.8 270.4 276.6 288.8
7 F 214.2 220.1 227.1 240.8
8 F 194.9 195.7 200.5 212.9
9 F 189.6 193.2 200.7 214.6
10 F 187.7 188.3 192.3 208.7
11 F 186.8 187.2 190.2 199.9
12 F 186.2 187.8 191.8 203.8

Table 6: Body Weight (g) of Individual Rat (Group GIII)

Rat No. Sex On Days

1 4 8 15
25 M 292.8 295.6 309.9 326.8
26 M 282.6 283.9 289.1 297.1
27 M 274.2 279.9 288.8 300.3
28 M 260.9 265.2 276.6 291.2
29 M 260.4 267.2 277.9 300.2
30 M 272.9 277.3 287.2 299.3
31 F 202.6 205.2 206.2 211.2
32 F 196.2 198.9 199.4 214.1
33 F 194.8 196.3 201.0 212.8
34 F 183.1 192.8 203.4 218.4
35 F 182.3 185.9 189.2 195.8
36 F 178.1 180.4 181.9 192.5
Table 7: Percent Body Weight Change of Individual Rat (Group GI)

Rat No. Sex Body Weight Change (%)

On Days

4 8 15
1 M 4.7 8.8 16.6

2 M 3.0 5.7 11.6
3 M 2.9 4.4 10.0
4 M 3.2 5.1 15.0
5 M 2.0 4.8 10.0
6 M 2.9 5.3 9.9
7 F 2.8 6.0 12.4
8 F 0.4 2.9 9.2
9 F 1.9 5.9 13.2
10 F 0.3 2.5 11.2
11 F 0.2 1.8 7.0
12 F 0.9 3.0 9.5
Table 8: Percent Body Weight Change of Individual Rat (Group GIII)

Rat No. Sex Body Weight Change (%)

On Days

4 8 15
25 M 1.0 5.8 11.6
26 M 0.5 2.3 5.1
27 M 2.1 5.3 9.5
28 M 1.6 6.0 11.6
29 M 2.6 6.7 15.3

30
M 1.6 5.2 9.7
31 F 1.3 1.8 4.2
32 F 1.4 1.6 9.1
33 F 0.8 3.2 9.2
34 F 5.3 11.1 19.3
35 F 2.0 3.8 7.4
36 F 1.3 2.1 8.1
Inference: It is evident from tables 5 to 8 that there was no statistical difference in body weight was observed during the evaluation period.
iii. Feed Consumption
The feed consumption of animals was calculated on day 4, day 8, and day 15.
The weekly feed consumption for each rat was calculated throughout the study using the below formula.
Total feed input – feed left over
Feed Consumption (g/rat/day) =
Number of rats per cage x Number of days
The details of the feed consumption are illustrated below in Tables 9 and 10
Table 9: Feed Consumption (g/rat/day) of Individual Rat (Group GI)

Cage No. A. No.
& Sex
1M On Days Cage No.
4 A. No.
& Sex
7F On Days

1-3 4-7 8-14

1-3 4-7 8-14
1
22.65 22.58 21.43

16.85 16.25 16.95

& 2M
3M & 8F
9F




2 & 4M
5M 21.35 18.91 18.08 5 & 10F
11F 14.18 15.24 16.13




3 & 6M 20.37 18.84 19.84 6 & 12F 13.37 13.06 13.32
Table 10: Feed Consumption (g/rat/day) of Individual Rat (Group GIII)

A. Week No. A. Week No .
Cage No. No.
& Sex Cage No. No.
& Sex
31F

1-3 4-7 8-14

1-3 4-7 8-14
25M


13 & 26M 20.65 19.85 18.74 16 & 32F
33F 14.42 15.71 14.46
27M


14 & 28M 18.77 19.05 16.44 17 & 34F
35F 14.40 16.33 14.91
29M


15 & 30M 20.12 18.79 19.68 18 & 36F 13.12 15.01 14.22
Inference: It is evident from table 9 and 10 that there was no statistical difference observed in the feed consumption during the evaluation period.
iv. Concentration of Ivermectin in rat plasma samples

The rats were administered with 0.27 mg/kg body weight of the dry powder inhalation formulation of Ivermectin in accordance with the present disclosure. The rat plasma was obtained on the 14th day of administration, the concentration of Ivermectin in the plasma was evaluated by LC-MS/MS.
1. Instrumental Parameters
HPLC Parameters
For Ivermectin
Instrument : LC-MS/MS [API 4000 mass spectrometer
coupled with Nexera X2 HPLC System]
Column : CSH Fluoro-phenyl, C18 (4.6 × 150 mm, 3.5µm)
Flow Rate : 0.8 mL/minute
Injection Volume : 10 µL
Column Temperature : 40° C
Cooler Temperature : 10° C
Mobile phase : Methanol: 5Mm Ammonium acetate in Milli-Q
water
Elution mode : Binary/Isocratic
Mass Spectrometer Parameters for Multiple Reaction Monitoring (Ivermectin) are given below in table 11:
Table 11:

Compound Name Ivermectin Rolipram
MRM [Multiple
Reaction Monitoring]
(Q1/Q3) 892.6>552. 1 892.6>307. 9 892.6>567.6 276.2>191. 1
Collision Energy (eV) 32 35 22 22
Collision Cell Exit 21 21 21 21

Potential (V)
De-clustering Potential (V) 65 65 65 75
Entrance Potential (V) 10
Ionization (ESI) Positive
Curtain Gas (psi) 10
Collision Gas (psi) CAD 10
Ion spray voltage (V) 5500
Gas-1 (psi) 50
Gas-2 (psi) 60
Temperature (°C) 300
Inference: Table 11 indicate the parameters for detection of Ivermectin using multiple reaction monitoring (MRM) through targeted mass spectrometry (MS) technique.
The analysed concentration of Ivermectin is illustrated in Table 12 below:
Table 12– Concentration of Ivermectin in rat plasma samples

Sample ID Analysed concentration (ng/mL)
GI M_1 BLQ
GI M_2 BLQ
GI M_3 BLQ
GI M_4 BLQ
GI M_5 BLQ
GI M_6 BLQ
GI F_7 BLQ
GI F_8 BLQ
GI F_9 BLQ
GI F_10 BLQ

GI F_11
BLQ
GI F_12 BLQ
GIII M_25 66.565
GIII M_26 85.860
GIII M_27 99.457
GIII M_28 59.523
GIII M_29 42.032
GIII M_30 42.040
GIII F_31 69.402
GIII F_32 62.461
GIII F_33 85.563
GIII F_34 71.511
GIII F_35 125.265
GIII F_36 121.681
BLQ = Below Limit of Quantification
v. Concentration of Ivermectin in Rat (Lung Homogenate) samples
The Ivermectin content in the lungs was analysed by measuring the Ivermectin content in lung homogenate by using LC-MS/MS, the concentration is as illustrated in Table 13.
Table 13 – Concentration of Ivermectin in lung homogenate

Sample ID Analysed concentration
in lung homogenate
(ng/mL) Concentration of Ivermectin in Lung homogenate
(ng/gram)
GI M_1 BLQ BLQ
GI M_2 BLQ BLQ
GI M_3 BLQ BLQ
GI M_4 BLQ BLQ
GI M_5 BLQ BLQ
GI M_6 BLQ BLQ

GI F_7 BLQ BLQ
GI F_8 BLQ BLQ
GI F_9 BLQ BLQ
GI F_10 BLQ BLQ
GI F_11 BLQ BLQ
GI F_12 BLQ BLQ
GIII M_25 39.032 78.064
GIII M_26 90.281 180.562
GIII M_27 56.721 113.442
GIII M_28 56.162 112.324
GIII M_29 35.830 71.660
GIII M_30 27.944 55.888
GIII F_31 52.333 104.666
GIII F_32 54.547 109.094
GIII F_33 61.136 122.272
GIII F_34 118.603 237.206
GIII F_35 127.274 254.548
GIII F_36 74.430 148.860
BLQ = Below Limit of Quantification
The concentration of Ivermectin in the lung homogenate was calculated using the formula below,
Concentration in homogenate [ng/g] = Concentration of drug in homogenate [ng/ml]/Concentration of lung in homogenate [g/ml]
vi. Broncho-Alveolar Lavage Analysis

The broncho-alveolar lavage was obtained from the right lung after euthanized by barbiturate overdose followed by exsanguination at scheduled sacrifices (day 15). The lavage was analyzed for cellular contents, the details are illustrated below in Table 14;
TABLE 14: Broncho-Alveolar Lavage Analysis - Group Mean Values
Sex: Male

Group N GI (N=6) GIII (N=6)
Group Control Ivermectin
Parameter Mean SD Mean SD
WBC (x103/µL) 0.43 0.19 0.65 0.35
Neutrophil (x103/µL) 0.11 0.07 0.18 0.14
Lymphocyte (x103/µL) 0.25 0.13 0.28 0.14
Monocyte (x103/µL) 0.01 0.01 0.03 0.03
Eosinophil (x103/µL) 0.00 0.00 0.00 0.00
Basophil (x103/µL) 0.03 0.02 0.06 0.07
LUC* (x103/µL) 0.07 0.05 0.16 0.13
LDH# (U/L) 81.67 30.08 64.83 22.96
Total protein (ug/mL) 170.07 53.29 135.51 44.46
* = Large unstained cells
# = Lactate dehydrogenase
Sex: Female

Group N GI (N=6) GIII (N=6)
Group Control Ivermectin
Parameter Mean SD Mean SD

WBC (x103/µL) 0.55 0.07 0.71 0.19
Neutrophil (x103/µL) 0.13 0.04 0.16 0.06
Lymphocyte (x103/µL) 0.31 0.06 0.41 0.28
Monocyte (x103/µL) 0.01 0.01 0.01 0.00
Eosinophil (x103/µL) 0.01 0.01 0.00 0.00
Basophil (x103/µL) 0.05 0.02 0.05 0.04
LUC* (x103/µL) 0.09 0.04 0.14 0.07
LDH# (U/L) 81.00 25.27 77.83 38.67
Total protein (ug/mL) 167.40 47.85 160.85 29.46
* = Large unstained cells
# = Lactate dehydrogenase
Inference: It is evident from table 14 that, the treatment by using the dry powder inhalation formulation did not lead to any treatment related alterations in broncho-alveolar lavage when compared with control.
vii. Organ Weight (g) – Group Mean Values
The rats were euthanized by barbiturate overdose, organs were removed intact, examined, and weighed. Organ weights of left lungs, liver, kidneys, spleen, adrenals, heart, testes, epididymides, thymus, ovaries, uterus with cervix, and brain were taken. All organs were preserved in 10% neutral buffered formalin solution except testes which was preserved in Modified Davidson’s fixative. The organ weights of treated groups (GIII) were compared well with the control group (GI), the results are illustrated below in Table 15.
TABLE 15: Organ Weight (g) – Group Mean Values Sex: Male

Group N GI (N=6) GIII (N=6)

Group
Control Ivermectin
Parameter Mean SD Mean SD
Liver 11.215 1.328 10.709 1.311
Heart 0.902 0.075 0.904 0.051
Spleen 0.632 0.150 0.620 0.070
Brain 2.046 0.153 2.039 0.048
Thymus 0.481 0.091 0.452 0.078
Kidneys 1.722 0.111 1.763 0.101
Adrenals 0.075 0.005 0.078 0.015
Testes 3.426 0.975 3.829 0.315
Epididymides 1.040 0.185 1.230 0.081
Left Lung 0.413 0.038 0.385 0.026
Sex: Female
Group N GI (N=6) GIII (N=6)
Group Control Ivermectin
Parameter Mean SD Mean SD
Liver 8.417 0.429 7.918 0.545
Heart 0.711 0.092 0.714 0.033
Spleen 0.520 0.110 0.511 0.036
Brain 1.920 0.074 1.977 0.065
Thymus 0.521 0.068 0.466 0.051
Kidneys 1.466 0.120 1.504 0.058
Adrenals 0.103 0.011 0.095 0.010
Ovaries 0.127 0.014 0.117 0.013

Inference: It was observed from table 15 that, a statistically significant decrease was observed in the weight of thymus in GIII females, which was not considered as an effect of test item treatment due to lack of consistency between sexes.
viii. Macroscopic findings
The rats were subjected to full gross necropsy, examined for external abnormalities. The thoracic and abdominal cavities were cut, opened and organs were examined to detect abnormalities. The findings are illustrated below in Table 16.
TABLE 16: Gross Findings – Summary by Groups Sex: Male

Group N GI (N=6) GIII (N=6)
Group Control Ivermectin
Organs and Lesions No. of Animals 6 6
MACROSCOPIC EXAMINATION
External
No Abnormality Detected 6 6
Internal
No Abnormality Detected 5 6
Testes: Reduced size, bilateral 1 0
Epididymides: Reduced size 1 0
Sex: Female

Group N GI (N=6) GIII (N=6)

Group
Control Ivermectin
Organs and Lesions No. of Animals 6 6
MACROSCOPIC EXAMINATION
External
No Abnormality Detected 6 6
Internal
No Abnormality Detected 6 6
Inference: The macroscopic observations on external and internal organs did not reveal any treatment related findings. However, it was noticed that the reduced size of testes and epididymides in one male rat of the control group was considered as incidental/spontaneous.
Example 6: In-silico drug discovery study of Ivermectin
The molecular docking study was performed by AutoDock 4.2.6 program, using the implemented empirical free energy function and the Lamarckian Genetic Algorithm (LGA). Molecular Docking is an important component of computer-assisted drug discovery. It helps in predicting the intermolecular framework formed between a protein and ligand and outputs the appropriate binding between the molecules. The best conformation with the lowest docked energy was chosen from the docking search. The interactions of complex protein-ligand conformations including hydrogen bonds and bond lengths were analyzed using Pymol software which is a cross-platform molecular graphics tool widely used for three-dimensional (3D) visualization of Proteins and Ligands, UCSF Chimera and Accelrys Discovery Studio Visualizer software.
The binding energy between the target (protein) and the ligand was calculated. Higher the value of free binding energy, the higher the requested energy to break that binding between two molecules. So, the negative values show a related binding between protein and ligand. Very negative score corresponds to a strong

binding and a less negative or even positive score corresponds to a weak or non-existing binding.
2-Deoxy-D-Glucose, IvermectinB1a and IvermectinB1b as a ligands were got from the PubChem drug databases and docked against hexokinase 1 protein (PDB: 1CZA) and hexokinase II (PDB: 2NZT) with high AutoDock V4.2 software. The findings were reported below in tables 16-17 and figures 2A to 2F that shows molecular docking of Ivermectin B1a, Ivermectin B1b, and 2-deoxy-D-glucose binding with hexokinase I and II along with 3D model of the interactions and the 2D interaction patterns and with or without H-bond interaction.
Table 17:

Vander Walls +
Protein Binding Energy No. of H Interacting Final Interm olecular hydrogen bonds +
desolvati
on
Energy Electrost atic Torsiona
l Free
Energy
(kcal/mol
)
s Name Ligand (kcal/mo Bonds residue Energy
Energy

Name l) (kcal/m ol)
(kcal/mol )

(kcal/mol )

03 (H1=2.1 TYR:461(H 1),
1CZA 2-Deoxy-D-Glucose -3.32 kcal/mol 3Å,
H2=2.39 Å, H3=2.74 LYS:147(H 2), -3.42
kcal/mo
l -3.31 kcal/mol -0.12 kcal/mol +2.68 kcal/mol

PRO:149,
Å, ASP:79(H3)
02 PHE:623,
1CZA IvermectinB 1A -6.72 kcal/mol (H1=2.8 5Å, LYS:621, GLY:532, -8.30 kcal/mo -8.07 -0.23 +3.28


l kcal/mol kcal/mol kcal/mol
H2=3.00 Å) MET:567, ILE:562,

ILE:564,
GLN:568(H 1),
ARG:779(H 2)
1CZA IvermectinB 1B -5.57 kcal/mol 00 ILE:781,
GLY:780,
LYS:785 -7.14
kcal/mo l -7.19 kcal/mol +0.05 kcal/mol +2.98 kcal/mol
Inference:
On performing the molecular docking of the protein name (PDB ID: 1CZA) with all five ligands, it was observed that the binding energy of Ivermectin B1A with 1CZA is good (-6.72 kcal/mol) followed by Ivermectin B1B shows -5.77 kcal/mol and -5.57 kcal/mol respectively. The study also shows the presence of H-bonds that indicates stable interaction between ligand and protein.
Table 18:

Vander
Protein s Name Ligand Name Binding
Energy
(kcal/mo
l) No. of H Bonds Interacting residue Final Interm olecular Energy (kcal/m ol) Walls +
hydrogen
bonds +
desolvati
on
Energy Electrost atic
Energy
(kcal/mol
) Torsiona
l Free
Energy
(kcal/mol
)

(kcal/mol )

2NZT 2-Deoxy-D-Glucose -3.40 kcal/mol 02
(H1=2.5
2Å,
H2=2.43
Å) LYS:147(H
1),
PRO:149,
GLU:79(H2
) -3.52 kcal/mo l -3.42 kcal/mol -0.09 kcal/mol +2.6 8 kcal/mol

GLY:780, PHE:623,
IvermectinB -7.36 02 MET:748 -8.98 -8.71 -0.26 +3.2
1 A kcal/mol (H1=3.0 3Å, ,MET :567,GL kcal/mo l l kcal/mol kcal/mol 8 kcal/mol
2NZT H2=2.45 Å) Y:747, ALA:561 ,ILE: 562(H1), ASN: 537(H2)
LYS:337,
IvermectinB -6.39 01 GLU:335 kcal/mo l l -8.24 -0.11 +2.9
2NZT 1 B kcal/mol (H1=2.4 ,MET:119,
kcal/mol kcal/mol 8
0Å) MET:300(H
kcal/mol
1)
Inference:
On performing the molecular docking of the protein name (PDB ID: 2NZT) with all five ligands, it was observed that the binding energy of IvermectinB1A with 2NZT is good (-7.36 kcal/mol) followed by IvermectinB1B shows -6.15 kcal/mol and -6.39 kcal/mol respectively. The study also shows the presence of H-bonds that indicates stable interaction between ligand and protein.
Comparative analysis:
The molecular docking of both the proteins (PDB ID: 1CZA) and (PDB ID: 2NZT) against hexokinase I and II is analyzed and compared below in table 19:
Table 19:

Ligand Name Hexokinase I (PDB ID: 1CZA) Hexokinase II (PDB ID: 2NZT)
2-Deoxy-D-Glucose -3.32 kcal/mol -3.40 kcal/mol
IvermectinB1A -6.72 kcal/mol -7.36 kcal/mol

IvermectinB1B -5.57 kcal/mol -6.39 kcal/mol
Inference:
It is evident from table 19 that the binding energy of IvermectinB1A with (PDB ID: 1CZA- -6.72 kcal/mol)and 2NZT is good (-7.36 kcal/mol) against hexokinase I and II respectively in comparison with 2DG has a binding energy of -3.3 kcal/mol.
Molecular docking studies of Ivermectin with 1WZY, 2BIN, 6S9X:
Since abnormal glycolytic metabolism contributes to joint inflammation and destruction in rheumatoid arthritis (RA), the present disclosure investigated the use of Ivermectin in the treatment of rheumatoid arthritis (RA). Rheumatoid arthritis (RA) is a chronic autoimmune disease in which imbalances in pro-anti-inflammatory cytokines promote the induction of autoimmunity, inflammation and joint destruction. Mitogen-activated protein kinases (MAPKs) have been implicated as playing key regulatory roles in the production of pro-inflammatory cytokines like IL-1, IL-6, IL-12, TNF and downstream signalling events leading to joint inflammation and destruction. The p53 tumor suppressor is abnormal in the synovial lining of RA, giving rise to the hypothesis that the p53-dependent apoptosis pathway is defective in RA and when overexpressed early, may ameliorate the course of the arthritis disease. AKT1 and the related AKT2 are activated by platelet-derived growth factors. AKT is a serine/threonine kinase that supports proliferation and survival in a variety of cells and is overexpressed in the rheumatoid synovial tissue.
Mitogen-activated protein kinase 1 (MAPK1)- (1WZY), TP53 Cellular Tumor Antigen P53 (2BIN) and AKT1 RAC-alpha serine/threonine-protein kinase (6S9X) as a protein were got from the Protein Data Bank (PDB) with PDB ID: -1WZY, 2BIN, 6S9X and docked against ligand Ivermectin with high AutoDock V4.2 software. The findings were reported below in table 20 and figures 4 to 4B shows molecular docking of proteins such as 1WZY, 2BIN, 6S9X binding with

Ivermectin along with 3D model of the interactions and the 2D interaction patterns and H-bond interaction and ligand interaction with distance and without distance.
Table 20:

Bindin No. H Interacting Final Vander Electro
g Bonds residue Intermo Waals + static
Protein Ligand energy (kcal/m lecular Energy (kcal/m hydroge
n bonds
+ Energy (kcal/m Torsio
nal
Free
Name Name ol) ol) desolvat
ion Energy (kcal/m
ol) ol) Energy
(kcal/m
ol)
01 THR:185(H1), TYR:187, -8.68 -8.62 -0.06 +3.28
1WZY Ivermectin -7.16 (H1=2 .10Å). LEU:170, ARG:67, VAL: 188.
LEU:114, PRO:142, -7.76 -7.43 -0.33 +3.28
2BIN Ivermectin -6.20 0 PRO:128, PHE:113, TYR: 126.
TYR:350, THR:443, -7.46 -7.41 -0.05 +3.28
6S9X Ivermectin -5.95 0 ARG:243, SER:240, PHE:236, GLU: 341.

Inference:
On performing the molecular docking of the protein names 1WZY, 2BIN, 6S9X with Ivermectin, it was observed that the binding energy of 1WZY with Ivermectin is good (-7.16 kcal/mol) followed by 2BIN shows -6.20 kcal/mol and followed by 6S9X shows -5.95 kcal/mol respectively. The study also shows the presence of H-bonds that indicates stable interaction between ligand and protein. Higher the value of free binding energy, the higher, and the requested energy to break that binding between two molecules. So, the negative values show a related binding between protein and ligand. A very negative score corresponds to a strong binding and a less negative or even positive score corresponds to a weak or non-existing binding. From the results, it was clear that Ivermectin could have a potential role in the alleviation of symptoms seen in rheumatoid arthritis (RA).
Example 7: Comparison of In-vitro activity of Ivermectin against 2-Deoxy-2-glucose (2DG) in COVID-19 by inhibiting hexokinase
In-vitro activity of formulation of the present disclosure was investigated against 2-Deoxy-2-glucose in COVID-19 by inhibiting hexokinase.
It is clearly evident from example 6 that Ivermectin acts as a potential hexokinase inhibitor. A Hexokinase is an enzyme that phosphorylates hexoses (six-carbon sugars), forming hexose phosphate. In most organisms, glucose is the most important substrate for hexokinases, and glucose-6-phosphate is the most important product. All hexokinases are capable of phosphorylating several hexoses but glucokinase acts with 50-fold lower substrate affinity and its main hexose substrate is glucose.
Hexokinases (HK) have been found in every organism ranging from bacteria, yeast, and plants, to humans and other vertebrates. They are grouped as actin fold proteins, sharing a common ATP binding site core and surrounded by more variable sequences that determine substrate affinities. In mammalian tissues, there are four major hexokinases which are HK1, HK2, HK3, and HK4. HK1 and

HK2 are the main hexokinases that have contribution in cell survival. Mammalian adult tissues mostly expressed HK1 isoform, while HK2 is present abundantly only in adult tissues such as cardiac muscles, skeletal, and adipose. A Hexokinase II also known as HK2 is an enzyme which in humans is encoded by the HK2 gene on chromosome 2. This gene encodes hexokinase 2, the predominant form found in skeletal muscle. It localizes to the outer membrane of mitochondria. Expression of this gene is insulin- responsive, and in rat it is involved in the increased rate of glycolysis seen in rapidly growing cancer cells. HK2 is one of four highly homologous hexokinase isoforms in mammalian cells. HK2 is highly expressed in several cancers, including breast cancer and colon cancer. Due to their action HK helps in promoting cancer cell growth through an increased glycolytic flux. There are three HK isoforms expressed in the lung, HK-I, HK-II, and HK-III. Hypoxia increased the steady-state levels of HK-II mRNA but did not increase the steady-state levels of HK-I. Hence, any molecule that will inhibit hexokinase would reduce the hyper-expressed HK and hence might address hypoxia state. As far as viral infection is concern, the infected viral cells require glycolysis and other associated pathways to gain substrates for their function and replication. Studies showed that to induce cell apoptosis, glycolysis can be target of interest. HK inhibition contributes to this apoptosis through intrinsic pathway that includes destabilization of mitochondrial complexes and also interfering with glycolysis results in autophagy.
Materials and methods: Chemicals:
Hexokinase kit was purchased from Sigma Aldrich (Product no: MAK91, Bangalore, India). 2-Deoxy-2-glucose, purchased from Sigma Aldrich (Bangalore, India). Other chemicals such as Ivermectin from Hoster biotech Pvt ltd were procured for the study.
Hexokinase assay:
The hexokinase (HK) assay was carried out by using hexokinase calorimetric assay kit (Sigma-Aldrich, MAK091). NADH standard solution from HK assay kit

was reconstituted with 400 µl water to generate 1.25 mM stock solution. From stock solution, 0, 2, 4, 6, 8, 10 µl aliquots were withdrawn into 96 well plate to achieve blank, 2.5, 5, 7.5, 10 and 12.5 nmol/well concentrations. One unit of HK is the amount of enzyme that will generate 1.0 µmole of NADH per minute at pH 8.0 at room temperature. To each well 50 µL of sample solution containing inhibitor solution and HK enzyme was added followed by 50 µL of appropriate reaction mix. For control samples, same procedure was followed without addition of inhibitor solution. The samples were mixed in well by pipetting. The plate was incubated in dark for 5 min at room temperature. After 5 min (Tinitial) incubation, initial absorbance [(A450)initial] was measured at 450 nm using ELISA plate reader (Erba LisaScan II). Plate was incubated for 60 min taking measurements every 10 min. Final measurement [(A450)final] and time of final reading (Tfinal) was used to calculate enzyme activity. Using these measurements, the change in measurement from Tinitial to Tfinal for the samples was calculated using following formula;
AA450 = (A450)final - (A450)initial The HK activity of a sample was determined by the following equation;
B X Sample Dilution Factor
HK Activity =
(Reaction Time^) X V
Where,
B = Amount (nmole) of NADH generated between Tinitial and Tfinal.
Reaction Time = Tfinal - Tinitial (minutes)
V = sample volume (mL) added to well
Screening for hexokinase inhibitors by in-vitro enzyme inhibition studies:
Screening for hexokinase inhibitors was carried out using hexokinase calorimetric assay kit (Sigma-Aldrich, MAK091). The stock solutions of 2-deoxy-2-glucose, was prepared with water whereas Ivermectin was prepared using buffer (10% DMSO+ 40% PEG 400+ 5% tween 80+ 45% saline). Reagent control for each solvent or diluent in which inhibitor solution was prepared was also kept to investigate the effect on hexokinase activity. The inhibitor stock solutions were

diluted in hexokinase assay buffer to get final volume of 50 µl with known units of hexokinase enzyme. The control reaction was carried out in hexokinase assay buffer without addition of any inhibitor solution or solvent. The relative hexokinase activity for each compound was calculated as per given below.
HK activity for compounds (Inhibitors)
Relative hexokinase activity = X 100
HK activity for control
Table no. 21: Composition of HK assay reaction mix

Sr. no Reagent Samples and standards Sample Blank
1 HK assay buffer 34µl 44 µ l
2 HK enzyme mix 2µl 2 µl
3 HK Developer 2µl 2 µl
4 HK Coenzyme 2µl 2 µl
5 HK substrate 10 -
Results and conclusion:
The study demonstrates HK inhibition activity of various antiviral compounds by measuring % residual HK activity with and without the presence of the compounds under testing. This coupled enzyme assay, based on conversion of glucose to glucose-6-phosphate by hexokinase, which is oxidized by glucose-6-phosphate dehydrogenase to form NADH. The resulting NADH reduces a colorless probe resulting in a colorimetric (450 nm) product proportional to the hexokinase activity present.
In this study, HK inhibitory activity of Ivermectin was evaluated by detecting generation of NADH. The lower amount of NADH generation confirms the

inhibitory potential of a compound. The anti-hexokinase activity of Ivermectin was compared with compound 1 i.e. 2-deoxy-2-glucose which have been proved for its HK inhibition activity and as antiviral in treatment of SARS-CoV-2.
Ivermectin was found to be 99% more stronger hexokinase inhibitor as compared to 2DG since it has similar level of inhibition when used at ten times less concentration to 2DG The inhibition of HK will induce the apoptosis and reduce the viral cell replication. So, it is evident that Ivermectin was found to be a more potent inhibitor of hexokinase as compared to 2DG as depicted in figure 3.

Sr. No. Compound name/ Concentration %
Hexokinase
activity % inhibition
of
hexokinase
1 2-Deoxy-D-Glucose (45 mM) 62.75 37.25
2 Ivermectin (3.43 mM) 69.04 30.96

TECHNICAL ADVANCES AND ECONOMIC SIGNIFICANCE
The present disclosure described hereinabove has several technical advantages including, but not limited to, the realization of
a dry powder inhalation (DPI) formulation, that
• has increased bioavailability of the Ivermectin, thus reduces dosage amount when compared to the oral dosage form;
• prevents/ reduces adverse effects in comparison to the oral dosage form;
• improved patient compliance; and
provides a simple and economical process for the preparation of the dry powder inhalation (DPI) formulation;
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results.
Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.
The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values

higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary.
While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

WE CLAIM:
1. A dry powder inhalation (DPI) formulation comprising:
• a micronized Ivermectin having a particle size in the range of 0.1 µm to 5 µm;
• a first lactose having a particle size in the range of 10 µm to 200 µm;
• a second lactose having a particle size in the range of 1 µm to 150 µm; and
• optionally at least one excipient;
wherein a mass ratio of the first lactose to the second lactose is in the range of 5:1 to 15:1.
2. The formulation as claimed in claim 1, wherein said micronized Ivermectin is in an amount in the range of 1 mass% to 15 mass% with respect to the total mass of the formulation.
3. The formulation as claimed in claim 1, wherein said first lactose is in an amount in the range of 70 mass% to 90 mass% with respect to the total mass of the formulation.
4. The formulation as claimed in claim 1, wherein said second lactose is in an amount in the range of 5 mass% to 15 mass% with respect to the total mass of the formulation.
5. The formulation as claimed in claim 1, wherein said excipient is in an amount in the range of 1 mass% to 5 mass% with respect to the total mass of the formulation.
6. The formulation as claimed in claim 1, wherein said excipient is selected from citric acid, magnesium stearate, and a combination thereof.
7. The formulation as claimed in claim 1, wherein the median mass aerodynamic diameter (MMAD) of said dry powder inhalation is in the range of 1.5 µm to 2.5 µm.

8. The formulation as claimed in claim 1, wherein the mean particle size (d10) of said first lactose is in the range of 10 µm to 50 µm.
9. The formulation as claimed in claim 1, wherein the mean particle size (d50) of said first lactose is in the range of 51 µm to 120 µm.
10. The formulation as claimed in claim 1, wherein the mean particle size (d90) of said first lactose is in the range of 121 µm to 200 µm.
11. The formulation as claimed in claim 1, wherein the mean particle size (d10) of said second lactose is in the range of 1 µm to 30 µm.
12. The formulation as claimed in claim 1, wherein the mean particle size (d50) of said second lactose is in the range of 31 µm to 90 µm.
13. The formulation as claimed in claim 1, wherein the mean particle size (d90) of said second lactose is in the range of 91 µm to 150 µm.
14. The formulation as claimed in claim 1, is used for in the treatment of cancer, bacterial infection, viral infection, fungal infection and autoimmune diseases.