Claims:CLAIMS
We claim;
1. A pharmaceutical composition of nanoparticle based drug delivery of pirfenidone comprising pirfenidone, biodegradable polymer, stabilizer, mucoadhesive polymer and a solvent.
2. The pharmaceutical composition of nanoparticle based drug delivery of pirfenidone as claimed in claim 1, wherein the biodegradable polymer is selected from poly glycolic acid, poly (lactic-co-glycolic acid), poly hydroxy butyrate and polycaprolactone.
3. The pharmaceutical composition of nanoparticle based drug delivery of pirfenidone as claimed in claim 1, wherein the stabilizer is selected from sodium alginate, sodium carboxymethyl cellulose (CMC), guar gum, poly vinyl alcohol and calcium stearate.
4. The pharmaceutical composition of nanoparticle based drug delivery of pirfenidone as claimed in claim 1, wherein the mucoadhesive polymer is selected from chitosan, poly acrylic acid, alginate, poly methacrylic acid, pectin, gelatin and sodium carboxymethyl cellulose.
5. The pharmaceutical composition of nanoparticle based drug delivery of pirfenidone as claimed in claim 1, wherein the solvent is selected from toluene, xylene, dichloromethane, chloroform and acetone.
6. The pharmaceutical composition of nanoparticle based drug delivery of pirfenidone as claimed in claim 1, wherein the pirfenidone used is in the range from 1 to 200 mg.
7. The pharmaceutical composition of nanoparticle based drug delivery of pirfenidone as claimed in claim 1, wherein the biodegradable polymer used is in the range from 1 to 200 mg, the stabilizer used is in the range from 1 to 5% w/v, the mucoadhesive polymer used is in the range from 1 to 300mg, the solvent is used in the range from 1 to 10 ml.
8. The pharmaceutical composition of nanoparticle based drug delivery of pirfenidone as claimed in claim 1, wherein the process of preparation comprises the steps of;
a) dissolving pirfenidone and poly (lactic-co-glycolic acid) in dichloromethane;
b) dissolving chitosan in the aqueous solution of polyvinyl alcohol with 4% acetic acid;
c) adding drop wise step (a) solution in the step (b) solution and stirring the solution for 5 min;
d) sonicating the step (c) solution over ice bath for 5 min and keeping on a magnetic stirrer under the atmospheric conditions at 400 rpm for 3 to 4 h to complete evaporation of dichloromethane and forming polymeric nanoparticles;
e) centrifuging the nanoparticles from step (e) at 15,000 rpm for 30 min and washing twice with phosphate buffer;
f) freezing the nanoparticles from step (e) in a glycol bath and lyophilizing at 790C for 24 hours to obtain dry powder;
g) filing the dry powder from step (f) in the vials and aerosolizing using a dry powder inhaler.
, Description:
FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. TITLE OF THE INVENTION – A PHARMACEUTICAL COMPOSITION OF NANOPARTICLE BASED DRUG DELIVERY OF PIRFENIDONE
2. Applicant (s)
NAME: R K UNIVERSITY
NATIONALITY: INDIAN
ADDRESS: R K UNIVERSITY, SCHOOL OF PHARMACY, BHAVNAGAR HIGHWAY, TRAMBA, RAJKOT-360020, GUJRAT, INDIA

3. PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and the manner in which it is to be performed.

A PHARMACEUTICAL COMPOSITION OF NANOPARTICLE BASED DRUG DELIVERY OF PIRFENIDONE

FIELD OF INVENTION
The present invention is related to a pharmaceutical composition of nanoparticle based drug delivery of pirfenidone. The present invention is about a pharmaceutical composition of nanoparticle based drug delivery of pirfenidone comprising pirfenidone, biodegradable polymer, stabilizer, mucoadhesive polymer and a solvent. The present invention is also related to a pharmaceutical composition of nanoparticle based drug delivery of pirfenidone and process of preparing the same.

BACKGROUND OF THE INVENTION
Idiopathic pulmonary fibrosis (IPF) is a destructive, chronic, irreversible, progressive, age-related lethal lung disease. It is a fatal disease in which the uncontrolled deposition of the extracellular matrix leads to progressive loss of lung function. Idiopathic pulmonary fibrosis is a chronic, progressive lung disease. This condition causes scar tissue (fibrosis) to build up in the lungs, which makes the lungs unable to transport oxygen into the bloodstream effectively. The disease usually affects people between the ages of 50 and 70.

Current pulmonary treatment against Idiopathic pulmonary fibrosis are with high mortality rate and less recovery with phototoxicity. IPF is reviewed as more harmful than many cancers. IPF commonly occurs with a male over middle-aged and elderly adults (range 55–75 years), with a history of cigarette smoking, and it is restricted to the lungs. The major introducing symptoms are shortness of breath and chronic cough. IPF has non-pharmacological treatments like oxygen therapy and lung transplantation is available, but it is costly and has a lower recovery rate. Pharmacological drug treatment with Pirfenidone is widely used and preferable.

Pirfenidone (PFD) is an orally administered pyridine that has an orphan drug for the treatment of mild to moderate IPF and was approved by the USFDA in 2014. Pirfenidone is chemically described as 5-methyl-1-phenyl-2-(1H)-pyridone (figure 2). It is a white to pale yellow powder that is aqueous solubility that is too high at 25ºC and non-hygroscopic.

Pirfenidone in the form of inhalation powder is an interesting concept to reduce side effects by targeting IPF. It is available in the market having a dose of 267 mg/capsule by oral conventional dosage form and administered repeatedly due to a short biological half-life of 2.5 h. The treatment started with 801 mg/day for the first 7 days and 1602 mg/day for the next 7 days. The recommended daily dose of Pirfenidone for IPF was found to be 2403 mg/day (3 tablets of 267 mg three times a day) after 15 days with food, but this large dose contains many adverse effects on the body. Patients treated with pirfenidone 2403 mg/day; Photosensitivity responses were more common observed (9%) in Phase 3 investigations. The other most common adverse reactions (=10%) are nausea, vomiting, diarrhea, rash, headache, dizziness, abdominal pain, upper respiratory tract infection, fatigue, dyspepsia, anorexia, insomnia, sinusitis, gastroesophageal reflux disease, weight decreased, and arthralgia.

Different types of nanoparticles have been used as pulmonary drug delivery systems. Among them, polymer-based systems can be the best candidate, because of their unique properties. Pulmonary drug delivery offers a vast variety of advantages over conventional oral drug delivery. Nanoparticle-mediated drug delivery systems open new perspectives by modifying the physical properties of the particles. The large surface area with faster absorption of drug molecules has been due to high vascularization and the elimination of the first-pass effect. This targeted drug delivery reduces the total daily dose quantity, hence, reduces the side effects.

The existing treatments have their drawbacks that they do not comply with the need of an appropriate treatment that is safe and effective to be used in the IPF.

Therefore, here in the present invention the inventors surprisingly found that the present invention can overcome the above stated problems associated with the existing treatment. The present invention is a stable formulation which is having polymeric nano particles used for targeting pulmonary drug delivery.

OBJECT OF THE INVENTION
The main object of the present invention is to provide a pharmaceutical composition of nano particle based drug delivery of pirfenidone.

The other main objective of the present invention is to provide a pharmaceutical composition of nano particle based drug delivery of pirfenidone which is having better patient compliance.

Another object of the present invention is to provide a pharmaceutical composition of nano particle based drug delivery of pirfenidone which is safe and effective.

Another object of the present invention is to provide a pharmaceutical composition of nano particle based drug delivery of pirfenidone which is having lower drug dose with minimize adverse effects.

The other object of the present invention is to provide a pharmaceutical composition of nano particle based drug delivery of pirfenidone which is having polymeric nano particles having high potential for the targeted drug delivery of of therapeutic agents to treat idiopathic pulmonary fibrosis.

SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a pharmaceutical composition of nano particle based drug delivery of pirfenidone.

The other main aspect of the present invention is to provide a pharmaceutical composition of nano particle based drug delivery of pirfenidone comprising pirfenidone, biodegradable polymer, stabilizer, mucoadhesive polymer and a solvent.

Another aspect of the present invention is to provide a pharmaceutical composition of nano particle based drug delivery of pirfenidone and the process of preparation of the same.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: In vitro drug release study of pure drug and chitosan coated pirfenidone loaded poly (lactic-co-glycolic acid) nano particles
Figure 2: In vitro drug release study of chitosan coated pirfenidone loaded poly (lactic-co-glycolic acid) nano particles after 180 days stability study
Figure 3: In vitro drug deposition of uncoated and chitosan coated NPs by Andersen cascade impactor
Figure 4.1: Surface morphology using scanning electron microscopy of raw pirfenidone drug powder
Figure 4.2: (a) SEM images of surface modified poly (lactic-co-glycolic acid) nanoparticles of pirfenidone (for optimized batch F100) at initial stage (b) SEM image after 6 months

DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

The main embodiment of the present invention is to provide a pharmaceutical composition of nano particle based drug delivery of pirfenidone.
As used herein, whether in a transitional phrase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps.

As used herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise.

The term “about” is used herein to means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” or “approximately” is used herein to 25 modify a numerical value above and below the stated value by a variance of 20%.

As used herein “dry powder inhalation pharmaceutical formulation or composition” is the use of particle engineering to produce low density particles that can be easily entrained in the patient's inhalation. The preferred modern treatment of respiratory diseases is with inhalation therapy, where the medicine is not taken as a tablet or liquid by mouth (orally), but is breathed in, or inhaled

As used herein “dry powder inhaler” is as mentioned here is defined as is a device that delivers medication to the lungs in the form of a dry powder. dry powder inhalers are commonly used to treat respiratory diseases.

As used herein “nanoparticles” means a small particles that ranges between 1 to 100 nanometres in size. Undetectable by the human eye, nanoparticles can exhibit significantly different physical and chemical properties to their larger material counterparts. Targeting Nanoparticles offer the possibility for specific tissue or cell targeting, which has the potential to greatly increase their therapeutic effect and decrease drug toxicity for the treatment of Idiopathic pulmonary fibrosis.
As used herein “muco adhesive polymer” is defined as low molecular weight polymer which reduced the exhalation rate of inhaled nanoparticles and prolong the residence time of it in the deep lung alveolar epithelial cell. The nanoparticles with mucoadhesion increases the drug release and the in vitro pirfenidone deposition activity of the formulation.

As used herein “biodegradable polymer” is defined as materials whose chemical and physical characteristics undergo deterioration and completely degrade when exposed to microorganisms, aerobic, and anaerobic processes. Biodegradable polymeric nanoparticles are widely used for targeting pulmonary delivery and may exhibit sustained release effects. Biodegradable polymer is used as a surface modification to create more mucoadhesive nanoparticles thus prolonging release rate.

As per one main embodiment the present invention is a pharmaceutical composition of nano particle based drug delivery of pirfenidone comprising pirfenidone, biodegradable polymer, stabilizer, mucoadhesive polymer and a solvent.

Pirfenidone:
The Pirfenidone used in the present invention is procured from ZCL Chemicals Ltd, Gujarat, India. It is not taken from any natural sources.

The Pirfenidone which is also known as 5-Methyl-1-phenyl-2-1(H)-pyridone and has the chemical structure depicted below:

Pirfenidone is a medication used for the treatment of idiopathic pulmonary fibrosis. It works by reducing lung fibrosis through downregulation of the production of growth factors and procollagens I and II. Pirfenidone has well-established antifibrotic and anti-inflammatory properties in various in vitro systems and animal models of fibrosis. Pirfenidone in the form of inhalation powder is an interesting concept to reduce side effects by targeting IPF.

As per one embodiment, the pirfenidone used in the present invention is in the range from 0.1 to 500 mg, preferably 1 to 400 mg, more preferably 1 to 300 mg, most preferably 1 to 200 mg.

As per one embodiment, the biodegradable polymer are used for targeting pulmonary delivery and may exhibit sustained release effects. Polymers increase surface properties and gives protection of the drug from degradation. Biodegradable polymer based nano particles are highly beneficial option for inhalation therapies.

As per one embodiment, the biodegradable polymer is selected from but not limited to poly glycolic acid, poly (lactic-co-glycolic acid), poly hydroxy butyrate and polycaprolactone. In the present invention, most preferably biodegradable polymers is poly (lactic-co-glycolic acid).

As per one embodiment, the poly (lactic-co-glycolic acid) used in the present invention is in the range from 0.1 to 500 mg, preferably 1 to 400 mg, more preferably 1 to 300 mg, most preferably 1 to 200 mg.

As per one embodiment, the mucoadhesive polymers are used for surface modification of the polymer and to create mucoadhesive nano particles for prolonged release of the drug. Surface modification of polymer nano particle using mucoadhesive polymer decrease the burst effect of the drug and increase the stability of the drug.

As per one embodiment the muco adhesive polymer is selected from but not limited to chitosan, poly acrylic acid, alginate, poly methacrylic acid, pectin, gelatin and sodium carboxymethyl cellulose. In the present invention, most preferably mucoadhesive polymers is chitosan.

As per one embodiment, the chitosan used in the present invention is in the range from 0.1 to 500 mg, preferably 1 to 500 mg, more preferably 1 to 400 mg, most preferably 1 to 300 mg.

As per one embodiment, the stabilizer used are in the aqueous phase. Increased concentration of stabilizer reduce the particle size due to more stabilizer molecules are adsorbed on the surface of emulsion droplets which provide an increased protection against coagulation of droplets, and resulting in smaller emulsion droplets formed after solvent evaporation. Nanoparticles will form if the stabilizer persists at the liquid–liquid interface during the diffusion process and its protective action is enough.

As per one embodiment, the stabilizer is selected from but not limited to sodium alginate, sodium carboxymethyl cellulose (CMC), guar gum, poly vinyl alcohol and calcium stearate. In the present invention, most preferably stabilizer is poly vinyl alcohol.

As per one embodiment, the poly vinyl alcohol used in the present invention is in the range from 0.1 to 10% w/v, preferably 1 to 8% w/v, more preferably 1 to 7% w/v, most preferably 1 to 5% w/v.

As per one other embodiment the solvent is selected from but not limited toluene, xylene, dichloromethane, chloroform and acetone. In the present invention, most preferably solvent is dichloromethane.
As per one embodiment, the dichloromethane used in the present invention is in the range from 0.1 to 50 ml, preferably 1 to 30 ml, more preferably 1 to 20 ml, most preferably 1 to 10 ml.

As per one preferred embodiment the process of preparation of the pharmaceutical composition of nanoparticle based drug delivery of pirfenidone comprises the steps of,
a) dissolving pirfenidone and poly (lactic-co-glycolic acid) in dichloromethane;
b) dissolving chitosan in the aqueous solution of polyvinyl alcohol with 4% acetic acid;
c) adding drop wise step (a) solution in the step (b) solution and stirring the solution for 5 min;
d) sonicating the step (c) solution over ice bath for 5 min and keeping on a magnetic stirrer under the atmospheric conditions at 400 rpm for 3 to 4 h to complete evaporation of dichloromethane and forming polymeric nanoparticles;
e) centrifuging the nanoparticles from step (e) at 15,000 rpm for 30 min and washing twice with phosphate buffer;
f) freezing the nanoparticles from step (e) in a glycol bath and lyophilizing at 790C for 24 hours to obtain dry powder;
g) filing the dry powder from step (f) in the vials and aerosolizing using a dry powder inhaler.

As per one embodiment, the present invention includes a dry power composition which is administered to the patient in the form of a dry powder by inhalation with an inhalation device termed as dry powder inhaler.

As per one embodiment the formulation of the invention includes polymeric nano particles. Increased drug concentration at the disease site, reduced drug degradation and loss, efficiency of designing inhalable formulations and the ability to target specific cells are all advantages of utilizing nanoparticles to deliver drugs.
As per one embodiment, the formulation of the invention includes a low molecular weight chitosan as a muco adhesive polymer wherein the chitosan is used to increase surface of the poly (lactic-co-glycolic acid) (polymer) nano particles. The modified nano particles exhibited a very high potential for the targeted delivery of therapeutic agents to treat idiopathic pulmonary fibrosis. The large surface area of the modified nano particles increase the fast absorption of the drug molecules due to high vascularization and the elimination of the first-pass effect. This targeted drug delivery minimize the daily dosage of the drug.

As per one embodiment advantages of the inhalation drug delivery are convenient and easy to inhale. Due to their prolonged action, the prepared formulations may increase patient compliance, resulting in a reduction in overall dose frequency and a decrease in side effect. The nano particles with mucoadhesion having improved effect and the drug release from the nano particles was prolonged after they were coated with chitosan. The drug deposition of the surface unmodified and modified by chitosan were revealed data the modification increases the size of the particle, so travel capacity reduced but increases deposition due to mucoadhesive properties of chitosan present on the surface of pirfenidone nanoparticles.

As per one main embodiment the inventors of the present invention have surprisingly found that a pharmaceutical composition of nanoparticle based drug delivery of pirfenidone comprising pirfenidone, biodegradable polymer, stabilizer, mucoadhesive polymer and a solvent can be used in the treatment of idiopathic pulmonary fibrosis with its safe and effectives advantages.

The invention is further illustrated by the following examples which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

EXAMPLE 1: COMPOSITION OF NANOPARTICLE BASED DRUG DELIVERY OF PIRFENIDONE
Ingredients
Batch
no. Pirfenidone (mg) Poly (lactic-co-glycolic acid) (mg) Chitosan (mg) Poly vinyl alcohol (%w/v) Dichloromethane
(ml)
PD 100 - - - -
BL 100 100 0 1% 3
F50 100 100 50 1% 3
F100 100 100 100 1% 3
F200 100 100 200 1% 3
F300 100 100 300 1% 3
Table: 1 Formulation of dry powder of pirfenidone for inhalation

Procedure:
a) pirfenidone and poly (lactic-co-glycolic acid) were dissolved in dichloromethane;
b) chitosan was dissolved in the aqueous solution of polyvinyl alcohol with 4% acetic acid;
c) step (a) solution was added drop wise in the step (b) solution and stirred the solution for 5 min;
d) the step (c) solution was sonicated over ice bath for 5 min and kept on a magnetic stirrer under the atmospheric conditions at 400 rpm for 3 to 4 h to complete evaporation of dichloromethane and polymeric nanoparticles were formed;
e) the nanoparticles from step (e) was centrifuged at 15,000 rpm for 30 min and washed twice with phosphate buffer;
f) the nanoparticles from step (e) were freezed in a glycol bath and lyophilized at 790C for 24 hours to obtain dry powder;
g) the dry powder from step (f) was filled in the vials and aerosolized using a dry powder inhaler.

EXAMPLE 2: IN VITRO DRUG RELEASE STUDIES
The in vitro drug release of pirfenidone from various nanoparticle formulations and pirfenidone from optimized formulation (as per example 1) was carried out using dialysis diffusion technique. Dry powder of 20 mg pirfenidone nanoparticles were taken into 100ml of phosphate buffer saline solution with pH 7.4. Dialysis membrane in molecular weight cut-off of 12 kDa was used for drug release manner study. The nanoparticles were poured in a dialysis bag and kept in a basket of USP type I dissolution apparatus. The system was stirred at 100 rpm and was kept at 37 ± 0.5°C. The sample was collected 1 mL from medium in time intervals of 0, 0.5, 1, 2, 4, 6, 8, 12 and 24 h and that were replaced by fresh phosphate buffer saline solution. The collected samples were passed through a 0.22 µm filter membrane and analyzed for PFD content by using UV-visible spectrophotometer at 311 nm.

The in vitro release of pirfenidone from different nanoparticle formulations is shown in Figure 1. The pure drug was completely available in the solution (98.89%) after 30 min. It is evident that the component of the nano particles affected the release of pirfenidone in vitro. All Nanoparticles showed biphasic release, with an initial burst of release arriving before the first 1 h, followed by a relatively slow drug release rate. In comparison to uncoated nano particles, the release of pirfenidone from coated nano particles was slower. After 24, the uncoated poly (lactic-co-glycolic acid) nanoparticles released 97.42±1.64% of the entrapped drug, while the chitosan coated poly (lactic-co-glycolic acid) nanoparticles released 94.34±2.46, 87.82±2.13, 77.87±2.34, and 72.81±2.22 for F50, F100, F200, and F300 respectively.

EXAMPLE 3: DRY POWDER CHARACTERISTICS OF CHITOSAN MODIFIED POLY (LACTIC-CO-GLYCOLIC ACID) NANO PARTICLES OF PIRFENIDONE
The bulk and tapped density values were attained for the both dried powder nano particles. The tapped density of each powder can be used to predict both its flow properties and respirable fraction. Lowering the tapped density of the dry powders considerably enhance the respirable fraction. The Carr’s Index, Hausner ratio and angle of repose commonly considered as proper criteria for estimation of the flow properties of solids. Dried powder nano particles showed good flow properties and low density. Chitosan modified poly (lactic-co-glycolic acid) nano particles of pirfenidone showed intermediate (fair) flow properties. It was observed that dry powder inhalation preparations composed of chitosan modified poly (lactic-co-glycolic acid) nano particles loaded with pirfenidone had relatively higher process yields because of the lower stickiness. The Dry Powder Characteristics of the inhalation formulations are summarized in Table 2.
The result are shown below:
Batch no. Bulk Density (g/ml) Tapped Density (g/ml) Carr’s index (%) Hausner ratio Angle of Repose (?) MPS (nm)
Pure
drug 0.655±
0.048 0.782±
0.016 16.24
±0.42 1.19±
0.038 35.7o
±0.87o 1343±
2.1
Blank 0.645±
0.044 0.778±
0.021 17.09
±1.15 1.20±
0.015 35.8o±
0.82o 269.6±
3.4
F50 0.63±
0.026 0.75±
0.034 16±
1. 68 1.19±
0.019 34.5o±
0.49o 250.8±
2.6
F100 0.605±
0.033 0.75±
0.017 19.33±
0.33 1.23±
0.021 33.6o±
0.27o 1761±
8.4
F200 0.659±
0.024 0.79±
0.026 16.58±
0.23 1.19±
0.039 34.6o±
0.37o 445.8±
5.2
F300 0.64±
0.019 0.78±
0.061 17.94±
0.87 1.21±
0.05 35.5o±
0.67o 287.3±
1.3
Table 2: Evaluation parameters

EXAMPLE 4: STABILITY STUDY
Accelerated stability studies of freeze-dried pirfenidone nano particles of optimized batch (F100) was conducted using particle size, zeta potential, % EE, PDI, In Vitro drug release 12 and 24 h as prime parameters. The results are reported in Table 3. It was observed from these results that there was slight but linear increase in average particle size, whereas slight but linear decrease in zeta potential and EE was observed on six-month storage. So, it can be concluded that, there were no significant alteration in average particle size, zeta potential, % EE, PDI, In Vitro drug release 12 and 24 h. Stability studies of 180 days with Particle Size and Zeta Potential of Chitosan coated pirfenidone loaded poly (lactic-co-glycolic acid) nano particles was indicated that the slight increase in the zeta potential and periodically increase the particle size. The in Vitro drug release study of chitosan coated pirfenidone loaded poly (lactic-co-glycolic acid) nano particles after 180 days was stable with no any changes as per shown in figure 2. Hence, they were found to be stable at 25±2oC/60±5% RH for a total period of six months. Freeze-drying improves the stability of nano particles and is significant for preserving their physical and chemical properties as well as ensuring long-term stability. Furthermore, the use of a cryoprotectant affects the size and size distribution of freeze-dried nano particles. The lack of a cryoprotectant during the freeze-drying process results in bigger, more prone to aggregation particles.
Evaluation
Parameter Time period for sampling
Initial After
1 month After
3 months After
6 months
Particle Size (nm) 404±9 422±9 431±10 435±5
Zeta potential (mV) 4.75±0.017 4.87±0.13 4.96±0.18 4.83±0.21
% Entrapment Efficiency 81.78% 82.11±1.19 81.35±2.19 81.47±1.28
PDI 0.695 ±0.053 0.641 ± 0.068 0.639± 0.083 0.647 ± 0.071
In Vitro drug release 12 h 83.12±1.53 82.29±2.72 82.92±2.20 80.16±2.66
In Vitro drug release 24 h 87.8±3.25 87.43±3.02 84.67±2.06 87.69±2.33
Table 3: Stability study of optimized batch (F100)

EXAMPLE 5: IN VITRO DRUG DEPOSITION STUDY
A very narrow range of aerodynamic diameter should be considered, usually accepted to vary between 0.5 to 1 µm are ideal for deep lung deposition. The deposition of particles in various sections of the trachea and lungs is determined by factors such as particle size and shape. Particles with a diameter of 0.5-5 µm can penetrate to alveoli. The dispersion and sedimentation patterns are influenced by the aerodynamic diameter, which is a combination of particle size and density. The prolonged residence of nano particles in the lungs due to ability to escape from the clearance mechanisms such as mucociliary escalator, macrophage uptake (a size of 1-2 µm is ideal for macrophage phagocytosis), and translocation to the systemic circulation is amongst the key advantages of nano particles. Nanoparticles were aerosolized as a dry powder inhaler (DPI) using Rotahaler VR with a flow rate of 28.3 L/min through the cascade impactor. After deposition of DPI in each chamber, pirfenidone content was measured, dissolved in methanol, and analyzed using HPLC.

Anderson Cascade Impactor scenario of the uncoated (BL batch) and chitosan-coated (F100-optimized batch) poly (lactic-co-glycolic acid) nanoparticles are shown in Figure 3. BL and F100 had the highest particle retention in stage 5 and lowest particle deposition at stage 8 of the impactor. The particles were deposited less than 10% in stages 0-2, 6-7. Stages 3 and 4 showed particle deposition above 10% but below 20%; whereas only stage 5 showed particle deposition above 20%.

Actuating the preparation through the device at a constant flow rate under vacuum, deposits the particles (Chitosan modified poly (lactic-co-glycolic acid) nanoparticles of pirfenidone) on different stages depending on the particle size-larger particles are retained on the initial stages of the impactor, while smaller ones are retained in the later stages, as determined by HPLC. The amount of chitosan modified poly (lactic-co-glycolic acid) nanoparticles deposited on these stages was measured, and significant particle parameters such as the FPF, MMAD, and GSD were calculated. FPF (fraction on ACI stages 3-F) is a measure of deposition efficiency in the deep lungs. MMAD is defined as the median of the airborne particle mass distribution with respect to the aerodynamic diameter. MMAD is always accompanied by GSD, which characterizes the variability of the particle size distribution.

A larger than expected particle diameter was obtained and this could be attributed to the tendency of nanoparticles to aggregate during inhalation study. The inhaled nanoparticles of pirfenidone had a geometric standard deviation varying between narrow ranges from 2.3 ± 1.25 µm to 2.8 ± 1.57 µm (Table 4). A widespread distribution of nanoparticles was attained with a substantial drug fraction being accumulated at various lower stages of impactor simulating the deep lung region, and a large mass fraction resided in the pre-separator which was acted as upper lung region to collect the non-inhalable powder. The deep lung inhalation of nanoparticles and drug deposition are required to target at damaged alveoli epithelial cell and macrophages.

The fine particle dose and to a lesser extent the fine particle fraction were directly related to the percent nanoparticle inhaled. The increase in percent nanoparticle inhaled was ascribed to the rise in percent nanoparticle dispersed following dry powder aerosolization. A higher fraction of nanoparticles and their microaggregates was released, inhaled and deposited at the lower stages of cascade impactor. This was reflected by a marked increase in the fine particle fraction of the prepared spherical nanoparticles. Hence, prepared dry powder inhaler can penetrate deep to lungs.
Evaluation Parameters % Deposition of
Uncoated PFD NPs (BL) % Deposition of
Coated PFD NPs (F100)
Fine particle fraction 85.63 ± 2.44% 83.4 ± 2.8%
Extra-fine particle fraction 14.37 ± 1.87 16.51 ± 1.48
Emitted dose 92.66% 93.72%
Mass Median Aerodynamic Diameter (MMAD) 3.2 ± 0.95 µm 4.7 ± 1.8 µm
Geometric standard deviation (GSD) 2.3 ± 1.25 µm 2.8 ± 1.57 µm
Table 4: Stability study of uncoated (BL batch) and chitosan-coated (F100-optimized batch) poly (lactic-co-glycolic acid) nanoparticles.

EXAMPLE 6: SCANNING ELECTRON MICROSCOPY
Surface morphology of the optimized nano particles was observed as being smoothly spherical in form using scanning electron microscopy study. The chitosan modified poly (lactic-co-glycolic acid) nano particles were mean 355.8±9.71 nm in size and figure 3.2 showed the largest size of nanoparticles of 739 nm and 697 nm having stability study period of 6 months (Figure 4.2). The scanning electron microscopy (SEM) study was conducted with initial time period and after six month study of stability. That indicated that prepared chitosan modified poly (lactic-co-glycolic acid) nano particles of pirfenidone were stable and there are no any aggregation or surface properties change. SEM images (Figure 4.1 and Figure 4.2) could mean that SEM shows the nano particles in a dry state. Because of the interaction between chitosan molecules, more noticeable adhesion between nanoparticles was observed as the amount of chitosan on the surface of nanoparticles increased. Furthermore, the surface of poly (lactic-co-glycolic acid) nano particles that had not been affected by chitosan was smoother than the surface of poly (lactic-co-glycolic acid) nano particles that had been modified by chitosan.