DESCRIPTION

Technical Field

This invention is related to the application of pirfenidone via electronic nicotine distribution systems by means of dragging from pirfenidone solution and to the usage of pirfenidone applied by this method in treating idiopathic pulmonary fibrosis.

Prior Art

Idiopathic pulmonary fibrosis (IPF) is chronic, progressive, interstitial pneumonia which progresses with idiopathic fibrosis, mostly seen in adults and it is a lung disease. The pathological findings that are dominant in IPF are fibroblastic foci and collagen accumulation, inflammation is minimal and this increases the possibility for antifibrotic drugs to reduce the progression rate of the disease. Pirfenidone which is an antifibrotic drug inhibits collagen synthesis that is stimulated by the transforming growth factor-beta (TGF-b); it reduces extracellular matrix and blocks in vitro fibroblast proliferation. The synthesis of pirfenidone has been initially disclosed in the patent document numbered US3839346 and the antifibrotic efficiency of pirfenidone has been disclosed in the patent numbered EP0458861. Its chemical name is 5-Methyl-l-phenyl-lH-pyridine-2-one and its structural formula is as shown below.

The first product that contained the active agent of pirfenidone was in 200 mg tablet form and it was legitimized in 2008 in Japan. The product in capsule form which comprises 267 mg of the pirfenidone active agent has been legitimized in Europe in the year 2011 to be used on mild- moderate IPF patients and in the year 2014 in America to be used in IPF treatment without weight specification. The objective of the Invention

Ideally, delivery methods of drugs that have treating effect on the body, that reach the area to be treated rapidly and directly, and therefore that is effective in lower doses, and which in turn enables to alleviate side effects and/or eliminates said side effects are preferred. Pirfenidone treatment is started as 1 capsule three times a day (total of 801 mg/day) via the oral route, and during a 14 day period, the application is continued as 3 capsules (2403 mg/day) per day. Doses above 2403 mg/day are not recommended for any patient.

In the clinical studies that were conducted, when pirfenidone was compared with placebo, it was reported that pirfenidone reduced the risk of death by 48% in the 1st year. However, in patients that used pirfenidone, side effects such as nausea (36%), rashes (32%), dyspepsia (19%), dizziness (18%), vomiting (14%), photosensitivity (12%), anorexia (11%), abdominal pain, upper respiratory tract infections, diarrhea, fatigue, headache, gastroesophageal reflux, sinusitis, insomnia, weight loss and arthralgia have been observed. Additionally, an increase in liver enzymes, photosensitive reactions, and gastrointestinal disorders have been reported. It is observed that these effects are dose based. These side effects are related to pirfenidone treatment increases when pirfenidone is taken orally at high doses.

In the case that one or more side effects are observed in individuals, close follow up and a dose reduction or discontinuing medication is recommended. In a study carried out recently, in order to reduce side effects that are 2nd degree or worse, the dose applied is gradually reduced. However, even though the dose is reduced to a minimum, the medicine is discontinued if the side effects continue or increase. The reduction of medical dose that is applied or discontinuing the medicine, eliminates the possibility to treat patients and/or to increase the lifetime of patients.

Pirfenidone delivery via inhalation has several advantages in comparison to delivery via the oral route. The reason for this is that pirfenidone is a low potency medicine. Due to this reason, a high dose application is required in the application via the oral route. However in applications via inhalation, it is also possible to receive the desired effect in low doses. In oral applications, high toxicity depending on high dose is observed, whereas in applications via inhalation toxicity due to low dose is reduced. While the gastrointestinal side effects with application by the oral route are severe, gastrointestinal side effects with applications by inhalation cannot be observed or they are reduced. In oral use, while the severity of the side effects and pirfenidone absorption depend on the patient having an empty or full stomach, pirfenidone use with inhalation is not affected by the patient being full or hungry. Tolerance to the medicine via inhalation application is higher than oral application and as a result dose adjustment is not required at the beginning of treatment. In the patent applications numbered W02015106150, WO2012106382 and WO2014018668 the application of pirfenidone formulations via inhalators comprising nebulizer, dry powder, and pressured gas has been disclosed.

Nebulizers are devices that vaporize liquid form medicine by means of methods such as sound waves or pressurized air and allow such medicines to be taken via inhalation. However, the compatibility of these devices with patients is difficult as nebulizer devices are generally large sized and as they are used in hospital environments. Although portable nebulizers have been developed based on the technology that has developed in recent years, particle size that is required for efficient treatment cannot be obtained with these devices.

Inhalators that comprise pressurized gas, are more advantageous than nebulizers as they are portable, however as the usage of these devices necessitates care, and hand-inhalation coordination, they are disadvantageous in terms of comfortable usage. Moreover, it is not highly possible to deliver the medicine to the targeted area efficiently and sufficiently with these devices. As particle size is not small enough in these devices, it has been noted that the majority of the dose accumulates at different points as said particles take part in systemic circulation before reaching the lungs.

Dry dose inhalators are more prominent as they are easier to use. Contrary to inhalators that comprise pressurized gas, hand and inhalation coordination is not required and by this means, it plays an important role for the patients in receiving the medicine efficiently. However the problem of not being able to obtain the required particle size for efficient treatment and in turn, the medicine not being able to be delivered to the exact desired area in the lungs continues with such devices.

As it is described in detail above, these methods that are used to deliver medicine via inhalation that is known in the prior art still have some shortcomings. Due to this reason, the development of different formulations and medicine delivery methods via inhalation that overcomes the shortcomings present in previous methods is required. The developed methods and formulations not only should be cheap, efficient and patient compatible, they should also be suitable for creating small aerosol particles.

Description of the Figures Figure 1 -Chromatogram for preventing the formation of combustion products

Description of the Invention

The inventors have surprisingly discovered as a result of the studies they have carried out, that electronic nicotine distribution systems could be used for higher specificity pirfenidone delivery.

The invention owners have noted that pirfenidone could be dragged by 100% from pirfenidone solution in electronic nicotine distribution systems.

The invention owners have reported following the studies they have conducted that, the oral application of pirfenidone and the application via inhalation of pirfenidone using electronic nicotine distribution systems had the same effect on fibrosis. However, the dose amount applied in order to provide the same effect is 20 times higher in oral application in comparison to application via inhalation. Due to this reason, it is believed that the application of pirfenidone via inhalation using electronic nicotine distribution systems shall be efficient in treating fibrosis and it is believed that the side effects experienced following oral application shall be experienced with low intensity or shall not be experienced at all.

Pirfenidone is used in aerosolized form in electronic nicotine distribution systems. It has been proved that negative side effects are experienced less frequently as the dose used in application of pirfenidone that has been aerosolized is lower, medicine delivery is more homogenous and optimized.

It has been enabled for drug delivery to lung alveoli to be more efficient and homogenous in electronic nicotine distribution systems, by means of being able to obtain smaller particles in comparison to aerosol particles produced with nebulizer devices.

Water is generally used as a solvent in nebulizers. During the application of pirfenidone with water, the problem of not being able to apply the required dose of the active agent is experienced as the solubility of the active agent is low. The solubility of pirfenidone in water is 10 mg/ml. The solubility of pirfenidone in propylene glycol solution used in electronic nicotine distribution systems subject to the invention is 300 mg/ml. Due to this reason, the desired amount of active agent is enabled. When evaluated according to this aspect, the delivery of pirfenidone via electronic nicotine distribution systems is more advantageous than both oral and other inhalation methods of the prior art.

Another advantage of the system is that electronic nicotine distribution systems enable usage by different patient groups. The daily required dose by patients can be easily adjusted by the puffing time, the number of puffs or by changing the amount of dose applied.

In electronic nicotine distribution systems, deficient /dead dose application problem is experienced in nebulizers are not encountered. Only 5-40% of the dose applied with nebulizers reaches the area of effect, whereas this rate is 100% when electronic nicotine distribution systems are used. The term pirfenidone encompasses all of the salts, hydrates, anhydrous forms, solvates, amorphous and crystal forms of pirfenidone.

The formulation subject to the invention comprises at least a pharmaceutically acceptable excipient in addition to pirfenidone. The term pharmaceutically acceptable excipient expresses all kinds of pharmaceutically acceptable excipient that can be applied without causing undesired toxicity. The excipients used in the formulation subject to the invention can be formed of a solvent and humectants or mixtures thereof.

The solvents and humectants that are used in the formulation subject to the invention can be selected from propylene glycol and/or glycerol and/or water or mixtures thereof. The solubility of pirfenidone in these mixtures is as presented in Table 1.

Table 1- Solubility of pirfenidone inside solutions that are used in ENDS

The propylene glycol: glycerin mixture ratio in the formulation subject to the present invention can be in the range of 1: 10 to 10: 1. The propylene glycol: water mixture ratio in the formulation subject to the present invention can be in the range of 1: 10 to 10: 1. The propylene glycol: glycerin: water mixture ratios in the formulation subject to the present invention can be in the range of 1: 1 : 10 to 1: 10: 1 to 10: 1: 1.

The D90 value of pirfenidone used in the formulation subject to the present invention is between the range of 1 pm to 150 pm. The particle size distribution of pirfenidone has been measured with the laser diffraction method. The basis of the laser diffraction method is dependent on the inverse ratio relation between the size of the particles and the diffraction angle of the rays. In the laser diffraction method, laser rays are delivered onto the particles and the rays that diffract after hitting the particles and that are forwardly reflected fall onto a detector after passing through a lens. The rays that fall onto the detector are digitized via a converter in order to calculate particle size and percentage thereof, by means of a computer. The particle size of pirfenidone used within the scope of the present invention has been measured with a Malvern Mastersizer 2000 device. A wet method where the active agent particles are measured (25°C, 2000 rpm, 30 sec.) by being dispersed inside a dispersant agent has been preferred.

The solution comprising pirfenidone that is to be used in ENDS needs to be stable, more particularly it needs to be thermally stable. It has been noted that the pirfenidone solution that is prepared to be used in END systems within the scope of the invention is stable. The inventors have noted that pirfenidone was thermally stable and therefore it was a candidate active agent that could be used in END systems. As a result of the studies carried out, it has been illustrated that pirfenidone was thermally stable, that it did not deteriorate in any way during application via electronic nicotine distribution systems and that combustion products were not created. Electronic nicotine distribution systems are personal vaporizers that basically operate with a power source. Different electronic nicotine distribution systems (ENDS) can be used within the scope of the invention. Generally ENDS is formed of 4 main components;

• A cartridge or housing inside which the solution is stored · A heating element (atomizer)

• A power source (battery)

• A nozzle.

The heating element that is operated with a battery is activated in electronic nicotine distribution systems following a puff. Following this activation, the solution inside the housing is transformed into aerosol/or it is vaporized. The users inhale the aerosol or vapor that is created.

The invention owners have proved that an electronic nicotine distribution system could be used as an electronic drug distribution system for the active agent of pirfenidone.

Electronic cigarettes are electronic nicotine distribution systems that operate with batteries. Presently around 500 electronic cigarette variations are available in the market. Please see. Shu- Hong Zhu and etc., Four hundred and sixty brands of e-cigarettes and counting: implications for product regulation, BMJ Journals, Volume 23, Issue suppl 3. Although Joyetech eVic-VTC Mini and Joyetech CUB IS Atomizers have been used in studies within the scope of the invention, all of the devices in the market can be used within the scope of the invention.

The formulation subject to the invention may comprise, in addition to the active agent pirfenidone, steroids (including but not limited to prednisolone), corticosteroids, nintedanib, cytotoxic agents (including but not limited to azathioprine and cyclophosphamide), bardoxolone,

LPA antagonists, for example LPA1 (including but not limited to AM152); Torisel

(temsirolimus); PI3K inhibitors; pentraxin (including but not limited to Pentraxin-2 (PTX-2 or

PRM-151)); MEK inhibitors (including but not limited to ARRY-162 and ARRY-300); p38 inhibitors; PAI-1 inhibitors (including but not limited to Tiplaxtinin); agents which reduce the activity of transforming growth factor beta (TGF-b) (including but not limited to TGF-b neutralizing antibodies such as GC- 1008 (Genzyme/Medlmmune)), anti-TGF-p2 mAbs such as lerdelimumab (CAT- 152; Trabio, Cambridge Antibody); anti-TGF-bI antibodies such as metelimumab (CAT- 192, Cambridge Antibody; small molecule TGF-PR1 inhibitors such as LY- 2157299 (Eli Lilly); ACU-HTR-028 (Opko Health)); antibodies that target one or more TGF-b isoforms; kinase inhibitors of TGF-b receptors TGFBR1 (ALK5) and TGFBR2 and modulators of post receptor signal pathways; chemokine receptor signal modulators; inhibitors which target both endothelin receptor A and B and which selectively target endothelin receptor A (including but not limited to ambrisentan; avosentan; bosentan; clazosentan; darusentan; BQ-153; FR- 139317, L-744453; macitentan; PD-145065; PD-156252; PD163610; PS-433540; S-0139; sitaxentan sodium; TBC-3711; zibotentan), agents which reduce connective tissue growth factor (CTGF) activity (including but not limited to FG-3019) additionally agents which comprise other CTGF-neutralizing antibodies such as FG-3019; matrix metaloproteinase (MMP) inhibitors (including but not limited to MMPI-12, PUP-1 and tigapotide triflutate and doxycycline, marimastat and sipemastat); agents which reduce the activity of epidermal growth factor receptor (EGFR) including but not limited to erlotinib, gefitinib, BMS-690514, cetuximab; antibodies that target EGF receptor; EGF receptor kinase inhibitors and modulators of post-receptor signal pathways; agents which reduce platelet sourced growth factor (PDGF) activity (including but not limited to imatinib mesylate (Novartis)); additionally PDGF neutralizing antibodies; Antibodies that target receptor (PDGFR), PDGFR kinase activity inhibitors and inhibitors of post -receptor signal pathways; agents that reduce vascular endothelial growth factor (VEGF) activity, (including but not limited to axitinib, bevacizumab, BIBF-1120, CDP-791, CT-322, IMC-18F1, PTC-299 and ramucirumab); additionally VEGF-neutralizing antibodies; antibodies that target VEGF receptor 1 (VEGFR1, Fit- 1 ) and VEGF receptor 2 (VEGFR2, KDR) and soluble form of VEGFR (sFlt) and derivatives that neutralize VEGF antibodies; multiple receptor kinase inhibitors such as BIBF-1120 which inhibits receptor kinase for vascular endothelial growth factor, fibroblast growth factor and platelet sourced growth factor; agents that interferes with integrin function (including but not limited to STX-100 and IMGN-388) and agents that comprise integrin targeted antibodies; Agents that interfere with pro-fibrotic activities of IL-4 (including but not limited to AER-001, AMG-317, APG-201 and sIL-4Ra) and IL-2 (AER-001, AMG- 317, anrukinzumab, CAT-354, sintredekin besudotox, MK-6105, QAX-576, SB-313, SL- 102 and TNX-650) and additionally antibodies that neutralize both cytokines and antibodies, antibodies that target IL-4 and IL-13 receptors, soluble forms of IL-4 receptor, agents that bind to IL-4 and IL-3 which neutralizes both, chimeric proteins that contain a part of IL-13 or all of it, and particularly which comprise pseudomonas endotoxins, agents that prevent epithelial mesenchymal passage including, JAK-STAT kinase pathway signaling regions, mTor inhibitors (including but not limited to AP-23573 or rapamycin); agents that reduce copper levels such as tetrathiomolybdate; agents that reduce oxidative stress including -acetyl cysteine and tetrathiomolybdate; and interferon gamma or combinations thereof. At the same time, it can comprise agents that are phosphodiesterase 4 (PDE4) (including but not limited to Roflumilast); phosphodiesterase 5 inhibitors (PDE5) (including but not limited to mirodenafil, PF-4480682, sildenafil citrate, SLx-2101, tadalafil, udenafil, UK-369003, vardenafil, and zaprinast) or arachidonic acid pathway modifiers or combinations thereof comprising cyclooxygenase and 5- lipoxygenase inhibitors (including but not limited to Zileuton). Moreover, it can comprise compounds that reduce tissue remodeling or fibrosis that comprises prolyl hydrolase inhibitors (including but not limited to 1016548, CG-0089, FG-2216, FG-4497, FG-5615, FG-6513, fibrostatin A (Takeda), lufironil, P-1894B and safironil) and receptor (PPAR)-gamma agonists (including but not limited to pioglitazone and rosiglitazone ) activated with peroxisome proliferators or combinations thereof. The agents can additionally be selected from, BG-12, chemokine activity modulators (including but not limited to CNTO 888 which is an antibody that targets CCL2), lysyl oxidase inhibitors (including but not limited to antibodies that target AB0024 / GS-6624, human lysis oxidase-2), NOX4 inhibitors (including but not limited to GKT137831, a selective NOX 1/4), angiotensin II receptor antagonists (including but not limited to lorsartan) or Wnt-beta catenin signalization agents (including but not limited to ICG-001); JNK inhibitors (including but not limited to CC930); IL-4 / IL-13 antibody / soluble receptors (including but not limited to SARI 56597) and a deuterium pirfenidone (for example as described in W02009035598, wherein it is substituted with one to fourteen deuterium atoms that replace a hydrogen atom in pirfenidone) or combinations thereof.

Drag Test:

The drag performance of pirfenidone in electronic nicotine distribution systems and the amount of pirfenidone that is dragged is measured.

In order to summarize this, the solutions comprising pirfenidone at concentrations of 2 mg/ml, 4 mg/ml, 8 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml within the scope of this test have been applied with a power level of 8, 10 and 12 Watts. The solution in each concentration has been applied as 3 separate puffs with 8, 10, 12 Watts of energy and each puff time has been determined as 3 seconds. The waiting time between 2 puffs has been determined to be 30 seconds. 60 ml aerosol has been collected for each different concentration in total. The aerosol that is obtained is then liquefied and the amount of pirfenidone it contains has been measured. The solutions used for comparison have been prepared such that they do not comprise the pirfenidone active agent and all conditions have been kept the same with the application conditions of the solutions that contained pirfenidone.

The active agent losses depending on the stability and surface adhesion of pirfenidone in the device have been measured. As a result, it has been proved that pirfenidone was dragged 100% through the device, that it was stable and it did not create combustion products. Table 2 is related to the drag test results of pirfenidone.

The details of the drag test and analysis study has been provided below.

Pirfenidone solution concentration and Atomizer Parameters

The amount of solution and blank amounts to be collected

Amount of vapor inhaled

Analytic Method Conditions, HPLC Equipment Parameters

Preparation of solutions:

Preparation of Diluted Ortho-Phosphoric Acid Solution 20 ml 85% ortho-phosphoric acid is poured into 100 ml volumetric flask. The volume is completed by adding water.

Preparation of Buffer Solution 0.9 ml triethylamine is added onto 650 ml distilled water and it is completely mixed. The pH is adjusted as 3.0 ± 0,05 with diluted ortho-phosphoric acid. Preparation of mobile phase solution: Tampon solution is mixed with methanol and acetonitrile at ratios of 650: 130:220 (h/h/h). The solution is filtered from a 0.45mhi HV membrane filter, and it is degassed.

Dilution Solution: Propylene glycokglycerin is prepared with a ratio of (80:20) and is mixed. Preparation of Pirfenidone Standard Solution: 30 mg pirfenidone was weighed into a 20 ml volumetric flask. It is dissolved by adding an amount of mobile phase solution. The volume is topped up with the mobile phase solution. (C pi _rfenidone = 1.5 mg/ml)

Preparation of the Sample Solution: 500 mg pirfenidone was weighed into a 25 ml volumetric flask. An amount of dilution solution is added. It is left to rest in an ultrasonic bath for 5 minutes and then it is dissolved. The volume is topped up with the dilution solution. This solution is dragged in the Electronic Device. The obtained vapor is condensed. Solution A, B, C, D, E, and F are prepared from the obtained stock solution.

Procedure · Baseline and pressure are conditioned with a dilution solution injection.

• After the system is balanced, 1 dilution solution injection is given.

• 6 injections of standard- 1 solution are given

• 2 injections of standard-2 solution are given.

• 3 sample solution is prepared and 2 injections are made from each of them. · Chromatograms of the injections given are recorded.

• Following 6 sample solution injections, 1 time of control injection is given from standard- 1 solution.

• The average, standard deviation and relative standard deviation of analysis results are calculated.

Acceptance Criteria

• The RSD% between the peak areas of pirfenidone obtained from 6 successive injections of Standard- 1 solution must be less than 2.0.

• The compatibility between the peak areas obtained from pirfenidone that have been obtained from standard-1 and standard-2 solutions must be between 98,0%- 102,0%. Calculation

AN x CS x P

Fixed volume pirfenidone percentage (% ) = - x 100

AS x CN

AN : Area of pirfenidone peak in the sample chromatogram

AS : Peak area of pirfenidone in standard chromatogram

CS : Standard Concentration (mg/ml) CN : Sample Concentration (mg/ml)

P : Potency of standard (as-is)

Determination of combustion product

The amount of the combusted product is determined, during the pirfenidone drag process carried out by means of the electronic nicotine distribution system. In order to determine this, the amount of pirfenidone that is dragged in 60ml for 3 seconds at different Watts is determined. This process is applied to the solution system that does not contain pirfenidone and to the solution system that contains pirfenidone. According to the analysis results, it was noted that pirfenidone did not produce a combustion product while it was being dragged in an electronic nicotine distribution system. The chromatogram indicating that a combustion product was not created has been presented in Figure 1.

Table 2- Drag Test Results

In vitro Study:

The particle distribution of the aerosols that were created by means of electronic nicotine distribution systems has been calculated using a Next Generation Impactor (NGI) and a Dekati® Low-Pressure Impactor (DLPI). The Dekati® Low-Pressure Impactor is a 13 digit impactor which allows measuring between 30 nm to 10 pm. It has been noted as a result of the measurements that the aerodynamic diameter of aerosol particles obtained by means of electronic nicotine distribution systems was between 500 nm to 1 pm. Particles having such a size can be delivered even to lung alveoli. The invention owners also measured the particle distribution of the aerosols that were formed by means of electronic nicotine distribution systems with a Malvern Mastersizer 2000 device. According to measurement results, it was noted that the D50 value was approximately 1,5 pm.

It is obvious that this value of particle size distribution is smaller than all of the particle size distributions obtained with delivery methods that utilize inhalation in the prior art. In vivo Study:

36 male Wistar-Albino rats weighing between 220-260 grams, were kept in normal laboratory conditions at 21-24 °C under a 12/12 hour day/night cycle. A single dose of intratracheal bleomycin (lOmg/kg) was applied for inducing pulmonary fibrosis. Following fibrosis induction, the rats were divided into six assay groups that have been mentioned below, such that 6 rats were randomly placed in each group.

• Control Group: In this group, pulmonary fibrosis is induced however, any kind of treatment is not given.

• The group that is to receive oral pirfenidone treatment: The animals in this group shall be subjected to 200 mg/kg/day oral pirfenidone treatment with gavage for 7 days.

• The group that is to receive lOmg/kg/day inhalation treatment: These groups of animals are left in a contained area and are given pirfenidone aerosols until the suitable dose medicine is given.

• The group that is to receive 20mg/kg/day inhalation treatment: These groups of animals are left in a contained area and are given pirfenidone aerosols until the suitable dose medicine is given.

• The group that is to receive 40mg/kg/day inhalation treatment: This group of animals is left in a contained area and is given pirfenidone aerosols until the suitable dose medicine is given.

• The group that is to receive only propylene glycol via inhalation: This group of animals was left in a contained area and is only given propylene glycol.

Pirfenidone was dissolved in propylene glycol having a concentration of lOOmg/ml for application via inhalation. The rats were subjected to aerosol pirfenidone or a fixed-dose pirfenidone once a day for 7 successive days in cages, using an electronic nicotine distribution system. For this application, for 7 days, 3-second puffs of 60 ml aerosol were given at 30-second intervals to animals that were placed into cages. The total amount of dose given in the cages was calculated.

The cage was maintained under normoxic and normocapnic conditions, wherein the volume thereof was approximately 0,5L. The selection of the amount of pirfenidone dose was carried out according to the results of Poulin et ah, and Rasooli et al. The application of propylene glycol via inhalation has been carried out similar to pirfenidone application. All of the rats were physically examined following the study, and the observations were as follows, in the treatment groups the rats were active, they had appetite and they were in a good mood. Moreover, it was noted that their furs were smooth and shiny and their body weight had increased. However, both the activity and the appetite of rats in the control group was decreased. It was noted that their furs were darker and they lost weight. Moreover, cyanoses were observed on the legs and around the lips of the rats and symptoms of coughing and difficulty in breathing was noted.

Within the scope of the study, the rats were sedated with intraperitoneal ketamine (100 mg/kg) and xylazine (10 mg/kg) injection and their right and left lungs were resected. A lobe of the lung is placed into 10% buffer formalin solution for histopathologic examination. The other lobe was frozen at -80 °C and supernatants of homogenates were prepared for cytokine ELISA analysis.

The results of the ELISA assay have been presented in Table 3. According to the results in the control groups TNF-a (pg/ml/mg), IL-6 (pg/ml/mg), LPO (ng/ml/mg), SOD (U/ml/mg,) TGF-b (pg/ml/mg), TIMP (ng/ml/mg), MMP-2 (ng/ml/mg) and hydroxyproline (pg/ml/mg) levels were significantly higher in comparison to treatment groups, however a difference was not found between the groups that were applied pirfenidone orally or via inhalation. The results show that pirfenidone treatment applied orally or via inhalation reduced inflammation and lung fibrosis.

Table 3- ELISA test results

Histologic examination results have been presented in Table 4. The presence of inflammation and fibrosis and its degree have been evaluated as described in previous studies. Please see. Sur S,

Wild JS, Choudhury BK, Sur N, Alam R, and Klinman DM: Long term prevention of allergic

5 lung inflammation in a mouse model of asthma by CpG oligodeoxynucleotides, J Immunol.

1999;162:6284-6293., Ashcroft T, Simpson JM, and Timbrell V: Simple method of estimating severity of pulmonary fibrosis on a numerical scale, J Clin Pathol. 1988;41 :467— 470., Chen M,

Cheung FW, Chan MH, Hui PK, Ip S-P, Ling YH, Che C-T, and Liu WK: Protective roles of

Cordyceps on lung fibrosis in cellular and rat models, J Ethnopharmacol. 2012; 143:448—454. Histopathological examination results conveyed that being subjected to bleomycin, caused an increase in significant inflammatory response and increase in collagen accumulation. The results show that pirfenidone treatment applied orally or via inhalation reduced collagen accumulation and inflammatory response induced by bleomycin. Moreover, it was noted that pirfenidone 5 applied via inhalation at much lower concentration reduced collagen accumulation and inflammatory response.

Table 4- Histopathologic examination results

Industrial application of the Invention

10 The formulation subject to the present invention is used in treating idiopathic pulmonary fibrosis treatment. Additionally, the formulation subject to the present invention can be used to treat pulmonary fibrosis, renal glomerulosclerosis, liver cirrhosis, benign prostate hypertrophy, hypertrophic scarring (keloids), rheumatoid arthritis, Hermansky-Pudlak Syndrome (HPS). Additionally, it can be used in preventing or eliminating the scar tissue in fibrosis, related to 15 tissues that have been damaged in the lungs, skin, joints, kidneys, prostate gland and liver. The studies that were conducted indicate that pirfenidone safely slowed down or stopped the gradual development of fibrotic lesions, and prevented the formation of new fibrotic lesions following tissue injuries.

For further detail the formulation subject to the invention encompasses the treatment of pulmonary fibrosis, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonia, autoimmune lung diseases, benign prostatic hypertrophy, coronary or myocardial infarction, atrial fibrillation, cerebral infarction, myocardial fibrosis, musculoskeletal fibrosis, post operation adhesions, liver cirrhosis, renal fibrotic disease, fibrotic vascular disease, scleroderma, Hermansky-Pudlak syndrome, neurofibromatosis, Alzheimer's disease, diabetic retinopathy, and/or skin lesions, lymph node fibrosis related with HIV, chronic obstructive pulmonary disease (COPD), inflammatory pulmonary fibrosis, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gut disease, other arthritic conditions, sepsis, septic shock, gram-negative sepsis, toxic shock syndrome, myofascial pain syndrome (MPS), shigellosis, asthma, respiratory distress syndrome seen in adults, inflammatory bowel disease, Crohn disease, psoriasis disease, eczema, ulcerative colitis, glomerulonephritis, scleroderma, chronic thyroid, Grave's disease, Ormond disease, autoimmune gastritis, myasthenia gravis, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, pancreatic fibrosis, active hepatitis including hepatic fibrosis, acute and chronic kidney disease, renal fibrosis, diabetic nephropathies, irritable bowel syndrome, pyresis, restenosis, cerebral malaria, stroke and ischemic damages, neural trauma, Alzheimer's disease, Huntington's disease, Parkinson's disease, acute and chronic pain, allergies including allergic rhinitis and allergic conjunctivitis, cardiac hypertrophy, chronic heart failure, acute coronary syndrome, cachexia, malaria, leprosy, leishmaniasis, Lyme disease, Reiter syndrome, acute synovitis, muscle degeneration, bursitis, tendonitis, tenosynovitis, hernia, rupture or prolapsed intervertebral disk syndrome, osteopetrosis, thrombosis, silicosis, pulmonary sarcoma, bone resorption, for example, osteoporosis or bone diseases related with multiple myeloma, metastatic breast carcinoma, colorectal carcinoma, malign melanoma, cancers including gastric cancer and non small cell lung cancer, graft-versus-host reaction and autoimmune diseases, for example multiple sclerosis, lupus and fibromyalgia, AIDS and other viral diseases, for example Herpes Zoster, Herpes Simplex I or II, influenza virus, Severe Acute Respiratory Syndrome (SARS), cytomegalovirus and diabetes. Example 1

The 10% pirfenidone solution in propylene glycol (weight/volume) comprises Pirfenidone 10 g

Propylene glycol (excipient ) 100 ml.