Claims:WE CLAIM
1. A dry powder inhalation (DPI) formulation comprising:
• a micronized favipiravir having a particle size in the range of 1 µm to 6 µm;
• first lactose having a particle size in the range of 19 µm to 93 µm;
• second lactose having a particle size in the range of 1 µm to 60 µm; and
• optionally at least one excipient;
wherein a weight ratio of the first lactose to the second lactose is in the range of 6:1 to 10:1.
2. The formulation as claimed in claim 1, wherein said micronized favipiravir is in an amount in the range of 35 wt% to 85 wt% with respect to the total weight of the formulation.
3. The formulation as claimed in claim 1, wherein said first lactose is t in an amount in the range of 17 wt% to 55 wt% with respect to the total weight of the formulation.
4. The formulation as claimed in claim 1, wherein said second lactose is in an amount in the range of 1 wt% to 8 wt% with respect to the total weight of the formulation.
5. The formulation as claimed in claim 1, wherein said excipient is in an amount in the range of 1 wt% to 7 wt% with respect to the total weight 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 weight ratio of said first lactose to second lactose is 9:1.
8. The formulation as claimed in claim 1, wherein the MMAD of said dry powder inhalation is in the range of 1 µm to 4 µm.
9. The formulation as claimed in claim 1, wherein the mean particle size (d10) of said first lactose is in the range of 19 µm to 43 µm.
10. The formulation as claimed in claim 1, wherein the mean particle size (d50) of said first lactose is in the range of 53 µm to 66 µm.
11. The formulation as claimed in claim 1, wherein the mean particle size (d90) of said first lactose is in the range of 75 µm to 93 µm.
12. 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 6 µm.
13. The formulation as claimed in claim 1, wherein the mean particle size (d50) of said second lactose is in the range of 10 µm to 30 µm.
14. The formulation as claimed in claim 1, wherein the mean particle size (d90) of said second lactose is in the range of 50 µm to 60 µm.
15. The formulation as claimed in claim 1, when administered the lung to plasma ratio of favipiravir is in the range of 0.15 to 0.49.

Dated this 20th day of April, 2021

_______________________________
MOHAN RAJKUMAR DEWAN, IN/PA – 25
of R.K.DEWAN & CO.
Authorized Agent of Applicant

TO,
THE CONTROLLER OF PATENTS
THE PATENT OFFICE, AT MUMBAI
, Description:
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.
BACKGROUND
The background information hereinbelow relates to the present disclosure but is not necessarily prior art.
Favipiravir is a broad-spectrum antiviral drug found to be active against various influenza viruses including seasonal strains A (H1N1), A (H3N2), influenza B, A (H1N1)pdm09, avian influenza virus A (H5N1) isolated from humans, A (H1N1), and A (H1N2) isolated from swine, A (H2N2), A (H4N2), and A (H7N2). Besides, it also inhibits the Ebola virus, bunyavirus, filovirus, West Nile virus, yellow fever virus, foot-and-mouth disease virus, and Lassa virus.
The drug inhibits the RNA-dependent RNA polymerase (RdRP) of 72 influenza and many other RNA viruses. Favipiravir acts by inhibiting this viral RNA dependent RNA polymerase (RdRp) enzyme, an enzyme impeding replication of RNA viruses and allowing facile insertion of Favipiravir into viral RNA while sparing human DNA.
Considering the COVID pandemic, absence of any approved medication for effective management and treatment, Favipiravir was preferred based on the available safety and efficacy details. The data suggest a high dose of Favipiravir was required to inhibit SARS-CoV-2 infection in Vero cells. The recommended dose for clinical use in the majority of the countries is 1800 mg bid on day 1, followed by 800 mg bid on days 2–14. It was observed that the high oral dosage leading to poor compliance of the patients. Further, it is also observed ethnic variations in the pharmacokinetic profile of the drug.
In an in vivo study, upon administration of single and multiple doses of Favipiravir lower concentration was observed in the lungs than in plasma. Further, a high dose of Favipiravir in animal models leads to mutations in the viral genomes (large number of G?A and C?U mutations). The mean number of mutations increased with an increase in the dosage of Favipiravir.
The oral administration of favipiravir 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 like ALT and AST.
Favipiravir administered at high dose of 1,000 mg•kg-1•d-1 in the hamster transmission model markedly blocked viral infection of sentinel hamsters that were in direct contact with infected hamsters. Favipiravir can be administered as a prophylactic before the onset of COVID symptoms, however administration of high doses can lead to adverse effects.
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 Favipiravir.
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 favipiravir having a particle size in the range of 1 µm to 6 µm; first lactose having a particle size in the range of 19 µm to 93 µm; second lactose having a particle size in the range of 1 µm to 60 µm, and optionally at least one excipient. The weight ratio of the first lactose to the second lactose is in the range of 6:1 to 10:1.
In another aspect, the process for the preparation of dry powder inhalation formulation comprises blending of first lactose and 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%). Additives such as magnesium stearate and citric acid are mixed with the first portion and blended to obtain a first resultant mixture. Separately, a predetermined amount of Favipiravir is mixed with the second portion 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 15 rpm to 30 rpm for 20 minutes to 40 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 HPLC chromatogram of diluent (water: methanol, 1:1)
Figure 1B illustrates a HPLC chromatogram of the standard solution of Favipiravir 100ppm;
Figure 1C illustrates a HPLC chromatogram of 100 ppm solution of Favipiravir 10 mg dry powder inhalation (DPI) formulation of the present disclosure stored for 3 months at 40 °C/75%RH;
Figure 1D illustrates a HPLC chromatogram of 100 ppm solution of Favipiravir 10 mg dry powder inhalation (DPI) formulation of the present disclosure stored for 3 months at 2-8 °C;
Figure 1E illustrates a HPLC chromatogram of 100 ppm solution of Favipiravir 20 mg dry powder inhalation (DPI) formulation of the present disclosure stored for 3 months at 40 °C/75%RH;
Figure 1F illustrates a HPLC chromatogram of 100 ppm solution of Favipiravir 20 mg dry powder inhalation (DPI) formulation stored for 3 months at 2-8 °C;
Figure 2A illustrates a graph depicting cumulative (% undersize) particle size distribution of dry powder inhalation (DPI) formulation of Favipiravir 10 mg of the present disclosure stored for 3 months at 40 °C/75%RH;
Figure 2B illustrates a graph depicting drug deposition of dry powder inhalation (DPI) formulation of Favipiravir 10 mg of the present disclosure stored for 3 months at 40 °C/75%RH;
Figure 2C illustrates a graph depicting cumulative (% undersize) particle size distribution of dry powder inhalation (DPI) formulation of Favipiravir 20 mg of the present disclosure stored for 3 months at 40 °C/75%RH; and
Figure 2D illustrates a graph depicting drug deposition of dry powder inhalation (DPI) formulation of Favipiravir 20 mg of the present disclosure stored for 3 months at 40 °C/75%RH.
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.
The recommended oral dose of favipiravir for clinical use is 1800 mg bid on day 1, followed by 800 mg bid on days 2 to 14. It is observed that due to high oral dosage, poor compliance of the patients is observed. Moreover, the oral administration of favipiravir is 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 an increased level of liver enzymes like 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 favipiravir having a particle size in the range of 1 µm to 6 µm;
• first lactose having a particle size in the range of 19 µm to 93 µm;
• second lactose having a particle size in the range of 1 µm to 60 µm; and
• optionally at least one excipient.
In an embodiment, the micronized favipiravir is in an amount in the range of 35 wt% to 85 wt% with respect to the total weight of the formulation.
In an embodiment, the first lactose is in an amount in the range of 17 wt% to 55 wt% with respect to the total weight of the formulation.
In an embodiment, the mean particle size (d10) of the first lactose is in the range of 19 µm to 43 µm.
In an embodiment, the mean particle size (d50) of the first lactose is in the range of 53 µm to 66 µm.
In an embodiment, the mean particle size (d90) of the first lactose is in the range of 75 µm to 93 µm.
In an embodiment, the second lactose is in an amount in the range of 1 wt% to 8 wt% with respect to the total weight of the formulation.
In an embodiment, the mean particle size (d10) of the second lactose is in the range of 1 µm to 6 µm.
In an embodiment, the mean particle size (d50) of the second lactose is in the range of 10 µm to 30 µm.
In an embodiment, the mean particle size (d90) of the second lactose is in the range of 50 µm to 60 µm.
In an embodiment, the weight ratio of the first lactose to the second lactose is in the range of 6:1 to 10:1. In an exemplary embodiment of the present disclosure, the weight ratio of first lactose to second lactose is 9:1.
In an embodiment, the MMAD of the dry powder inhalation is in the range of 1 µm to 4 µm.
In an embodiment, the excipient is in an amount in the range of 1 wt% to 7 wt% with respect to the total weight of the formulation.
In an embodiment, the excipient is selected from citric acid, magnesium stearate, and a combination thereof.
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 the first step, the first lactose and second lactose are blended to obtain the lactose mixture. The first lactose mixture is sifted through 80 mesh in controlled temperature and humidity conditions to obtain a homogeneous lactose mixture. The homogeneous lactose mixture is divided into two portions i.e. first portion (~75%) and the second portion (~25%).
In a second step, additives magnesium stearate and citric acid are mixed with the first portion and blended to obtain the first resultant mixture.
In a third step, separately Favipiravir is mixed with the second portion, the mixture is sifted through 80 mesh 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 15 rpm to 30 rpm for a period of 20 minutes to 40 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 Favipiravir is >2.13 mg a.i./kg b.wt. when administered through inhalation for up to 2 weeks.
The dry powder inhalation formulation of the present disclosure when administered to female Wistar rats, it is observed that the lung to plasma ratio of favipiravir is in the range of 0.15 to 0.49.
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: Favipiravir - Excipient Compatibility Study
The compatibility of Favipiravir (API) has been evaluated with different carriers i.e., lactose and mannitol. The favipiravir and carrier were mixed in equal proportions in the ratio of 1:1 to obtain API-Carrier mixture and evaluated in packed conditions (amber coloured glass vials (10 ml) with a rubber closure and flip off seal) and open conditions. The formulations were subjected to accelerated stability studies. The ratio of favipiravir (API) and carrier, stability conditions, and impurity profile of API-Carrier mixture are illustrated below in Tables 1-3.
Table 1: Ratio of favipiravir (API) to carrier
Sr. No. Code API Excipients Drug: Excipient Ratio Quantity
(In duplicate)
1 1170-04-A Favipiravir NA 1 2 gm
2 1170-04-B Favipiravir Lactose 1:1 2 gm
3 1170-04-C Favipiravir Mannitol 1:1 2 gm
Table 2: Stability conditions of API-Carrier mixture
S. No. Stability Conditions Period (4 Weeks)
Open Closed
1 Initial x ?
2 25°C/60%RH ? ?
3 40°C/75%RH ? ?
4 60oC ? (15 days) ? (15 days)
Table 3: Impurity profile of API-Carrier mixture
Sr. No. API Excipients Condition Total Impurities %
(Closed vials) Total Impurities %
(Open vials)
1 Favipiravir NA 25°C/60% RH 0.081 0.088
2 Favipiravir NA 40°C/75% RH 0.064 0.079
3 Favipiravir NA 60°C (15 days) 0.066 0.537
4 Favipiravir Lactose 25°C/60% RH 0.079 0.085
5 Favipiravir Lactose 40°C/75% RH 0.062 0.064
6 Favipiravir Lactose 60°C (15 days) 0.059 0.389
7 Favipiravir Mannitol 25°C/60% RH 0.077 0.085
8 Favipiravir Mannitol 40°C/75% RH 0.064 0.061
9 Favipiravir Mannitol 60°C (15 days) 0.066 0.466
RH= Relative humidity
It is evident from the above data that Favipiravir (API) is compatible with lactose and mannitol, at 25 °C/60% RH, and 40 °C/75% RH conditions, wherein increase in impurities were not observed. The API-Carrier mixture samples were kept open at 60 oC for 15 days, wherein it was observed that the Favipiravir API when kept open has higher impurity levels when compared to samples stored at 25 °C/60% RH, and 40 °C/75% RH. However, the Favipiravir lactose mixture has lower impurity levels when compared to the Favipiravir mannitol mixture and favipiravir alone.
Example 2: Preparation of DPI formulation
Dry mixing:
12.15 gm of first lactose (respitose SV003) and 1.35 gm of second lactose (respitose ML006) were mixed using double lined polybags and sifted using 80 mesh to obtain a homogeneous lactose mixture. The homogeneous lactose mixture was divided into two portions i.e. first portion (~75%) and a second portion (~25%). 1.25_gm of magnesium stearate and 0.25 gm of citric acid were added to the first portion and blended to obtain the first resultant mixture. Separately 10 gm of Favipiravir (particle size of 6 microns was mixed with the second portion to obtain the mixture, the mixture was sifted through 80 mesh to obtain the second resultant mixture. The first resultant mixture and the second resultant mixture mixtures are transferred to a double cone blender and blended at a speed of 15 rpm for a period of 20 minutes to obtain the dry powder inhalation (DPI) formulation of Favipiravir (Formulation 1).
The so obtained dry powder inhalation (DPI) formulation of Favipiravir was loaded into size 3 HPMC capsules using a capsule filler.
Example 3:
The dry powder inhalation formulations were prepared in the similar manner as disclosed in Example 2, varying the concentration of ingredients according to the formulations as illustrated in Table 4.

Table 4:
Ingredient (mg) Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9
Favipiravir (micronized) 10 - 10 - 20 - - -
Favipiravir (repeat micronized) - 10 - 10 - 20 20 20
First lactose 13.5 13.5 13.5 12.15 4.5 4.5 4.3875 3.15
Second lactose 1.5 1.5 1.5 1.35 0.5 0.5 0.4875 0.35
Citric acid - - - 0.25 - - 0.125 0.25
Magnesium Stearate - - - 1.25 - - - 1.25

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.
APSD emitted from each dose of the dry powder inhalation formulation of the present disclosure was evaluated using Next Generation Impactor (NGI) instrument. The instrument was loaded with 100 mg of the dry powder formulation, the flow rate through the instrument during testing was regulated at 83 L/minute. The dry powder inhalation formulation collected in the filter was analyzed for fine particle fraction and MMAD. The procedure was repeated for the various formulations (Favipiravir 10 mg and 20 mg), the results as illustrated in Tables 5 and 6.
Table 5:
Favipiravir 10 mg Powder for Inhalation
Batch No. Composition FPF (%) MMAD (µm)
1172-02 In-house micronized API +Respitose SV003+ML006 (9:1) 25.753 4.829
1172-03 In-house micronized API (second cycle)+Respitose SV003+ML006 (9:1) 27.876 4.463
1172-07 Micronized API without excipient 29.159 4.904
1172-08A Blend filled in HG capsules 26.59 4.987
1172-08B Blend filled in Mg stearate coated HG capsules 28.94 4.913

Table 6:
Favipiravir 20 mg Powder for Inhalation
Batch No. Composition FPF (%) MMAD (µm)
1173-01 In-house Micronized API + Respitose SV003+ML006 (9:1) 20.493 5.307
1173-02 API from Microfine + Respitose SV003+ML006 (9:1) 17.843 1.841
1173-03 API + Magnesium Stearate (0.5%) 22.902 1.821
1173-04 API + Magnesium Stearate (5%) + Citric acid (1.0%) 22.732 5.402
1173-07 API without excipients 23.013 4.857

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 (GII) was administered Favipiravir at a dose of 2.13 mg/kg body weight once daily for 14 days.
The Favipiravir was loaded in the rotating brush generator and dust was generated. The Favipiravir 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 26 litres per minute for GII and 39 litres per minute for GI. Rats belonging to GI and GII were exposed for 15 minutes.
The mean delivered doses were 2.13 mg a.i./kg b. wt. for Favipiravir (mean measured value). The mass median aerodynamic diameter (MMAD) of Favipiravir aerosols was found within a range of 3.54 and 3.93 µm with a geometric standard deviation (GSD) within a range of 1.59 and 1.62.
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 analysed.
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 3.54 and 3.93 µm with a geometric standard deviation (GSD) within a range of 1.59 and 1.62.
In GII rats were treated with dry powder inhalation formulation of the present disclosure, showed 121.490 to 164.484 ng/mL of Favipiravir in plasma for male rats and 279.334 to 816.285 ng/mL of Favipiravir in plasma for female rats and 28.918 to 80.998 ng/gram of Favipiravir in lungs for male rats and 73.640 to 161.280 ng/gram of Favipiravir in lungs for female rats.
The No Observed Effect Level (NOEL) of Favipiravir was >2.13 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 7 and 8, and Tables 9 and 10.
Table 7: 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 8: Body Weight (g) of Individual Rat (Group GII)
Rat No. Sex On Days
1 4 8 15
13 M 295.4 298.7 308.1 322.9
14 M 294.3 297.2 304.6 328.6
15 M 274.7 276.2 282.9 299.7
16 M 269.2 273.9 283.3 296.8
17 M 267.8 275.2 289.1 303.9
18 M 260.8 269.4 284.8 300.5
19 F 204.8 210.8 218.3 229.1
20 F 202.1 204.2 207.2 212.5
21 F 194.7 197.8 202.8 212.4
22 F 183.2 192.5 193.1 199.0
23 F 179.6 190.2 198.8 210.4
24 F 175.8 178.8 181.9 190.6

Table 9: 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 10: Percent Body Weight Change of Individual Rat (Group GII)
Rat No. Sex Body Weight Change (%)
On Days
4 8 15
13 M 1.1 4.3 9.3
14 M 1.0 3.5 11.7
15 M 0.5 3.0 9.1
16 M 1.7 5.2 10.3
17 M 2.8 8.0 13.5
18 M 3.3 9.2 15.2
19 F 2.9 6.6 11.9
20 F 1.0 2.5 5.1
21 F 1.6 4.2 9.1
22 F 5.1 5.4 8.6
23 F 5.9 10.7 17.1
24 F 1.7 3.5 8.4
It is evident from the above data 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 leftover
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 11 and 12

Table 11: Feed Consumption (g/rat/day) of Individual Rat (Group GI)
Cage No. A. No. & Sex On Days Cage No. A. No. & Sex On Days
1-3 4-7 8-14 1-3 4-7 8-14
1 1M and 2M 22.65 22.58 21.43 4 7F and 8F 16.85 16.25 16.95
2 3M and 4M 21.35 18.91 18.08 5 9F and 10F 14.18 15.24 16.13
3 5M and 6M 20.37 18.84 19.84 6 11F and12F 13.37 13.06 13.32
Table 12: Feed Consumption (g/rat/day) of Individual Rat (Group GII)
Cage No. A. No. & Sex On Days Cage No. A. No. & Sex On Days
1-3 4-7 8-14 1-3 4-7 8-14
7 13M and
14M 21.58 22.48 20.09 10 19F and
20F 16.58 16.71 18.28
8 15M and 16M 19.23 20.21 20.11 11 21F and 22F 14.83 14.88 14.11
9 17M and 18M 22.08 22.99 20.31 12 23F and
24F 13.88 13.46 13.21
It is evident from the above data that there was no statistical difference observed in the feed consumption during the evaluation period.
iv. Concentration of Favipiravir in rat plasma samples
The rats were administered with 2.13 mg/kg body weight of the dry powder inhalation formulation of the present disclosure. The rat plasma was obtained on the 14th day of administration, the concentration of Favipiravir in the plasma was evaluated by LC-MS/MS.
The analysed concentration of Favipiravir is illustrated in Table 13 below:
Table 13– Concentration of Favipiravir 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
GII M_13 154.274
GII M_14 133.246
GII M_15 125.775
GII M_16 121.490
GII M_17 145.156
GII M_18 164.484
GII F_19 279.334
GII F_20 313.787
GII F_21 452.530
GII F_22 622.428
GII F_23 816.285
GII F_24 300.023
BLQ = Below Limit of Quantification
v. Concentration of Favipiravir in Rat (Lung Homogenate) samples
The Favipiravir content in the lungs was analysed by measuring the Favipiravir content in lung homogenate by using LC-MS/MS, the concentration is as illustrated in Table 14.
Table 14 – Concentration of Favipiravir in lung homogenate
Sample ID Analysed concentration in lung homogenate (ng/mL) Concentration of Favipiravir 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
GII M_13 40.344 80.688
GII M_14 30.308 60.616
GII M_15 19.074 38.148
GII M_16 27.592 55.184
GII M_17 14.459 28.918
GII M_18 40.499 80.998
GII F_19 55.545 111.090
GII F_20 36.820 73.640
GII F_21 42.425 84.850
GII F_22 48.427 96.854
GII F_23 80.640 161.280
GII F_24 73.701 147.402
BLQ = Below Limit of Quantification
The concentration of Favipiravir 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 analysed for cellular contents, the details are illustrated below in Table 15;
TABLE 15: Broncho-Alveolar Lavage Analysis - Group Mean Values
Sex: Male
Group N GI (N=6) GII (N=6)
Group Control Favipiravir
Parameter Mean SD Mean SD
WBC (x103/µL) 0.43 0.19 0.48 0.11
Neutrophil (x103/µL) 0.11 0.07 0.10 0.02
Lymphocyte (x103/µL) 0.25 0.13 0.28 0.09
Monocyte (x103/µL) 0.01 0.01 0.01 0.01
Eosinophil (x103/µL) 0.00 0.00 0.00 0.00
Basophil (x103/µL) 0.03 0.02 0.04 0.02
LUC* (x103/µL) 0.07 0.05 0.09 0.03
LDH# (U/L) 81.67 30.08 82.83 37.85
Total protein (ug/mL) 170.07 53.29 173.25 39.90
* = Large unstained cells
# = Lactate dehydrogenase

Sex: Female
Group N GI (N=6) GII (N=6)
Group Control Favipiravir
Parameter Mean SD Mean SD
WBC (x103/µL) 0.55 0.07 0.66 0.26
Neutrophil (x103/µL) 0.13 0.04 0.13 0.07
Lymphocyte (x103/µL) 0.31 0.06 0.39 0.20
Monocyte (x103/µL) 0.01 0.01 0.01 0.01
Eosinophil (x103/µL) 0.01 0.01 0.00 0.01
Basophil (x103/µL) 0.05 0.02 0.07 0.08
LUC* (x103/µL) 0.09 0.04 0.12 0.03
LDH# (U/L) 81.00 25.27 121.67 48.67
Total protein (ug/mL) 167.40 47.85 182.43 24.66
* = Large unstained cells
# = Lactate dehydrogenase

It is evident from the above data 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 (GII) were compared well with the control group (GI), the results are illustrated below in Table 16.
TABLE 16: Organ Weight (g) – Group Mean Values
Sex: Male
Group N GI (N=6) GII (N=6)
Group Control Favipiravir
Parameter Mean SD Mean SD
Liver 11.215 1.328 11.581 0.844
Heart 0.902 0.075 0.939 0.070
Spleen 0.632 0.150 0.615 0.023
Brain 2.046 0.153 2.002 0.055
Thymus 0.481 0.091 0.522 0.018
Kidneys 1.722 0.111 1.804 0.172
Adrenals 0.075 0.005 0.085 0.011
Testes 3.426 0.975 3.689 0.226
Epididymides 1.040 0.185 1.124 0.139
Left Lung 0.413 0.038 0.439 0.027
Sex: Female
Group N GI (N=6) GII (N=6)
Group Control Favipiravir
Parameter Mean SD Mean SD
Liver 8.417 0.429 7.929 0.631
Heart 0.711 0.092 0.763 0.081
Spleen 0.520 0.110 0.525 0.076
Brain 1.920 0.074 1.922 0.054
Thymus 0.521 0.068 0.448? 0.040
Kidneys 1.466 0.120 1.448 0.090
Adrenals 0.103 0.011 0.094 0.020
Ovaries 0.127 0.014 0.111 0.016
Uterus with cervix 0.715 0.156 0.604 0.210
Left Lung 0.321 0.022 0.309 0.016
It was observed from the above data that, a statistically significant decrease was observed in the weight of thymus in GII 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 17.

TABLE 17: Gross Findings – Summary by Groups
Sex: Male
Group N GI (N=6) GII (N=6)
Group Control Favipiravir
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) GII (N=6)
Group Control Favipiravir
Organs and Lesions No. of Animals 6 6
MACROSCOPIC EXAMINATION
External
No Abnormality Detected 6 6
Internal
No Abnormality Detected 6 6
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 an in vivo study (Gowen BB et al., 2015), the female Syrian golden hamsters were administered a single 0.2 ml oral (p.o.) treatment of 100 mg/kg favipiravir on day 7 post-challenge with PICV (strain An 4763). It was observed that the lung to plasma ratio is 0.29.
The dry powder inhalation formulation of the present disclosure was administered at a dose of 2.13 mg/kg/day in the female Wistar rats. It was observed that the lung to plasma ratio of favipiravir is in the range of 0.15 to 0.49.
It is evident from the above data, that the dry powder inhalation formulation of the present disclosure achieves similar lung concentration with a low dose of administration in comparison to conventional oral administration.
Example 7:
The dry powder inhalation (DPI) formulation of Favipiravir in capsules of the present disclosure was subjected to 3 months accelerated stability testing at 40 °C/75%RH and 2-8 °C. The content of Favipiravir in the capsules after 3 months of accelerated stability testing was evaluated by HPLC chromatography. The chromatographic conditions are provided below,
Buffer solution: The buffer solution was prepared by dissolving 1 mL of orthophosphoric acid in 1000 mL of water, mixed well, and filtered through a 0.45 µm filter.
Mobile Phase: The mobile phase was prepared by mixing buffer solution and acetonitrile in the ratio of 75:25 % v/v.
Diluent: The diluent was prepared by mixing water and methanol in the ratio of 1:1 % v/v. The chromatogram of diluent was illustrated in Fig. 1A.
Standard solution: 25 mg of Favipiravir was mixed with 35 mL of diluent and sonicated to dissolve, cooled to room temperature and diluted to 50 mL with diluent to obtain a solution. Further, 5 ml of the solution is diluted to 25 ml with diluent to obtain a standard solution containing 100 ppm of Favipiravir. The chromatogram of the standard solution was illustrated in Fig. 1B.
Sample preparation (Favipiravir 10mg capsules): Collect dry powder inhalation (DPI) formulation from 20 capsules and the average weight of the content was calculated. The contents of the capsule i.e. 25 mg of dry powder inhalation formulation (equivalent to about 10 mg of Favipiravir) was mixed with 70 mL of diluent and sonicated for 15 minutes to obtain a sonicated DPI solution. The sonicated DPI solution was cooled and diluted to 100 mL with diluent and mix well to obtain a DPI solution. The DPI solution was filtered through a 0.45 µm nylon filter to obtain a filtered DPI solution. The first 5mL of the filtered DPI solution was discarded and the remaining filtered DPI solution (100 ppm solution) was used for evaluation.
Sample preparation (Favipiravir 20 mg capsules): Collect dry powder inhalation (DPI) formulation from 20 capsules and the average weight of the content was calculated. The contents of the capsule i.e. 25 mg of sample (equivalent to about 20 mg of Favipiravir) was mixed with 140 mL of diluent and sonicated for 15 minutes to obtain a sonicated DPI solution. The sonicated DPI solution was cooled and diluted to 200 mL with diluent and mix well to obtain a DPI solution. The DPI solution was filtered through a 0.45 µm nylon filter to obtain a filtered DPI solution. The first 5mL of the filtered DPI solution was discarding and the remaining filtered DPI solution (100 ppm solution) was used for evaluation.
The standard solution was subjected to chromatography and peak response was recorded. The % RSD of the area of the analyte of five replicate standard injections should not be more than 2.0%, the column efficiency should not be less than 2000 theoretical plates, and the tailing factor should not be more than 2.0.
The Favipiravir content was evaluated by separately injecting an equal volume of the diluent, standard, and sample solutions into the chromatograph, recorded the chromatograms, and measured the responses for the analyte peak, in standard and sample, and determining the amount of Favipiravir in % assay by the following formulae.
For Favipiravir Inhalation Powder 10 mg
Favipiravir (mg/Capsule) = AT x Wt.Std x 5 x 100 x P x 1000 x Avg wt
As 50 25 Wt.Spl 100
For Favipiravir Inhalation Powder 20 mg
Favipiravir (mg/Capsule) = AT x Wt.Std x 5 x 200 x P x 1000 x Avg wt
As 50 25 Wt.Spl 100

Assay (%) = (mg/capsule found) x 100
L.C.
Where,
AT : Average area of analyte peak in the chromatogram of sample solution;
As : Average area of analyte peak in the chromatogram of standard solution;
Wt. std.: Weight of standard in mg;
Wt. spl.: Weight of sample in mg;
Avg.wt.: Average content weight of sample powder in mg;
P : Potency of Standard (on as is basis);
L.C. : Label Claim in mg per capsule.
The chromatograms of dry powder inhalation (DPI) formulation of Favipiravir 10 mg stored for 3 months at 40 °C/75%RH and 2-8 °C were illustrated in Fig. 1C and 1D respectively.
The chromatograms of dry powder inhalation (DPI) formulation of Favipiravir 20 mg stored for 3 months at 40 °C/75%RH and 2-8 °C were illustrated in Fig. 1E and 1F respectively.
It is evident from the chromatograms that there was no change in the Favipiravir content in dry powder inhalation (DPI) formulations of Favipiravir 10 mg and Favipiravir 20 mg stored for 3 months at 40 °C/75%RH and 2-8 °C.
Further, dry powder inhalation (DPI) formulation of Favipiravir 10 mg stored for 3 months at 40 °C/75%RH 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) instrument. The instrument was loaded with content of the 10 mg capsule of the dry powder formulation, the flow rate through the instrument during testing was regulated at 83 L/minute. The procedure was repeated for Favipiravir 20 mg. The dry powder inhalation formulation collected in the filter was analyzed for Favipiravir content in lungs, fine particle fraction, MMAD, and geometric standard deviation (GSD), results are provided below in Table 18. The cumulative (% undersize) particle size distribution, drug deposition for Favipiravir 10 mg and Favipiravir 20 mg was illustrated in Fig. 2A, 2B, 2C, and 2D respectively.
TABLE 18:
DPI formulation Favipiravir content in lungs FPF (%) MMAD (µm) GSD
Favipiravir 10 mg 2.2 mg 25.923 5.126 1.901
Favipiravir 20 mg 4.2 mg 26.512 5.040 1.865

It is evident that irrespective of Favipiravir dose, nearly 20% reaches the lungs.

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 Favipiravir, thus reduces dosage amount when compared to 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.