Niclosamide was discovered in 1958 and it was approved by the US Food and Drug Administration (FDA) for use in humans to treat tapeworm infection in 1982 and is included in the World Health Organization's list of essential medicines.

As mentioned above, niclosamide is indicated for intestinal infections caused by taenia saginata, taenia solium, diphyllobothrium latum, hymenolepis nana in adults and children. Niclosamide is marketed under the brand name Yomesan®as a tablet formulation. Niclosamide has been reported as a potential agent for host defense during viral infections. Wu et al. found that niclosamide inhibited Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) replication. (Wu et al. “Inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide” 2004 Jul;48(7):2693-6. doi: 10.1128/AAC.48.7.2693- 2696.2004.)

College of Pharmacy researchers at The University of Texas at Austin are studying on new drug delivery formulation technologies to increase niclosamide’s absorption into the body, designing the drug so that it can be inhaled directly into the lungs, which could prove effective at treating and preventing serious COVID-19 virus symptoms in patients. (“New Delivery Method Could Make Niclosamide an Effective Antiviral to Treat COVID-19”, Apr 06, 2020)

In the present invention, the low absorption problem of orally administered niclosamide is overcome by using a new drug delivery formulation that allows niclosamide to reach the target tissue effectively. Taking a lower dose of niclosamide by inhalation from the lungs instead of taking a high dose orally is beneficial for patient compliance.

Among the inhalation techniques, nebul products are easier to use. Nebulizers have a relatively simple usage technique compared to metered dose inhaler and dry powder inhaler devices. A metered-dose inhaler requires hand-breath coordination and an adequate flow rate for a dry powder inhaler. Nebulizer devices can be selected for patients who cannot use these two devices effectively. The medicine produced in the form of nebules from these devices can be delivered to the lungs by inhaling and exhaling with a mouthpiece or mask.

Efficient access of the inhaler formulations to the lungs is achieved by optimizing the formulation and process steps with the device, active and auxiliary components. In order to ensure effective delivery of niclosamide to the lungs, the excipients included in the drug formulation must be properly selected, quantified, and included in the process in determining steps.

Besides, one of the most important factors in order to ensure an effective penetration into the low respiratory tract of the patient is that the particle size of the active agent, which must be equal or lower than 10 pm.

The dispersing or suspending agents, buffering agents, isotonic agents and preservatives used in a certain order in the process steps and their weight ratio are important in terms of increased stability, delivery rate, and total active agent values. Also, the type of all these excipients has a critical effect on these quality parameters and, accordingly, the performance of the suspension product.

The steps of adding dispersing or suspending agents, buffering agents, isotonic agents and preservatives used in the process to the process and their weight ratios of applied are of great importance in order to ensure homogenization and prevent losses in the process.

In order to ensure an effective and save penetration into the low respiratory tract of the patient, another important factor that must be met by pharmaceutical formulations for inhalation is sterility. This requirement is becoming more and more mandatory as confirmed by the FDA final rule "Sterility Requirement for Aqueous-Based Drug Products for Oral Inhalation" published in the Federal Register of May 26, 2000 (65 FR 34082) governing the quality and safety of pharmaceutical products for a number of reasons, including the fact that the lungs are a particularly vulnerable organ of the human body, and many patients who use inhaled drugs have general health problems.

There are two types of sterilization can be used to manufacture sterile pharmaceutical formulations for inhalation: chemical and physical.

Chemical sterilization, for the most part, has been based on exposure to toxic compounds, for example, formaldehyde, ethylene oxide.

Physical methods include a sun-light method, heat methods (dry heat which includes red heat, flaming, incineration, hot air oven, infra-red and moist heat which includes below 100°C, at 100°C, above 100°C), vibration methods, filtration methods and radiation methods.

Autoclaving is one of the most effective methods for sterilization in in several industries, especially in the pharmaceutical industry.

The autoclaving process takes advantage of the fact that the boiling point of water increases when it is under high pressure.

Sterilization is achieved by exposing pharmaceutical active agents or excipients to saturated steam at high temperature for desired period of time. Among the most efficient methods of transferring thermal energy is by humidity found in steam. Owing to its capacity to infiltrate bacterial cells using steam, autoclaving has been observed to be more efficient at destroying a wide variety of microorganisms than sterilization by other methods. Because of increased pressure and steam production inside an autoclave, the temperature inside the vessel may be raised above 100°C. This process enables the eradication of a wider variety of bacteria and other microbes. In comparison with other sterilization methods, autoclaving is nontoxic. Since steam that comes from boiling water requires no additional chemicals, it reduces the need for using dangerous chemicals. Autoclaving, therefore, is safer because it easily eliminates bacteria and other pathogens while still being a relatively safe procedure to use. Along with being readily usable, steam is often cost-efficient. Because autoclaves don’t need any other chemicals, operating it is reasonably affordable. This makes it the most economical method of sterilization.

Considering the state of art, there is still a need for innovative processes that will solve the homogenization and sterilization problems, and which will provide a standardized method for the fast, robust and reproducible production of sterile, homogeneous suspensions for inhalation.

Therefore, production processes consisting of optimized steps for the preparation of a sterile, homogeneous suspension comprising niclosamide as an active agent for inhalation by nebulization will provide a development and improvement in the technical field.

Objects and Brief Description of the Invention

The main object of the present invention is to provide a process for the preparation a sterile, homogeneous suspension of niclosamide or a pharmaceutically acceptable salt thereof for inhalation by nebulization which eliminates all aforesaid problems and brings additional advantages to the relevant prior art.

Another object of the present invention is to provide a process for the preparation of a sterile, homogeneous suspension of niclosamide or a pharmaceutically acceptable salt thereof for inhalation by nebulization for use in the prevention, treatment, or the alleviation of the symptoms of Covid-19.

Another object of the present invention is to provide a process for the preparation of a sterile, homogeneous suspension of niclosamide or a pharmaceutically acceptable salt thereof for inhalation by nebulization with enhanced uniformity and homogeneity.

A further object of the present invention is to obtain a suspension comprising niclosamide or a pharmaceutically acceptable salt thereof. Another object of the present invention is to obtain a suspension comprising niclosamide, distilled water, dispersing or suspending agents, buffering agents, isotonic agents and preservatives.

Another object of the present invention is to a new drug delivery formulation that allows niclosamide to reach the target tissue effectively provided by the above-mentioned process.

Another object of the present invention is to provide a suspension for inhalation by nebulization, sterilized by autoclaving.

Detailed description of the invention

In accordance with the objects outlined above, detailed features of the present invention are given herein.

The present invention relates to a process for the preparation of a sterile, homogeneous suspension of niclosamide for inhalation by nebulization, which comprises the following steps; a- obtaining Solvent A by:

(i) mixing at least two buffering agents and an isotonic agent,

(ii) filtering the mixture through a 0.2 pm pore size membrane filter, b- obtaining Solvent B by:

(i) mixing distilled water and at least two dispersing or suspending agents,

(ii) adding niclosamide and mixing,

(iii) homogenizing the mixture,

(iv) autoclaving, c- mixing the Solvent A and Solvent B, d- filling the final mixture into vials.

An important factor is the preparation of a suitable dispersing medium by including the dispersing or suspending agents in the process before the active agents to help disperse the active agents that is insoluble in water such as niclosamide.

The main purpose of dispersing a powder in a liquid is to separate primary particles from aggregates and agglomerates and then stabilize them in the liquid medium. This requires an effective dispersing or suspending agent that adsorbs very quickly at the solid/liquid interface. The main criterion for an effective dispersing or suspending agent is strong adsorption or attachment to the particle surface. This requires the dispersing or suspending agents to be attached to the active agent particle surface at multiple points.

In addition, another role of the dispersing or suspending agents in suspension is to reduce the surface energy of the powder. This facilitates the decomposition and homogeneous dispersion stages of the agglomerates of the particles in the subsequent homogenization.

Although a single dispersing or suspending agent could not provide the medium to suspend the active agent, the desired quality profile was achieved when the second dispersing or suspending agent was added. Likewise, although the second dispersing or suspending agent alone could not provide the medium to suspend the active agent, the desired quality profile was achieved when the first dispersing or suspending agent was added. The inventors have surprisingly been found that when at least two dispersing or suspending agents are added together to the mixture together, a homogeneous final product can be obtained.

The applied process steps have a direct effect on the blend uniformity, which is one of the first chemical indicators of the product's quality profile. It has been observed that if the above steps are not followed and are added only one dispersing or suspending agent into the present process, the blend uniformity cannot be achieved.

The steps of adding dispersing or suspending agents and their weight ratios of great importance in order to ensure homogenization and prevent losses in the process.

According to the one embodiment, the dispersing or suspending agents are selected from the group comprising polysorbate 20, sorbitan monolaurate, sorbitan trioleate (SpanR85), sorbitan mono-oleate, polyoxyethylene (20) sorbitan monooleate, natural lecithin, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of oxyethylene and oxypropylene, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, glyceryl mono-oleate, polyethylene glycol 400 and glyceryl monolaurate or mixtures thereof.

According to the preferred embodiment, the dispersing or suspending agents are polysorbate 20 and sorbitan monolaurate.

Typically, the pharmaceutical suspension comprises one or more buffering agents. The buffering agents are pharmaceutically acceptable buffering agents. The buffering agents may be any buffering agents suitable for use in a liquid pharmaceutical composition suitable for inhalation. According to one embodiment, the pharmaceutical composition comprises at least two buffering agents in the present invention.

According to one embodiment, the buffering agents are selected from the group comprising monosodium phosphate dihydrate, dibasic sodium phosphate anhydrous, citric acid, phosphate buffers, acetate buffers, acetic acid, sulfuric acid, fumaric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, sodium carbonate, potassium carbonate, ammonium carbonate, sodium sulfate, sodium hydroxide, potassium hydroxide, sodium citrate or mixtures thereof.

According to the preferred embodiment, the buffering agents are monosodium phosphate dihydrate and dibasic sodium phosphate anhydrous.

The pharmaceutical suspension typically comprises isotonic agents. The isotonic agents may be any pharmaceutically acceptable isotonic agents. Suspensions will desirably be isotonic. The formulations which are used present process may be adjusted to desired isotonicity by the addition of suitable isotonic agents.

According to a preferred embodiment, the isotonic agent is selected from the group comprising sodium chloride, potassium chloride and sodium bromide or mixtures thereof.

According to the preferred embodiment, the isotonic agent is sodium chloride.

The pharmaceutical suspension typically comprises preservatives to the control of microbial proliferation in inhalation products in aqueous form. The simplest approach is to use preservatives that protect the product against certain microorganisms.

According to a preferred embodiment, the preservative is selected from the group comprising benzyl alcohol, methyl paraben, propyl paraben, benzalkonium chloride or mixtures thereof.

According to one embodiment, the pharmaceutical compositions subjected to the invention are prepared by these steps: a- obtaining Solvent A by:

(i) mixing monosodium phosphate dihydrate, dibasic sodium phosphate anhydrous and sodium chloride,

(i) mixing distilled water and polysorbate 20, sorbitan monolaurate,

(ii) adding niclosamide and mixing,

(iii) homogenizing the mixture,

The invention also defines a sterile, homogeneous suspension for inhalation by nebulization obtained by the process subjected to the invention.

According to the preferred embodiment, a sterile, homogeneous suspension for inhalation by nebulization comprising niclosamide, distilled water, dispersing or suspending agents, isotonic agents, buffering agents and preservatives.

According to one embodiment, the concentration of polysorbate 20 in the suspension is between 0.01-1 mg/ml.

According to one embodiment, the concentration of sorbitan monolaurate in the suspension is between 0.005-3 mg/ml.

According to one embodiment, the concentration of monosodium phosphate dihydrate in the suspension is between 5-15 mg/ml.

According to one embodiment, the concentration of dibasic sodium phosphate anhydrous in the suspension is between 0.1-5 mg/ml.

According to one embodiment, the concentration of sodium chloride in the suspension is between 3-9 mg/ml.

According to the one embodiment, the temperature of the mixture in the step numbered a (i) and b (i), (ii), (iii) is 30°C-40°C, preferably 40°C. The reason for this specific temperature is to increase the solubility or distribution of the excipients to be added.

According to the one embodiment, the duration of the step numbered a (i) and b (i) is 15-30 minutes. According to the one embodiment, the duration of the step numbered b (ii), (iii) is 45-75 minutes, preferably 50-70 minutes, more preferably 55-65 minutes.

If a high rotational speed of homogenization is used, the particles may be damaged and the product may degrade due to heating. On the other hand, a low rotational speed of homogenization is used, mixing uniformity may not be achieved. If the mixture cannot be homogeneous, a suitable product with desired therapeutic properties cannot be produced. Moreover, the increase in temperature during homogenization causes an increase in the impurities in the product. Therefore, rotational speed and time of homogenization are very important during homogenization.

According to one embodiment, the rotational speed in the step numbered a (i) and b (i), (ii) is 200-5000 rpm, preferably 200-4000 rpm, more preferably 200-2000 rpm.

According to one embodiment, the rotational speed in the step numbered b (iii) is 3000-25000 rpm, preferably 3200-15000 rpm, more preferably 3400-8000 rpm.

Particle size distribution of the active agent plays a critical role for the qualification of the composition subjected to the invention.

As used herein, ‘particle size distribution’ means the cumulative volume size distribution as tested by any conventionally accepted method such as the laser diffraction method (Malvern analysis).

Laser diffraction measures particle size distributions by measuring the angular variation in intensity of light scattered as a laser beam passes through a dispersed particulate sample. Large particles scatter light at small angles relative to the laser beam and small particles scatter light at large angles. The angular scattering intensity data is then analyzed to calculate the size of the particles responsible for creating the scattering. The particle size is reported as a volume equivalent sphere diameter.

In the preferred embodiment of the invention, the active agent particles have a particle size (Dg₀ value) lower than 10 pm.

According to the preferred embodiment, the active agent is niclosamide or pharmaceutically acceptable salt thereof. According to one embodiment, the concentration of niclosamide in the suspension is 0.1-5 mg/ml, preferably 0.1-3 mg/ml, more preferably 0.1-2.5 mg/ml.

According to one embodiment, sterilization of process for the preparation of suspensions for inhalation by nebulization comprises autoclaving.

In the preferred embodiment of the invention, autoclaving is carried out at a temperature of 121 °C for about 15 to about 30 minutes.

The inventors have been observed physical and chemical degradations when they applied the various sterilization types specified in the state of the art to this process. One of the most important aspects of the invention is the use of autoclaving, among the sterilization types mentioned in the state of the art. On the other hand, the inventors have surprisingly been found that the problems of chemical and physical degradation of the product have been solved when they applied autoclaving. Because steam is a kind of vapor that is formed when water is boiled, it effectively is water but in a different state of matter so it is a simple sterilization method without using chemicals etc.. The problems of chemical and physical degradation of the product, which occur in other sterilization methods, are solved by autoclaving.

As seen above, there are various processes for the preparation of a sterile, homogeneous suspension. The active and excipients used in the process steps in a certain order in the process steps and using certain mixing speeds in certain steps, the time and temperature of the process steps are important in terms of increased stability, delivery rate and total active agent values.

According to all these embodiments, the below-given formulation can be used process for the preparation of a sterile, homogeneous suspension subjected to the invention. This example is not limiting the scope of the present invention and should be considered under the light of the foregoing detailed disclosure. Example 1 :

According to a preferred embodiment, a sterile, homogeneous suspension subjected to the invention is used in the treatment or the prophylaxis of different infections especially, SARS- CoV-2, the coronavirus that causes COVID-19.

Claims

1 . A process for the preparation of a sterile, homogeneous suspension of niclosamide for inhalation by nebulization, which comprises the following steps; a- obtaining Solvent A by:

(i) mixing at least two buffering agents and an isotonic agent,

(i) mixing distilled water and at least two dispersing or suspending agents,

(ii) adding niclosamide and mixing,

(iii) homogenizing the mixture,

2. The process according to claim 1 , wherein the dispersing or suspending agents are selected from the group comprising polysorbate 20, sorbitan monolaurate, sorbitan trioleate (SpanR85), sorbitan mono-oleate, polyoxyethylene (20) sorbitan monooleate, natural lecithin, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of oxyethylene and oxypropylene, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, glyceryl monooleate, polyethylene glycol 400 and glyceryl monolaurate or mixtures thereof.

3. The process according to claim 2, the dispersing or suspending agents are polysorbate 20 and sorbitan monolaurate.

4. The process according to any one of the preceding claims, wherein the buffering agents are selected from the group comprising monosodium phosphate dihydrate, dibasic sodium phosphate anhydrous, citric acid, phosphate buffers, acetate buffers, acetic acid, sulfuric acid, fumaric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, sodium carbonate, potassium carbonate, ammonium carbonate, sodium sulfate, sodium hydroxide, potassium hydroxide, sodium citrate or mixtures thereof.

5. The process according to claim 4, wherein the buffering agents are monosodium phosphate dihydrate and dibasic sodium phosphate anhydrous.

6. The process according to any one of the preceding claims, wherein the isotonic agent is selected from the group comprising sodium chloride, potassium chloride and sodium bromide or mixtures thereof.

7. The process according to claim 6, the isotonic agent is sodium chloride.

8. The process according to any one of the preceding claims, wherein the composition further comprises a preservative.

9. The process according to claim 8, wherein the preservative is selected from the group comprising benzyl alcohol, methyl paraben, propyl paraben, benzalkonium chloride or mixtures thereof.

10. A process according to any one of the preceding claims, wherein the temperature of the mixture in the step numbered a (i) and b (i), (ii), (iii) is 30°C-40°C.

11. A process according to any one of the preceding claims, wherein duration of the step numbered a (i) and b (i) is 15-30 minutes.

12. A process according to any one of the preceding claims, wherein duration of the step numbered b (ii), (iii) is 45-75 minutes, preferably 50-70 minutes, more preferably 55-65 minutes.

13. A process according to any one of the preceding claims, wherein the rotational speed in the step numbered a (i) and b (i), (ii) is 200-5000 rpm, preferably 200-4000 rpm, more preferably 200-2000 rpm.

14. A process according to any one of the preceding claims, wherein the rotational speed in the step numbered b (iii) is 3000-25000 rpm, preferably 3200-15000 rpm, more preferably 3400-8000 rpm.

15. A process according to any one of the preceding claims, wherein the concentration of niclosamide in the pharmaceutical composition is 0.1-5 mg/ml, preferably 0.1-3 mg/ml, more preferably 0.1 -2.5 mg/ml.