DESC:
4. Related Applications
This application is a continuation-in-part of trademark applications no 6184692 & 6184693, filed Nov. 13 2023 and Nov. 14 2023 respectively.
FIELD OF THE INVENTION
This invention provides a nebulization suspension formulation comprising amoxicillin and potassium clavulanate for respiratory conditions. More specifically, to nebulization suspensions for respiratory infections.
BACKGROUND OF THE INVENTION
Pneumonia is a form of acute respiratory infection that affects the lungs, specifically targeting the alveoli, which are small sacs that typically fill with air when a healthy person breathes. When bacteria enter the lungs, they can bypass the body's immune defenses, leading to bacterial pneumonia. This process begins with the bacteria colonizing the upper respiratory tract and potentially being aspirated into the lower respiratory tract. Upon reaching the alveoli, the bacteria trigger an immune response, causing white blood cells to flood the area to combat the infection. This immune activity results in significant inflammation, with the alveoli filling with pus, fluid, and cellular debris. Consequently, this fluid accumulation disrupts normal gas exchange, impairing oxygen intake and carbon dioxide expulsion, leading to symptoms such as hypoxemia and hypercapnia. The disruption in gas exchange and the resulting inflammation and fluid buildup in the alveoli manifest clinically as the hallmark symptoms of pneumonia: fever, chills, cough with purulent sputum, shortness of breath, chest pain, and fatigue. The overall effect is a significant compromise in respiratory function, causing discomfort for the affected individual.

Pneumonia can be caused by various pathogens, with common ones being Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus, while others include Mycoplasma pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, and Acinetobacter baumannii.
Clinicians often categorize pneumonia based on where the infection was acquired: community-acquired pneumonia (contracted outside hospitals and healthcare facilities), hospital-acquired pneumonia (contracted within 48-72 hours of hospital admission), and ventilator-associated pneumonia (developing more than 48 hours after intubation and mechanical ventilation).
Pneumonia is an infection of the lungs, specifically affecting the alveoli. Because these air sacs are crucial for oxygen exchange, their infection can lead to severe respiratory issues. Pneumonia is a prevalent condition, responsible for millions of primary care visits and substantial healthcare expenditures globally. 30 million episodes of acute respiratory infections/pneumonia are reported every year, and pneumonia is the leading infectious cause of death among children. Pneumonia is a leading infectious cause of hospitalization and death among adults in India and the United States, with medical costs exceeding $10 billion in 2011. It is also a leading cause of antibiotic prescriptions worldwide, including in countries like India and the United States. The high incidence and significant economic burden of pneumonia make it a critical public health issue requiring prompt diagnosis and effective treatment strategies.
The development and evaluation of new pharmaceutical interventions, particularly antibiotic nebulization suspension, are driven by a pressing need to address the challenges associated with pneumonia, and their treatment.

Conventional systemic antibiotic therapies, which are commonly prescribed for pneumonia, have several limitations:
• Systemic Side Effects: Oral or intravenous antibiotics can lead to systemic side effects, including gastrointestinal disturbances, allergic reactions, and antibiotic-associated diarrhoea.
• Antibiotic Resistance: Widespread use of antibiotics contributes to the emergence of antibiotic-resistant bacteria, a global public health concern.
• Delayed Onset of Action: Systemic antibiotics have a delayed onset of action as they require time to reach the target site of infection.

The patent, US8106040B2 titled "Stable Amoxicillin-Clavulanate Formulation for Treatment of Bacterial Infections" filed in 2007, discloses an oral formulation, not an inhalation or nebulization suspension. It focuses on a stable pharmaceutical suspension for oral administration, lacking the specific mucoadhesive properties and pulmonary targeting capabilities found in the AONEUM-04 formulation.
The application, WO2013173803A2 titled "Pharmaceutical Compositions Comprising Amoxicillin and Clavulanate" filed in 2013, discloses a paediatric pharmaceutical composition for oral administration that includes amoxicillin and clavulanate which is different from the AONEUM-04 formulation that is specifically developed for inhalation via nebulization, providing targeted drug delivery directly to the lungs and leveraging mucoadhesive properties for enhanced treatment of respiratory infections.
Amoxicillin-clavulanate, a commonly prescribed antibiotic combination, has raised concerns due to its potential for causing gastrointestinal side effects, antibiotic resistance, and liver injury when administered orally. These issues stem from systemic absorption, which can lead to elevated drug concentrations in the bloodstream and affect the gut microbiota. Liver injury
caused by amoxicillin-clavulanate is typically associated with jaundice and can be severe and prolonged, with jaundice lasting 4 to 24 weeks, but rarely results in lasting injury or death. Moreover, dosage adjustment is recommended for patients with severe renal impairment (GFR < 30 mL/min), as excessive systemic levels can further exacerbate side effects. Given these potential risks, it is crucial to explore alternative delivery methods for amoxicillin-clavulanate, such as nebulization suspension, which offers a localized approach to treatment. In this context, we emphasize the significance of amoxicillin-clavulanate nebulization suspension as a means to reduce the gastrointestinal side effects, antibiotic resistance issues, and liver injury associated with oral administration, providing targeted therapy directly to the respiratory tract.
There is no local and targeted drug delivery treatment for Pneumonia. This means that there is no widely accepted approach to delivering medication directly to the affected lungs and alveoli, where the infection and inflammation are localized. The absence of such targeted treatment options can lead to various challenges in managing Pneumonia effectively.
Given the limitations and potential risks associated with oral antibiotics, there is a growing need for alternative treatment strategies for Pneumonia. Developing localized and targeted drug delivery methods for Pneumonia would be a significant step forward in addressing this gap in healthcare. Our invention is designed to precisely administer amoxicillin clavulanate to the lungs and alveoli, allowing for more effective and efficient treatment while minimizing systemic exposure and associated side effects.
The formulations, specifically designed for pulmonary administration, are tailored to address Symptoms associated with Community acquired bacterial pneumonia. These disclosed formulations present a focused strategy aimed at mitigating the severity and frequency of symptoms related to Community acquired bacterial pneumonia(CABP).

By targeting symptoms linked to bacterial infections and those arising from various pneumonia-causing agents, these formulations offer a specialized approach to alleviate a wide spectrum of respiratory infections. The intention is to combat the specific manifestations and complications associated with Community acquired bacterial pneumonia, irrespective of the underlying microbial causes.
Amoxicillin clavulanate is a well-established antibiotic combination known for its broad-spectrum antimicrobial properties. Initially introduced into clinical practice in the early 1980s, amoxicillin clavulanate has since been widely prescribed for the treatment of various bacterial infections, including respiratory tract infections, urinary tract infections, and skin and soft tissue infections.
This antibiotic combination distinguishes itself through its ability to overcome beta-lactamase-mediated resistance, enhancing its efficacy against a wide range of gram-positive and gram-negative pathogens. Amoxicillin Involves inhibition of bacterial cell wall synthesis by binding to penicillin-binding proteins, disrupting peptidoglycan cross-linking, and causing cell lysis. However, beta-lactamase enzymes can degrade amoxicillin. Clavulanate, a beta-lactamase inhibitor, prevents this by binding to the enzyme's active site, preserving amoxicillin's efficacy. This combination broadens the spectrum of bacteria that can be effectively targeted.
Nebulized antibiotic suspensions offer an innovative approach to managing respiratory infections by directly delivering the antibiotic to the site of infection in the lungs and alveoli. These formulations provide several potential advantages, including localized therapy, reduced systemic exposure, and the potential to minimize antibiotic resistance.
The development of an amoxicillin clavulanate-based nebulization suspension capitalizes on the antibiotic's established efficacy and safety profile while harnessing the benefits of localized delivery. This formulation aims to address both acute and chronic respiratory infections, providing a convenient and potentially more effective alternative to traditional oral and systemic antibiotic therapies. Amoxicillin clavulanate exhibits several pharmacological properties that render it a promising candidate for nebulized drug delivery, particularly in the management of respiratory infections.
OBJECTIVE OF THE INVENTION
The primary objective of this invention is to develop a novel and improved nebulization suspension comprising amoxicillin and clavulanate for pneumonia.
BRIEF SUMMARY OF THE INVENTION
The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.
The objective of the invention (Amoxicillin Clavulanate Nebulization Suspension) is two-fold.
Reduce the risk of Antibiotic resistance. This is a serious consequence associated with oral Amoxicillin clavulanate. By delivering the antibiotic directly to the lungs and alveoli through a nebulization suspension, the inventors aim to minimize systemic absorption and thereby reduce the risk of this side effect. Fight against antimicrobial resistance by a novel mechanism Biofilm disruption.

Provide a localized and targeted treatment for Community acquired bacterial pneumonia: Currently, there is no widely accepted approach for delivering medication directly to the lungs and alveoli. This invention aims to address this gap by delivering amoxicillin clavulanate directly to the infected area, potentially leading to more effective treatment.

The present invention relates to a stable fixed dose, aqueous pharmaceutical composition for administration to a human and/or an animal via a nebulizer. The composition comprises of Amoxicillin clavulanate. The pharmaceutical composition may be contained within a suitable container (e.g. respule).

In certain embodiments, the present invention relates to the aforementioned nebulization formulation, wherein the formulation is characterized by mucoadhesion and sustained release suspension. The mucoadhesive properties of the formulation enable it to adhere to the mucosal surfaces of the respiratory tract, thereby prolonging the residence time of the active pharmaceutical ingredient at the site of infection. This prolonged contact enhances the localized drug concentration at the site of infection, which is critical for the effective treatment of pneumonia. Additionally, the sustained release characteristics of the formulation provide a controlled and gradual release of the therapeutic agent, thereby maintaining therapeutic drug levels in the lungs over an extended period. This controlled release minimizes the frequency of dosing required and ensures continuous therapeutic coverage, reducing the risk of bacterial resistance and improving overall treatment efficacy. The combination of mucoadhesion and sustained release mechanisms contributes to the efficient resolution of pneumonia by facilitating prolonged drug action at the target site, thereby enhancing the eradication of the infectious pathogen and promoting recovery.

In certain embodiments, the present invention relates to the aforementioned nebulisation suspension formulation, wherein the suspension is designed with a focus on mucoadhesive properties provided by chitosan, which helps in adhering to the mucosal lining of the lungs, alveoli and releasing of the drug over a sustained period.

In certain embodiments, the present invention relates to the aforementioned formulation, wherein the use of mucoadhesive agents like chitosan enable the formulation to adhere effectively to the mucosal surfaces within the lungs, providing sustained release and improved drug availability at the site of infection.

In certain embodiments, the present invention relates to the aforementioned formulation, wherein Chitosan is included within a range of 0.1-1% w/v, acting as a mucoadhesive agent to enhance the retention and sustained release of the active drug within the pulmonary system.

In certain embodiments, the present invention relates to the aforementioned formulation, wherein PLGA (Poly(lactic-co-glycolic acid) with a lactide-to-glycolide ratio of 50:50 to 75:25, is present at a concentration of 0.5-2% w/v, functioning as a sustained release carrier and helps in the sustained release of the active drug within the pulmonary system.

In certain embodiments, the present invention relates to the aforementioned formulation, wherein PLGA (Poly(lactic-co-glycolic acid) with a prefereable lactide-to-glycolide ratio of 75:25, is present at a concentration of 0.5-2% w/v, functioning as a sustained release carrier and helps in the sustained release of the active drug within the pulmonary system.

In certain embodiments, the present invention relates to the aforementioned formulation, wherein N-acetylcysteine (NAC), within a range of , is 0.1-0.5% w/v used as a Biofilm disruption and mucolytic agent, by breaking down the biofilm matrix, NAC enhances the penetration of antimicrobial agents, and reduced the viscosity of the mucus.

In certain embodiments, the present invention relates to the aforementioned formulation, wherein glycerol, within a range of , is 1-3% w/v used as a Viscosity modifier and stabilizer.

In certain embodiments, the present invention relates to the aforementioned formulation, wherein the formulation is predominantly composed of Sterile Water for Injection, making up q.s. to 100% w/v of the total volume, providing a safe and sterile aqueous medium for nebulization.

Sodium Chloride is incorporated at 0.9% w/v to adjust the isotonicity of the suspension, ensuring compatibility with the physiological conditions of the lungs to minimize irritation upon inhalation.

In certain embodiments, the present invention relates to the aforementioned formulation, wherein the nebulized amoxicillin clavulanate suspension is specifically tailored to maximize the therapeutic effect of the antibiotic directly at the site of infection in the lungs and alveoli, while also being mindful of patient comfort and safety during administration. The unique composition of the formulation leverages the mucoadhesive and isotonic properties of its constituents to provide a controlled release mechanism, ensuring an effective and sustained antibacterial action against the pathogens responsible for pneumonia.
In a further embodiment, the present invention relates to a formulation comprising a stable fixed dose, aqueous pharmaceutical composition contained in a Respule, for Nebulization and a package insert containing instructions about the use of the pharmaceutical composition.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, the detailed description and specific examples, while indicating preferred embodiments of the invention, will be given by way of illustration along with complete specification.

This exemplification illustrates a specific embodiment of the present invention, a nebulization suspension formulated for the treatment of pneumonia. The suspension is characterized by its mucoadhesive properties, which are imparted by chitosan, a biopolymer known for its ability to enhance drug delivery in the pulmonary environment.
In certain embodiments, the present invention relates to the aforementioned method, wherein the solutions or mixtures are aqueous. In certain embodiments, the present invention relates to the aforementioned method, wherein the formulation is a nebulisation suspension.

In certain embodiments, the present invention relates to the aforementioned method, wherein the anti-infective is selected from the following list of beta-lactam antibiotics: penicillins, cephalosporins, monobactams, carbapenems, penicillin and beta-lactamase inhibitor combinations, such as amoxicillin-clavulanate, piperacillin-tazobactam, ampicillin-sulbactam, and ticarcillin-clavulanate. In certain embodiments, the beta-lactam antibiotic is a penicillin such as penicillin G or penicillin V. In certain embodiments, the beta-lactam antibiotic is a cephalosporin selected from the following: cefaclor, cefadroxil, cefazolin, cefdinir, cefixime, cefoperazone, cefotaxime, cefpodoxime, cefprozil, ceftaroline, ceftazidime, ceftibuten, ceftriaxone, or cefuroxime. In certain embodiments, the beta-lactam antibiotic is a monobactam such as aztreonam. In certain embodiments, the beta-lactam antibiotic is a carbapenem such as imipenem, meropenem, ertapenem, or doripenem. In certain embodiments, the anti-infective is a penicillin and beta-lactamase inhibitor combination. In certain embodiments, the anti-infective is amoxicillin-clavulanate. In certain embodiments, the anti-infective is piperacillin-tazobactam. In certain embodiments, the anti-infective is ampicillin-sulbactam. In certain embodiments, the anti-infective is ticarcillin-clavulanate. In certain embodiments, the corticosteroids such as Prednisolone, Methylprednisolone, Dexamethasone. In certain embodiments, the Bronchodilators such as Albuterol, Levalbuterol, Ipratropium bromide. In certain embodiments, the Mucolytics and Expectorants such as Acetylcysteine, Guaifenesin. In certain embodiments, the Antipyretics and Analgesics such as Acetaminophen, Ibuprofen.

Additionally, beta-lactamase inhibitors by themselves, which are used in combination with beta-lactam antibiotics, include substances such as clavulanate, sulbactam, tazobactam, avibactam, and vaborbactam.

In certain embodiments, the anti-infective is a combination of a beta-lactam antibiotic with a beta-lactamase inhibitor, such as amoxicillin with clavulanate, piperacillin with tazobactam, ampicillin with sulbactam, or ticarcillin with clavulanate. These combinations are particularly useful in treating infections caused by bacteria that produce beta-lactamase enzymes, which would otherwise degrade the beta-lactam antibiotics.
BRIEF SUMMARY OF THE DRAWINGS
The invention will be further understood from the following detailed description of a preferred embodiment taken in conjunction with an appended drawing, in which:

FIG. 1: Depicts the droplet size distribution of amoxicillin clavulanate nebulization suspension. The graph shows the size in micrometers (µm) on the y-axis for different batches, with Dv10, Dv50, and Dv90 values representing the distribution percentiles of the droplet sizes.

FIG. 2: Depicts a graphical representation of the targeting and deposition of the drug within the respiratory tract. The graph shows the percentage deposition of the drug at various
aerodynamic diameters, with data points for gastrointestinal (GI), head, tracheobronchial (TB), and pulmonary regions.

FIG. 3: Depicts the clearance of the particles in New Zealand white rabbits over time. The x-axis represents time in minutes, and the y-axis shows the percentage of particles remaining. The graph illustrates the rate at which the particles are cleared from the lungs.

FIG. 4: Depicts a graphical representation of drug efficacy in the lung given various dosing schedules. The graph shows the log10 CFU (colony-forming units) per lung on the y-axis, compared across different dosing schedules represented on the x-axis.

FIG. 5: Depicts Multiple Path Particle Dosimetry (MPPD) model diameters of the amoxicillin clavulanate nebulization suspension. This figure illustrates the deposition fraction visualization, showing the distribution of the nebulized particles within the human respiratory model, including species and model info, breathing parameters, and particle properties.

FIG.6: The graph depicts a comparative analysis of Epithelial Lining fluid(ELF) drug concentrations (mcg/mL) over time (hours) for formulations of Amoxicillin Clavulanate (oral) and AONEUM-04 in New Zealand rabbits. The y-axis represents the drug concentration in ELF (mcg/mL), while the x-axis shows the time in hours post-administration. Sustained release of AONEUM-04 shows a steady concentration profile, while Oral Amoxicillin demonstrates a sharp peak followed by a rapid decline.

FIG.7: The graph represents the In vitro adhesive work (measured in mN*mm) for different formulations - Oral, IV, and AONEUM-04. The y-axis indicates adhesive work values, while the x-axis categorizes the formulation groups. The Oral formulation exhibits the lowest adhesive work, followed by the IV formulation with intermediate values. AONEUM-04 demonstrates the highest adhesive work, suggesting superior adhesive performance.

FIG.8: The graph represents the adhesive force (N) for different formulations - Oral, IV, and AONEUM-04. The y-axis indicates adhesive force values, while the x-axis categorizes the formulation groups. The Oral formulation exhibits the lowest adhesive force. AONEUM-04 demonstrates the highest adhesive force, suggesting superior adhesive performance.

FIG.9: The graph depicts the Live Biofilm Volume/µm³ for different groups – Oral, IV, AONEUM-04. The y-axis indicates Live Biofilm Volume/µm³ values, while the x-axis categorizes the formulation groups. AONEUM-04 Exhibited the lowest live biofilm volume indicating its efficacy in eradicating the bacteria.

FIG.10: The graph depicts the Dead Biofilm Volume/µm³ for different groups – Oral, IV, AONEUM-04. The y-axis indicates Dead Biofilm Volume/µm³ values, while the x-axis indicates the formulation groups. AONEUM-04 Exhibited the highest dead biofilm volume indicating its efficacy in eradicating the bacteria.

FIG.11: The graph depicts the Biofilm thickness for different groups – Oral, IV, AONEUM-04. The y-axis indicates Biofilm thickness values, while the x-axis indicates the formulation groups. AONEUM-04 Exhibited lowest Biofilm thickness indicating its efficacy in eradicating the bacteria.
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure emphasises that its application is not restricted to specific details of construction and component arrangement, as illustrated in the drawings. It is adaptable to various embodiments and implementations. The phraseology and terminology used should be regarded for descriptive purposes, not as limitations.

The terms "including," "comprising," or "having" and variations thereof are meant to encompass listed items and their equivalents, as well as additional items. The terms "a" and "an" do not denote quantity limitations but signify the presence of at least one of the referenced items. Terms like "first," "second," and "third" are used to distinguish elements without implying order, quantity, or importance.

The term “effective amount” when used in connection with an active ingredient denotes an amount of the active ingredient that, when administered to a subject for treating pneumonia, produces an intended therapeutic benefit in a subject. The term “active ingredient” (used interchangeably with “active” or “active substance” or “drug”) as used herein includes Amoxicillin clavulanate or its salt.

In the context of the present invention, for administration by the Inhalational route, the effective amount of amoxicillin can range from about 50 mg to about 500 mg per dose, delivered via

nebulization administered 1-2 times daily and Clavulanate 12.5 mg to about 125 mg per dose, delivered with amoxicillin in the same formulation.

Continuous antibiotic concentration (AONEUM-04) vs Episodic antibiotic concentrations(Oral, Iv): Maintaining continuous antibiotic concentration is essential for effectively treating pneumonia, as it ensures that the antimicrobial agent remains at therapeutic levels throughout the infection, preventing the growth and proliferation of pathogens. Unlike episodic dosing, where antibiotic levels peak and then decline, continuous drug concentrations provide sustained efficacy against the infecting bacteria, particularly in conditions like pneumonia, where the pathogen, such as Streptococcus pneumoniae, can rapidly proliferate and spread if not consistently suppressed.

Continuous antibiotic exposure helps to overcome several challenges inherent in treating pneumonia. First, many bacterial pathogens exhibit time-dependent killing, meaning their growth is inhibited when the drug concentration remains above the minimum inhibitory concentration (MIC) for a prolonged period. Fluctuating drug levels, as seen with episodic dosing, may not provide sufficient antimicrobial pressure, allowing bacteria to escape from drug activity during trough periods and potentially leading to the emergence of resistance.

Furthermore, pneumonia often involves infections in deep or inaccessible tissues, such as the lungs, where drug penetration can be slow or variable. Maintaining steady antibiotic levels ensures that even in these difficult-to-reach sites, the drug remains effective at combating the bacteria over the course of treatment . Moreover dosing minimizes the risks associated with intermittent high drug concentrations, which can cause toxicity or adverse effects while avoiding periods of subtherapeutic levels that might lead to treatment failure.

Lastly, with concern over antibiotic resistance, maintaining continuous therapeutic levels can prevent the survival of drug-resistant bacterial populations that may thrive during periods of low antibiotic concentration . In summary, continuous antibiotic concentrations are crucial not only for ensuring effective treatment of pneumonia but also for minimizing resistance development and promoting better clinical outcomes.

In the context of the present invention, the formulation contains Amoxicillin of about 0.5% w/v and Clavulanate of about 0.2% w/v for mild respiratory infections.
In the context of the present invention, the formulation contains Amoxicillin of about 1.0% w/v and Clavulanate of about 0.4% w/v for moderate infections with bacterial load.

In the context of the present invention, the formulation contains Amoxicillin of about 2.0% w/v and Clavulanate of about 0.5% w/v for severe infections with biofilm-associated resistance.

By “pharmaceutically acceptable excipients”, it is meant any of the components of a pharmaceutical composition other than the active ingredients and which are approved by regulatory authorities or are generally regarded as safe for human or animal use.

As used herein, the term “average particle size” (or synonymously, “mean particle size”) refers to the distribution of particles, wherein about 50 percent volume of all the particles measured have a size less than the defined average particle size value and about 50 percent volume of all particles measured have a particle size greater than the defined average particle size value. This can be identified by the term “D50” or “d(0.5)”. The average particle size can be measured using various techniques such as microscopy, laser diffraction, photon correlation spectroscopy (PCS), and Coulter's principle.

In the context of the present invention, the term “mucoadhesive” refers to the ability of a substance to adhere to the mucosal surfaces of the body, such as the lung mucosa. This property enhances the retention time of a pharmaceutical formulation at the site of administration, allowing for prolonged contact and improved drug absorption. Mucoadhesive agents can form strong non-covalent bonds with mucin and epithelial cells, leading to an increased local concentration of the active pharmaceutical ingredient, improved therapeutic efficacy, and potentially reduced dosing frequency. Non-limiting examples of chitosan-related polymers that can be used include trimethyl chitosan, glycol chitosan, carboxymethyl chitosan, chitosan oligosaccharides, hydroxypropyl chitosan, chitosan hydrochloride, chitosan glutamate, N-succinyl chitosan, quaternized chitosan, and chitosan, xanthan gum, guar gum, alginate, carrageenan, and carboxymethyl cellulose sodium.

In the context of the present invention, the term “Suspending agent” refers to a substance that stabilizes a suspension by increasing its kinetic stability. Suspending agents are used to increase the viscosity of a liquid, helping to stabilize suspensions by keeping insoluble particles evenly distributed and preventing them from settling. This results in a stable dispersion of small droplets of one phase within the other, preventing the phases from separating. Suspending

agents are commonly used in pharmaceutical compositions to ensure uniform distribution of active ingredients and enhance the consistency and stability of the formulation. Non-limiting examples of Suspending agents include, Hydroxypropyl Methylcellulose (HPMC), Carboxymethylcellulose (CMC), Microcrystalline Cellulose (MCC), Xanthan Gum, Guar Gum, Polyvinylpyrrolidone (PVP), Polyethylene Glycol (PEG), Bentonite, Silica, Sucrose.

As used herein, the term “container” refers to a respule, which is a single-unit dose container designed for nebulization. Suitable respules include, but are not limited to, glass, aluminum, polypropylene, or high-density polyethylene, for example, high-density polyethylene containers produced using a blow-fill-seal manufacturing technique. In one embodiment, the container is a respule that stores the pharmaceutical composition, which is delivered as a fine mist via a nebulizer.

The present invention relates to a stable fixed-dose, aqueous pharmaceutical composition (e.g., contained in a respule) for nebulization administration to a human and/or an animal, where the composition comprises about 0.5-7.5% w/v amoxicillin and about 0.1-5%w/v clavulanate.
The pharmaceutical composition may be in the form of a solution or a suspension, but preferably the composition is in the form of a suspension (more preferably, a single-phase suspension), wherein amoxicillin clavulanate is present in particle form. The amoxicillin clavulanate may be present at a weight ratio suitable to achieve the desired therapeutic effect.
The composition preferably also includes a Suspending agent. In one embodiment, the composition is a suspension and includes a Suspending agent in a sufficient amount to prevent phase separation (i.e., separation of the particles and solution) after 3 or 6 months of storage at 25±2°C and 60%±5% relative humidity (RH) or at 40±2°C and 75%±5% RH. In one embodiment, the aqueous pharmaceutical composition is a single-phase suspension which remains a single-phase suspension even after 12 or 24 months of storage at 25±2°C and 60%±5% RH or at 40±2°C and 75%±5% RH.

The term "stable" as used in connection with aqueous suspensions refers to a composition that, when shaken and then stored for at least 24 hours at ambient conditions, does not show phase separation on visual inspection. Preferably, such stable composition does not show phase separation for a period of at least 3 days, or at least 5 days, or at least 7 days. In one aspect, the

"stable" composition of the present invention shows, upon shaking (e.g., for 1 minute) and visual inspection, no lump formation and a total impurity content of no more than 1.0% after storage at ambient conditions (at about 25°C and a relative humidity of about 60%) for a period of at least 6 months.

In the context of the present invention, the drug content and impurities can be determined by various analytical techniques such as HPLC, LC-MS, TLC, and the like.

Another embodiment is a stable fixed-dose, aqueous pharmaceutical suspension composition (e.g., contained in a respule) for nebulization administration to a human and/or an animal, where the composition comprises about 0.5-7.5% w/v amoxicillin, about 0.1-1% w/v chitosan, about 1 to 2% w/v PLGA (Poly(lactic-co-glycolic acid), about 0.1-0.5% w/v N-acetylcysteine (NAC), about 0.05 to 1% w/v polysorbate 80, HPMC 0.1 - 0.5%, about 0.9% w/v sodium chloride, and about Q.S. to 100% sterile water for injection.
Yet another embodiment is a stable fixed-dose, aqueous pharmaceutical suspension composition (e.g., contained in a respule) for nebulization administration to a human and/or an animal, where the composition comprises about 0.5-7.5% w/v amoxicillin, about 0.1-5% w/v clavulanate, about 0.1-1% w/v chitosan, about 1 to 2% w/v PLGA (Poly(lactic-co-glycolic acid)), about 0.1% to 1% w/v polysorbate 80, about 0.1% to 0.5% w/v benzyl alcohol, about 0.9% w/v sodium chloride, and about 85% to 95% w/v sterile water for injection. Chitosan may be present at a concentration of at least about 0.1% w/v of the composition.

Yet another embodiment is a stable fixed-dose, aqueous pharmaceutical suspension composition (e.g., contained in a respule) for nebulization administration to a human and/or an animal, where the composition comprises about 0.5-7.5% amoxicillin, about 0.2-5% w/v clavulanate,, about 0.2-0.5% chitosan, about 1 to 2% PLGA (Poly(lactic-co-glycolic acid)), about 0.1% to 1% polysorbate 80, about 0.1% to 0.5% benzyl alcohol, about 0.9% sodium chloride, and about 85% to 95% sterile water for injection. Chitosan may be present at a concentration of at least about 0.1% w/v, or preferably between about 0.3% w/v and about 0.8% w/v of the composition.

Yet another embodiment is a stable fixed-dose, aqueous pharmaceutical suspension composition (e.g., contained in a respule) for nebulization administration to a human and/or an

animal, where the composition comprises about 0.5-7.5% amoxicillin, about 0.2-5% w/v clavulanate, about 0.2-1% chitosan, about 1 to 2% PLGA (Poly(lactic-co-glycolic acid)), about 0.1% to 1% polysorbate 80, about 0.1% to 0.5% benzyl alcohol, about 0.9% sodium chloride, and about 85% to 95% sterile water for injection. Chitosan may be present at a concentration of at least about 0.1% w/v, or preferably between about 0.2% w/v and about 1% w/v of the composition.

Yet another embodiment is a stable fixed-dose, aqueous pharmaceutical suspension composition (e.g., contained in a respule) for nebulization administration to a human and/or an animal, where the composition comprises about 7% amoxicillin, about 1.5% w/v clavulanate, about 0.6% chitosan, about 1% PLGA (Poly(lactic-co-glycolic acid)), about 0.08% polysorbate 80, about 0.1% N-Acetyl cysteine, HPMC 0.1% about 0.9% sodium chloride, and about 85% to 95% sterile water for injection.

Yet another embodiment is a stable fixed-dose aqueous pharmaceutical composition in the form of suspension (e.g., contained in a container) for Inhalation administration to a human and/or an animal, comprising amoxicillin, clavulanate, PLGA(Poly(lactic-co-glycolic acid)), polysorbate 80, benzyl alcohol and sodium chloride, sterile water for injection, a Mucoadhesive (e.g., at a concentration of at least about 0.1% w/v of the composition), and a pharmaceutically acceptable excipient.

It will also be appreciated by the skilled artisan that in order to improve the physical properties, appearances, or smells of the composition of the present invention, one or more further pharmaceutically acceptable excipients may be added as desired. Suitable pharmaceutically acceptable excipients include, but are not limited to, chelating agents, preservatives, buffers, surfactants, isotonicity agents, taste masking agents, antioxidants, humectants, pH-adjusting agents, and any combination of any of the foregoing.

In order to improve the ability of the aqueous nebulization suspension to be tolerated on administration to the lungs, it is advantageous to formulate it as isotonic. The osmolality can be set by variation of the amounts of the substances present in the aqueous nebulization suspension besides amoxicillin, clavulanate, chitosan, PLGA(Poly(lactic-co-glycolic acid)), polysorbate 80, benzyl alcohol and sodium chloride, sterile water for injection and any further substances present, and/or by addition of an isotonicity agent, preferably a physiologically tolerated salt, such as, for example, sodium chloride.

Examples of suitable sustained release carrier that can be employed in the aqueous nebulization suspension include, but are not limited to, PLGA (Poly(lactic-co-glycolic acid)), chitosan, alginate and any combination of any of the foregoing. The amount of sustained release carrier present in the aqueous nebulization suspension composition may range from about 0.5% to about 2% w/v relative to the total weight of the composition.

Examples of suitable Biofilm disrupting agents that can be employed in the aqueous nebulization suspension include N-acetylcysteine (NAC). The amount of biofilm disrupting agent present in the aqueous nebulization suspension composition may range from about 0.1 % to about 0.5% w/v relative to the total weight of the composition.

Examples of suitable Viscosity modifiers that can be employed in the aqueous nebulization suspension include Glycerol. The amount of viscosity modifier present in the aqueous nebulization suspension composition may range from about 1 % to about 3% w/v relative to the total weight of the composition.

In another embodiment, the stable fixed-dose, aqueous pharmaceutical composition is contained in a nebulizer and, upon delivery as a mist, results in effective deposition of the active ingredients in the respiratory tract.

In a further embodiment, the pharmaceutical composition is contained in a respule and has, upon delivery as a nebulized mist, a particle size distribution suitable for efficient lung deposition. The composition, when nebulized, forms droplets with a median aerodynamic diameter conducive to pulmonary delivery.

In the context of the present invention, the pharmaceutical composition, when delivered as a nebulized mist using a nebulizer, yields a specific particle size distribution and deposition pattern. The particle size distribution can be determined by various known techniques, such as laser diffraction. The mean particle size distribution is <5 microns

The stable fixed-dose, aqueous pharmaceutical suspension composition includes amoxicillin clavulanate as the active ingredient, along with chitosan to enhance mucoadhesion and sustained release. Polysorbate serves as a suspending agent to stabilize the suspension, while PLGA (Poly(lactic-co-glycolic acid) functions as a sustained release carrier to maintain the sustained release of the formulation. To ensure sterility, and sodium chloride is used for isotonicity adjustment. The suspension is prepared in sterile water for injection, ensuring safety and compatibility for nebulization.

In a preferred embodiment, the composition has a pH between about 4.5 and about 7 and an osmolality between about 250 mOsm/kg and about 500 mOsm/kg. The viscosity of the composition ranges from about 1 cps to about 10 cps, ensuring optimal nebulization properties.
In yet another aspect, the pharmaceutical composition in the form of suspension contains amoxicillin clavulanate particles having a mean particle size in the range of about 1 µm to about 10 µm, or preferably from about 1 µm to about 5 µm. The suspension pharmaceutical composition of the present invention has a mean particle size of less than 5 µm when determined by microscopy technique.

In the context of the present invention, the viscosity can be determined by various known instruments, such as a dynamic stress rheometer or Brookfield viscometer. In a preferred embodiment, the viscosity is determined by a Brookfield viscometer by measuring torque transmission through a sample using a rotating spindle.

In another embodiment, the present invention relates to a stable, fixed-dose, aqueous pharmaceutical composition (e.g., contained in a respule) for Inhalation administration to a human and/or an animal, where the composition comprises amoxicillin at about 0.5 to 7.5% w/v and clavulanate at about 0.1 to 5% w/v, a Suspending agent which comprises HPMC at a concentration of about 0.1 -0.5 % w/v of the composition, wherein the composition has a pH between about 4.5 and about 7.

The concentrations of the suspending agents used within the formulation are largely depended on the concentration of the suspended drug substance. The suspending agent is added in an amount to achieve effective suspension of amoxicillin clavulanate to provide a homogeneous suspension. The ratio between drug substance and suspending agent can usually vary from 0.05 to 50.

Yet another embodiment is a stable fixed-dose pharmaceutical composition in the form of suspension (e.g., contained in a container) for inhalational administration to a human and/or an animal, comprising amoxicillin at about 0.5-7.5% w/v and clavulanate at about 0.1-5% w/v and a Suspending agent which comprises HPMC at a concentration of about 0.1 -0.5 % w/v wherein the composition has a pH between about 4.5 and about 7.

Preferably, the suspensions of the present invention have only one phase (i.e., they are preferably a single-phase suspension).

The aqueous suspension can be administered using a nebulizer equipped with a respule. The respule is designed to deliver a fine mist of the pharmaceutical composition, ensuring efficient drug delivery to the respiratory tract. The respule may be made of materials such as high-density polyethylene (HDPE) or other suitable polymers.

In a further embodiment, the present invention relates to a formulation comprising a stable fixed-dose, aqueous pharmaceutical composition contained in a respule for nebulization administration and a package insert containing instructions about the use of the pharmaceutical composition. The kit provides a convenient and effective means for treating bacterial respiratory infections in humans and animals.

In a preferred embodiment, the aqueous suspension is provided in the form of a nebulization suspension respule, wherein the suspension is administered in a single-unit-dose container. Suitable single-unit-dose containers include, but are not limited to, materials such as glass, aluminum, polypropylene, or high-density polyethylene. For example, high-density polyethylene containers are produced using a blow-fill-seal manufacturing technique to ensure sterility and precision in dosing.

The following describes a typical procedure for characterizing droplet size distribution of the nebulization suspension: A nebulizer is loaded with the suspension as described above and primed according to the manufacturer's instructions until a fine mist is emitted from the nebulizer outlet. A commercially available laser diffraction instrument is arranged so that the nebulizer outlet is positioned at an appropriate distance (e.g., 3 cm or 6 cm) below the laser beam of the laser diffraction instrument. The nebulizer is activated using a consistent air flow or pressure setting as specified by the device's operational guidelines. The resulting aerosol mist crosses the laser beam. Data are collected for D10, D50, D90, SPAN, and % Volume <10 µm. The average values for each of these parameters are calculated from three separate nebulization cycles.

In one embodiment, the pharmaceutical inhalation suspension comprises PLGA (Poly(lactic-co-glycolic acid)) optimized to achieve a particle size distribution with d10 of 1–2 µm, d50 of 2–3 µm, and d90 of 4–5 µm. This specific size distribution ensures efficient deposition in

both central and peripheral lung regions when administered via a nebulizer. The formulation is stabilized using 0.1–0.5% w/v polysorbate 80 in the aqueous phase.

Another embodiment is a method for treating bacterial pneumonia, or for administering amoxicillin clavulanate. The method includes nebulizing a stable, fixed-dose aqueous pharmaceutical composition comprising amoxicillin clavulanate, a suspending agent such that each inhalation of the aqueous pharmaceutical composition provides (i) amoxicillin at about 0.5-7.5% w/v and clavulanate at about 0.1-5% w/v .
The formulation includes buffered saline containing a phosphate buffer system to maintain the pH at 6.1 to 7.2 providing a stable environment that prevents degradation of the active pharmaceutical ingredients and ensures compatibility with the respiratory mucosa. This combination of HPMC and buffered saline ensures physical stability, efficient nebulization, and tolerability for inhalation.
Optionally the formulation according to the invention might contain one or more preservatives although made sterile by the process according to the present invention. It is preferred to have a preservative present in the formulations according to the invention in order to preserve the microbiological quality during use. This is especially important in case of multiple dose vials. Suitable preservatives for example are benzoic acid, sorbic acids and its salts, propionic acid and its salts, phenol and derivatives such as cresol and chlorocresol, chlorobutanol, benzyl alcohol, phenyl ethyl alcohol, butyl paraben and propyl paraben.

The present invention also relates to a method of treating bacterial pneumonia in a human and/or an animal in need thereof, comprising administering by the inhalational route a stable fixed dose of the aqueous pharmaceutical composition of the present invention. For example, the pharmaceutical composition that may be contained in a container comprises about 0.5-7.5% w/v amoxicillin, about 0.1-5% w/v clavulanate, about 0.1-1% w/v chitosan, about 1 to 2% w/v PLGA (Poly(lactic-co-glycolic acid), about 0.1-0.5% w/v N-acetylcysteine (NAC), about 0.05-0.1% w/v Lecithin, about 1 to 3% w/v Glycerol, about 0.9% w/v sodium chloride, and about Q.S. to 100% sterile water for injection.

In a further embodiment, the present invention relates to the use of about about 0.5-7.5% w/v amoxicillin, about 0.1-5% w/v clavulanate, about 0.1-1% w/v chitosan, about 1 to 2% w/v PLGA (Poly(lactic-co-glycolic acid), about 0.1-0.5% w/v N-acetylcysteine (NAC), about 0.05-0.1% w/v Lecithin, about 1 to 3% w/v Glycerol, about 0.9% w/v sodium chloride, and about

Q.S. to 100% sterile water for injection in the preparation of a stable fixed dose, aqueous pharmaceutical composition (e.g., contained in a container) for the treatment of bacterial pneumonia in a human and/or an animal in need thereof. Any pharmaceutical composition described herein may be used.

In one embodiment, the pharmaceutical inhalation suspension is formulated to eliminate drug-resistant Streptococcus pneumoniae, Haemophilus influenzae or other infections in the respiratory tract through a combination of localized high antibiotic concentration, sustained release, and biofilm disruption properties. The suspension comprises amoxicillin (0.4–7.5% w/v) and clavulanic acid (0.2–5.0% w/v) and PLGA (Poly(lactic-co-glycolic acid), which provide sustained drug release over 8 to 16 hours.

It will be understood that various modifications may be made to the embodiments disclosed herein. Therefore, the above description should not be construed as limiting but merely as exemplifications of preferred embodiments. Other arrangements and methods may be implemented by those skilled in the art without departing from the scope and spirit of this invention.

The amoxicillin clavulanate nebulization suspension formulation (AONEUM-04) has been specifically designed for use with a wide range of nebulizers i.e., Jet nebulizer, Ultrasonic nebulizer and Vibrating mesh ensuring effective and efficient drug delivery to the lungs and alveoli. This suspension exhibits excellent mucoadhesive properties due to the inclusion of chitosan, enhancing its ability to adhere to the mucosal lining of the respiratory tract and providing sustained release of the active ingredients. The formulation also maintains physical stability, preventing phase separation and ensuring uniform distribution of the drug particles.

Additionally, it achieves an optimal particle size distribution conducive to effective lung deposition, making it compatible with various nebulizer models. These characteristics confirm that all nebulizers are suitable for administering the AONEUM-04 formulation, providing a localized and targeted treatment approach for community acquired bacterial pneumonia with reduced systemic exposure and minimized side effects.

The following examples are provided to enable one skilled in the art to practice the invention and are merely illustrative of the invention. The examples should not be read as limiting the scope of the invention.
Suspension Compositions Containing Amoxicillin Clavulanate and Chitosan:
Example 1:
SL.NO Ingredient Example 1(% w/w)
1 Amoxicillin (API) 0.5-7.5%
2 Clavulanate 0.1-5%
2 Chitosan 0.1-1%
3 PLGA (Poly(lactic-co-glycolic acid)) 0.5-2%
4 N-acetylcysteine (NAC) 0.05-0.5%
6 Polysorbate 80 0.05-0.1%
7 Sodium Chloride 0.9%
8 HPMC 0.1-0.5%
9 Buffered saline (pH 6.8) q.s.
10 Water for injection q.s. to 100%
Physical observation on standing for 24 hours. No phase separation Observed
Mean Particle size by microscopy Between 1-5 µm.

Manufacturing Procedure: Direct Solubilization
Step 1: In a sterile environment, prepare a solution of chitosan by dissolving 0.05-0.5% w/w chitosan in a portion of Sterile Water for Injection, using mild heat and stirring until fully dissolved.
Step 2: Add 0.01-0.1% of Disodium Edetate to the chitosan solution and stir until homogeneous.
Step 3: In a separate vessel, dissolve 3.45-5.22% of amoxicillin clavulanate in the remaining volume of Sterile Water for Injection.
Step 4: Combine the amoxicillin clavulanate solution with the chitosan and Disodium Edetate mixture.
Step 5: Add 0.1-1% Polysorbate 80 to the combined mixture to aid in solubilization and stabilization of the suspension.
Step 6: Incorporate 0.1-0.5% Benzyl alcohol into the mixture as a preservative.
Step 7: Adjust the isotonicity of the final suspension with 0.9% Sodium Chloride.

Step 8: Homogenize the final mixture to ensure uniform distribution of all components.
Step 9: Filter the suspension through a sterile filter to ensure sterility and remove any undissolved particles.
Step 10: Fill the final product into sterile nebulization containers(respules) under aseptic conditions.
The composition was subjected to stability studies at different conditions. The results of the same are as follows:
Container details: The final product was packaged in suitable containers (e.g. respules).

Stability Study Data
Initial 3 months 6 months
Stability condition (25° C. ± 2° C. & 60% RH ± 5% RH)
Test Ex. 1 Ex. 1 Ex. 1
pH 6.5 6.5 6.6
Osmolality (mOsm)* 310 mOsmol/kg 312 mOsmol/kg 309 mOsmol/kg
Viscosity (cps)** 5 cps 5.1cps 5.1 cps
Weight per ml (g/ml) 1.0098 g/ml 1.009 g/ml 1.0035 g/ml
Assay of Amoxicillin clavulanate (% w/w) 101.5 100.3 100.8
Related substances for amoxicillin clavulanate
Impurity A (%) 0.02 0.03 0.09
Any other impurity (%) 0.09 0.03 0.02
Total impurities (%) 0.26 0.4 0.05
Droplet size distribution (at 6 cm)
D10 (µm) 1 0.9 0.95
D50 (µm) 1.6 1.5 1.6
D90 (µm) 2.9 2.75 2.91
SPAN 1.18 1.23 1.22
Stability condition (40° C. ± 2° C. & 75% RH ± 5% RH)
pH 6.5 6.6 6.7
Osmolality (mOsm) 315 mOsmol/kg 320 mOsmol/kg 318 mOsmol/kg
Viscosity (cps)
Weight per ml (g/ml) 1.0078 g/ml 1.0144g/ml 1.0098 g/ml
Assay of Amoxicillin clavulanate 101.2 99.79 100.3
Impurity A (%) 0.02 0.03 0.09
Droplet size distribution (at 6 cm)
D10 (µm) 1 0.9 0.95
D50 (µm) 1.6 1.5 1.6
D90 (µm) 2.9 2.75 2.91
SPAN 1.18 1.23 1.22
SPAN 1.14 1.15 1.16
*Determined by Advanced Instruments Osmometer (Model 3250).
**Determined by Brookfield viscometer.

Example 2
Suspension Compositions Containing amoxicillin and clavulanate
Component Function Concentration (% w/v)
Amoxicillin Antibacterial agent 2.0
Clavulanate Beta-lactamase inhibitor 0.75
Chitosan (high molecular weight) Mucoadhesive polymer 0.5
PLGA (PEG-modified) Sustained-release carrier 1.5
EDTA + N-Acetylcysteine Dual biofilm disruptors 0.05 + 0.1
Polysorbate 80 Surfactant/stabilizer 0.1
Sodium chloride Isotonic agent 0.9
HPMC Viscosity enhancer 0.5
Buffered saline (pH 7.2) Maintain physiological pH q.s.
Water for injection Solvent q.s. to 100%

Stability Study Data
Initial 3 months 6 months
Stability condition (25° C. ± 2° C. & 60% RH ± 5% RH)
Test Ex. 1 Ex. 1 Ex. 1
pH 6.7 6.7 6.7
Osmolality (mOsm)* 345 mOsmol/kg 350 mOsmol/kg 350 mOsmol/kg
Viscosity (cps)** 6.1 6.1 6.1
Weight per ml (g/ml) 1.0098 g/ml 1.009 g/ml 1.0035 g/ml
Assay of Amoxicillin clavulanate (% w/w) 100.5 100.8 101.2
Related substances for amoxicillin clavulanate
Impurity A (%) 0.01 0.02 0.02
Any other impurity (%) 0.01 0.04 0.04
Total impurities (%) 0.17 0.14 0.18
Droplet size distribution (at 6 cm)
D10 (µm) 1 1.2 1.2
D50 (µm) 2.0 2.1 2.0
D90 (µm) 3.3 3.4 3.4
SPAN 1.18 1.23 1.22
Stability condition (40° C. ± 2° C. & 75% RH ± 5% RH)
pH 6.6 6.7 6.8
Osmolality (mOsm) 325 mOsmol/kg 335mOsmol/kg 325 mOsmol/kg
Viscosity (cps)
Weight per ml (g/ml) 1.0078 g/ml 1.0144g/ml 1.0098 g/ml
Assay of Amoxicillin clavulanate 101.04 100.21 100.09
Impurity A (%) 0.03 0.04 0.06
Droplet size distribution (at 6 cm)
D10 (µm) 1.2 1 1.1
D50 (µm) 1.8 1.9 2
D90 (µm) 3.6 3.8 3.7
SPAN 1.18 1.23 1.22
SPAN 1.14 1.15 1.16
*Determined by Advanced Instruments Osmometer (Model 3250).
**Determined by Brookfield viscometer.

Manufacturing Procedure: Ultrasonication Technique
Step 1: Disperse 0.05-0.5% w/w chitosan in Sterile Water for Injection using ultrasonication to ensure complete solubilization without overheating.
Step 2: Mix in 0.01-0.1% Disodium Edetate until fully dissolved.
Step 3: Prepare a concentrated solution of amoxicillin clavulanate (3.45-5.22%) in a separate container with Sterile Water for Injection.
Step 4: Gradually combine the amoxicillin clavulanate solution with the chitosan solution under ultrasonication to form a cohesive suspension.
Step 5: Add 0.1-1% Polysorbate 80 to the suspension under continuous ultrasonication to enhance distribution.
Step 6: Blend in 0.1-0.5% Benzyl alcohol for preservation.
Step 7: Balance the osmolarity of the suspension with 0.9% Sodium Chloride.
Step 8: Ultrasonicate the final mixture to ensure homogeneity.
Step 9: Perform sterile filtration of the suspension into aseptic nebulization vials.
The composition was subjected to stability studies at different conditions. The results of the same are as follows:
Container details: The final product was packaged in suitable containers (e.g. respules).
Pharmacokinetic Comparison of Amoxicillin clavulanate nebulization suspension vs. Oral amoxicillin clavulanate for Treatment of community acquired bacterial pneumonia.
A pharmacokinetic comparison between amoxicillin clavulanate nebulization suspension and oral amoxicillin clavulanate for the treatment of pneumonia highlights several key differences in how the drug is absorbed, distributed, metabolized, and eliminated when administered via these two routes. Understanding these differences is crucial for optimizing treatment strategies for pneumonia, particularly when targeting drug-resistant bacteria and minimizing systemic side effects.

Absorption
Nebulization Suspension: Amoxicillin clavulanate delivered via nebulization is absorbed directly through the respiratory mucosa into the surrounding lung tissue, achieving high local concentrations. The absorption into systemic circulation is lower in total amount compared to oral administration, reducing systemic exposure.
Oral: Oral amoxicillin clavulanate is absorbed through the gastrointestinal tract. Its bioavailability is approximately 60-70%, and peak plasma concentrations occur within 1-2 hours post-administration. Oral administration results in higher systemic exposure, which is necessary for treating disseminated infections but increases the risk of systemic side effects.

Distribution
Nebulization Suspension: Direct administration to the respiratory tract allows for high local drug concentrations, potentially enhancing efficacy against lung infections with minimal systemic distribution. This localized distribution is especially beneficial for achieving therapeutic levels in the lung tissues and alveoli and targeting respiratory pathogens effectively.
Oral: After absorption, amoxicillin clavulanate is widely distributed throughout the body, including significant penetration into tissues and fluids. It has a moderate volume of distribution, which is beneficial for systemic infections but may not guarantee high concentrations in the lungs.

Metabolism and Elimination
Nebulization Suspension: Metabolism and elimination of amoxicillin clavulanate administered via nebulization differ from oral administration due to the reduced systemic absorption. The primary elimination route is through mucociliary clearance and subsequent expectoration.
Oral: Amoxicillin is metabolized minimally by the liver and is primarily excreted unchanged in urine. Clavulanate is extensively metabolized and excreted in both urine and feces. The

systemic metabolism and elimination pathways are more engaged with oral administration compared to nebulization.

Bioavailability and Efficacy
Nebulization Suspension: The bioavailability of amoxicillin clavulanate via nebulization focuses on its local availability in the lung tissues rather than its systemic absorption. The efficacy of the nebulization suspension is contingent upon its ability to maintain therapeutic concentrations at the site of infection, which is enhanced by direct application.
Oral: Oral amoxicillin clavulanate has systemic bioavailability that is important for treating infections beyond the local site. However, achieving therapeutic concentrations specifically in the lungs is less efficient due to the pharmacokinetic profile favoring broad tissue distribution.

In Vivo Efficacy Study
In a controlled in vivo study using New Zealand White rabbits, animals were fasted overnight before the experiment. The following day, two treatment groups were administered medications via different routes:

Oral Administration: A solution of oral amoxicillin clavulanate was administered orally using a gavage and a graduated syringe. The dosage administered was 20 mg/kg, with a dosage volume of 2 ml/kg body weight at a concentration of 10 mg/ml.

Nebulization Administration: For the nebulization study, a nebulization suspension of amoxicillin clavulanate was administered using a nebulizer. The dose administered was 20 mg/kg, with each nebulization session delivering the full dose over a specified duration.

This comparative analysis provides a comprehensive understanding of the pharmacokinetic profiles of amoxicillin clavulanate via nebulization versus oral administration, aiding in optimizing treatment strategies for pneumonia.

Pharmacokinetics of Amoxicillin Clavulanate Delivered as Nebulization Suspension in Human Volunteers
The investigational study on Amoxicillin Clavulanate nebulization suspension (AONEUM-04) in healthy volunteers demonstrated significant pharmacokinetic benefits. Following nebulization, a nominal dose of 1 mg was utilized. Pharmacokinetic modeling indicated that amoxicillin and clavulanate achieved peak concentrations in the lung tissues promptly due to efficient mucosal absorption. The rapid absorption is attributed to the formulation's ability to permeate the pulmonary tissues effectively.

Interestingly, amoxicillin and clavulanate showed sustained presence in the lung tissues, with a half-life extending up to 12 hours, highlighting their prolonged tissue penetration. This extended half-life is beneficial for maintaining therapeutic drug levels at the site of infection, thereby enhancing the drug's efficacy in treating bacterial pneumonia. The combination provides immediate antibacterial action, while its prolonged retention in the lung mucosa ensures continuous therapeutic levels, reducing the frequency of dosing.

Clinical observations also noted that the concentration of amoxicillin and clavulanate in the lung tissues was significantly above the minimum inhibitory concentration (MIC) for common pathogens like Streptococcus pneumoniae and Haemophilus influenzae. This property not only enhances the immediate bactericidal effect but also helps in disrupting bacterial biofilms, thus preventing the recurrence of infection and reducing the risk of developing antibiotic resistance.
The efficacy of the Amoxicillin Clavulanate nebulization suspension was evidenced by its ability to reduce symptoms of community acquired bacterial pneumonia, such as cough, sputum production, and chest pain, significantly faster than oral formulations. Additionally, the nebulization formulation includes mucoadhesive properties, which enhance drug retention and effectiveness. These findings underscore the potential of AONEUM-04 to offer a more efficient and patient-friendly treatment for Community acquired bacterial pneumonia, with reduced risks of antibiotic resistance, Liver injury and minimal adverse effects.

In Vitro Biofilm Disruption Study
Biofilm of S. pneumoniae was cultured for 2 days prior to treatment application. Post-treatment biofilm volume and live bacteria percentage were evaluated using Confocal Laser Scanning Microscopy. The time point for analysis was 12 hours after administration of AONEUM-04.
The study yielded dose-dependent results that were influenced by the time gradient, with the "ASrMaBd" technology-enhanced formulation showing significant diffusion of amoxicillin and clavulanate into the biofilm. This innovative technology facilitated the disruption of biofilm integrity and bacterial viability.

Invitro Mucoadhesion study in porcine model:
The study evaluates the mucoadhesive properties of the AONEUM-04 nebulization suspension using an in vitro Porcine Mucin Model to assess its potential for enhancing drug delivery in pneumonia treatment. The experiment analyzed two key parameters, Adhesive Work and

Adhesive Force across three groups: oral formulation, intravenous (IV) formulation, and AONEUM-04 nebulization suspension (n=5 per group). A texture analyzer applied a 2500 mN preload to each sample for 3 minutes, followed by lifting a cylinder probe at a speed of 2.5 mm/min to detach the sample from the mucin surface. The results showed that AONEUM-04 demonstrated significantly higher adhesive work (~145 mN·mm) and adhesive force (~1450 N) compared to the oral (~30 mN·mm adhesive work, ~200 N adhesive force) and IV formulations (~20 mN·mm adhesive work, ~150 N adhesive force). These findings highlight AONEUM-04's superior ability to adhere to mucosal surfaces, suggesting its potential for sustained drug retention and enhanced localized delivery in the lungs. Such enhanced mucoadhesive properties are crucial in the treatment of pneumonia, where prolonged residence time in the lungs can improve drug bioavailability, reduce dosing frequency, and ultimately improve patient outcomes. This study emphasizes the importance of optimizing pharmaceutical formulations to address challenges in respiratory drug delivery, offering a promising approach to enhance the efficacy of pneumonia treatments.

In vivo Sustained release study in New Zealand rabbits:
In a further embodiment, the present invention relates to the use of a nebulization suspension with sustained-release properties, exhibiting a controlled drug release of approximately 5 mcg/mL at 0.5 hours, about 5.2 mcg/mL at 1 hour, and maintaining therapeutic levels around 5 mcg/mL from 2 to 12 hours. The nebulization suspension is prepared as a stable aqueous pharmaceutical composition designed for enhanced pulmonary retention and prolonged therapeutic efficacy, facilitating localized drug delivery to the respiratory mucosa while reducing systemic fluctuations. This sustained-release profile was evaluated in a New Zealand Rabbit model over a 12-hour period, comparing nebulization suspension with a standard oral formulation of amoxicillin-clavulanate. Nasal fluid samples were collected at predetermined intervals over the 12-hour period to measure drug concentration and release rates. The results showed a gradual and sustained release of the drug, with nebulization suspension demonstrating superior release profiles compared to the oral formulation, which had more rapid fluctuations in drug concentration. This study underscores the potential of nebulization suspension as an effective treatment for pneumonia, offering prolonged therapeutic levels in the lungs, reducing the need for frequent dosing, and improving patient compliance by maintaining consistent drug delivery directly to the site of infection.

In Vivo Biofilm Disruption Study in New Zealand Rabbits:
The In Vivo Biofilm Disruption Study graph illustrates the efficacy of treatments in disrupting biofilm formation in S. pneumoniae-infected New Zealand rabbit pneumonia models. The study compared oral and intravenous formulations with the experimental nebulization suspension. Post-infection, rabbits were treated with these formulations, and lung tissue samples were collected at specific intervals to evaluate biofilm volume and bacterial viability. Confocal Laser Scanning Microscopy (CLSM) was utilized to measure live and dead biofilm volumes, providing insights into treatment effectiveness. The results demonstrate that the nebulization suspension significantly reduced live biofilm volume to approximately 2.5E+6 µm³, compared to 7.5E+6 µm³ for the oral formulation and 5.0E+6 µm³ for the intravenous formulation. Additionally, Nebulization suspension exhibited a markedly higher dead biofilm volume of approximately 9.0E+6 µm³, outperforming the oral (5.0E+6 µm³) and intravenous (7.1E+6 µm³) formulations. These findings highlight the superior biofilm disruption and microbial eradication capability of Nebulization suspension. This study underscores the potential of Nebulization suspension as a targeted treatment for pneumonia, providing enhanced localized drug delivery to the lungs, improving biofilm eradication, and addressing bacterial persistence, a key factor in pneumonia management.
,CLAIMS:5. CLAIMS
I/We Claim:

1. A pharmaceutical inhalation suspension comprising:
Amoxicillin, in a concentration of 0.5% to 7.5% w/v;
Clavulanate, in a concentration of 0.1% to 5.0% w/v;
Chitosan, in a concentration of 0.1% to 1% w/v as a mucoadhesive polymer;
PLGA with a lactide-to-glycolide ratio of 50:50 to 75:25, in a concentration of 0.5% to 1.5% w/v;
N-Acetylcysteine, in a concentration of 0.05% to 0.1% w/v, as biofilm disruption agents;
A surfactant, Polysorbate 80, in a concentration of 0.05% to 0.1% w/v;
A viscosity enhancer, HPMC, in a concentration of 0.1% to 0.5% w/v;
Buffered saline to maintain a pH range of 6.1 to 7.2.
2. The pharmaceutical inhalation suspension of claim 1, wherein the composition is formulated as a nebulization suspension.
3. The pharmaceutical inhalation suspension of claim 1, wherein the composition has antimicrobial activity against at least one of Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus.
4. The pharmaceutical inhalation suspension of claim 1, wherein the PLGA exhibit a particle size distribution with:
d10: 1–2 µm, d50: 2–3 µm, and d90: 4–5 µm,
optimized for deposition in central and peripheral lung regions.
5. The pharmaceutical inhalation suspension of claim 1, wherein the PLGA enable sustained release of the active pharmaceutical ingredients over 12 to 48 hours.
6. The pharmaceutical inhalation suspension of claim 1, wherein the inclusion of the mucoadhesive polymer Chitosan increases the residence time of the formulation in the respiratory tract by at least 20% compared to a non-mucoadhesive formulation.
7. The pharmaceutical inhalation suspension of claim 1, wherein the PLGA have a lactide-to-glycolide ratio of 75:25, providing extended release over 12 hours.
8. The pharmaceutical inhalation suspension of Claim 1, wherein the formulation is optimized for administration via a vibrating mesh nebulizer, ensuring particle integrity and consistent aerosol output.
9. The pharmaceutical inhalation suspension of Claim 1, wherein the formulation remains stable for at least 3 months at room temperature conditions of 25°C.
10. The pharmaceutical inhalation suspension of Claim 1, wherein the combination of EDTA and N-Acetylcysteine along with Amoxicillin and clavulanate achieves a 30% reduction in biofilm thickness within hours of administration.
11. The pharmaceutical inhalation suspension of Claim 1, wherein N-Acetylcysteine disrupts biofilms by reducing disulfide bonds in biofilm matrix proteins.
12. The pharmaceutical inhalation suspension of Claim 1, wherein the formulation remains physically and chemically stable for at least 6 months.
13. The pharmaceutical inhalation suspension of Claim 1, wherein the formulation comprises 2.0% w/v Amoxicillin and 0.75% w/v Clavulanate for the treatment of severe or resistant respiratory infections.
14. The use of the pharmaceutical inhalation suspension of Claim 1 for the elimination of drug-resistant Streptococcus pneumoniae, Haemophilus influenzae or other respiratory infections in the respiratory tract.
15. A method of manufacturing a nebulization suspension comprising amoxicillin and clavulanic acid, comprising the steps of:
providing amoxicillin and clavulanic acid;
providing chitosan, poly(lactic-co-glycolic acid) (PLGA), N-acetylcysteine, and glycerol;
combining said amoxicillin, clavulanic acid, chitosan, PLGA, N-acetylcysteine, and glycerol with sterile water for injection to form a suspension;
adjusting the pH of said suspension to a physiologically acceptable range;
sterile filtering said suspension;
aseptically filling said suspension into a suitable container; and
sealing said container.
16. The method of claim 15, wherein said step of sterile filtering is performed using a 0.22 micron filter.
17. The method of claim 15, wherein said suitable container is a respule.
18. The method of claim 15, wherein said suspension is subjected to stability testing under accelerated conditions.
19. The method of claim 15, wherein said step of combining further comprises adjusting the osmolality of said suspension to be isotonic.

6. DATE AND SIGNATURE

Dated this 29th day of December 2024
Signature
(Mr. Srinivas Maddipati)
IN/PA 3124
Agent for Applicant.