GB2565941A

GB2565941A - A process for preparing a formulation

Info

Publication number: GB2565941A
Application number: GB1819292.2A
Authority: GB
Inventors: Hawkins Kevin; Barnett Michelle; Smith Nichola
Original assignee: Norton Healthcare Ltd
Current assignee: Norton Healthcare Ltd
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2019-02-27
Also published as: GB201819292D0

Abstract

A process for preparing a tobramycin formulation is provided, wherein the process comprises the following steps in order: (i) providing a bulk solution consisting of tobramycin; water; sodium chloride, sodium citrate and a pH adjusting agent, preferably sulfuric acid or a combination of sulfuric acid and sodium hydroxide, wherein the bulk solution has a pH of 5.0 and an osmolality of 248 mOsmol/kg; (ii) sparging the bulk solution with nitrogen; (iii) filling, under sterile air, the bulk solution into ampoules, wherein each ampoule contains 4ml of the bulk solution and 300mg tobramycin. Preferably the sodium citrate is trisodium citrate dihydrate. A 4ml formulation for inhalation, the formulation consisting of: 300mg tobramycin; water; 13.5mg sodium chloride; 12.0mg sodium citrate; and an amount of sulfuric acid to a achieve a pH of 5.0, wherein the osmolality of the formulation is 248 mOsmol/kg is also provided. Preferably the formulation is for use in the treatment of a Pseudomonas aeruginosa infection in patients with cystic fibrosis.

Description

A process for preparing a formulation

The present invention relates to a process for preparing a formulation and in particular, a tobramycin formulation for inhalation.

Tobramycin is an aminoglycoside antibiotic having the following chemical structure:

It is specifically active against Pseudomonas aeruginosa and is indicated for the long-term management of chronic pulmonary infection caused by Pseudomonas aeruginosa in cystic fibrosis (CF) patients aged 6 years and older.

Tobramycin may be administered parenterally for the treatment of chronic pulmonary infection. However, aminoglycoside penetration into the bronchial secretions is poor and to achieve a therapeutic concentration in sputum, high doses are required. Such high doses increase the risk of systemic toxicity.

To avoid the problem of toxicity via parenteral administration, tobramycin may also be administered via inhalation. The preferred approach for administration by inhalation for tobramycin is by nebulisation of an aqueous solution of tobramycin. Typically, active ingredients are dissolved in saline for nebulisation. However, jet and ultrasonic nebulisers are highly sensitive to the osmolality of the formulation and hence careful control of the tonicity is required. Moreover, CF patients and other patients with chronic endobronchial infections are sensitive to hypo- or hypertonic aerosols.

Tobramycin is marketed as TOBI® by Novartis in the EU and Bramitob®/Bethkis® by Chiesi in the EU and US. TOBI® is sold in the form of a 300 mg/5 mL nebuliser solution and Bramitob®/Bethkis® is sold in the form of a 300 mg/4 mL nebuliser solution. Both 5 mL and 4 mL nebules contain tobramycin 300 mg as a single dose. They are supplied for use via inhalation. Both TOBI® and Bramitob®/Bethkis® contain tobramycin, sodium chloride, water for injection, and sulfuric acid/sodium hydroxide for pH adjustment.

Tobramycin formulations are known to be chemically unstable. Both TOBI® and Bramitob®/Bethkis® are supplied with special precautions for storage, namely that they should be stored at 2-8°C in their original packaging and protected from sunlight. Some yellowing of the formulations nevertheless occurs overtime due to degradation.

Further discussion of the 5 mL formulation may be found in WO 96/12471. This document is not specifically concerned with addressing the problem of storage stability but it does explain that the tobramycin formulation should be treated by nitrogen sparging (see the paragraph bridging pages 15 and 16) in order to minimise hydrolysis and oxidative degradation, although it does not elaborate further.

WO 2013/167233 also relates to a 5 mL tobramycin formulation. This document is concerned with providing a stable formulation. The formulation contains sodium citrate in order to minimise the formation of the degradation product, nebramine. Nebramine is (1/?,2/?,3S,4/?,6S)-4,6-diamino-2,3dihydroxycyclohexyl 2,6-diamino-2,3,6-trideoxy-a-D-ribo-hexopyranoside and has the following chemical structure:

WO 2013/167233 also discusses how, on account of the effect of sodium citrate, the formulation may be prepared without sparging with an inert gas.

Tobramycin formulations having a volume of 4 mL are advantageous over those having a volume of 5 mL in that the reduction of 1 mL in the total volume reduces the average inhalation time by about 12% for adults, e.g. from 847 seconds to 748 seconds, and by about 28% for children, e.g. from 1059 seconds to 759 seconds, which is important when considering the amount of time CF patients spend inhaling medicines.

However, the presence of impurities such as nebramine and other impurities in tobramycin formulations remains a concern, particularly for the 4 mL tobramycin formulation, which is of course more concentrated than the 5 mL tobramycin formulation. Typically, the amount of impurities in any pharmaceutical product tends to increase over time, and even more in unstable products such as tobramycin. Degradation of the drug before aerosolisation results in a smaller amount of the drug being delivered to the lung thus impairing the treatment as well as provoking conditions which could lead to the development of resistance to tobramycin because the delivered dose would be too low. Moreover, tobramycin degradation products may provoke bronchospasm and a cough. To preclude this degradation, the 4 mL tobramycin formulation BramitobO/Bethkis® is prepared by not only sparging a bulk solution of the tobramycin formulation with nitrogen but also by filling under nitrogen the bulk solution into ampoules.

However, there remains a need in the art for an improved process for preparing a chemically stable 4 ml. tobramycin formulation.

Accordingly, the present invention provides a process for preparing a tobramycin formulation, the process comprising the following steps, in order: (i) providing a bulk solution consisting of tobramycin, water, sodium chloride, sodium citrate and a pH adjusting agent, wherein the bulk solution has a pH of 5.0 and an osmolality of 248 mOsmol/Kg; (ii) sparging the bulk solution with nitrogen; (iii) filling, under sterile air, the bulk solution into ampoules, wherein each ampoule contains 4 ml. of the bulk solution and 300 mg tobramycin.

The inventors have surprisingly found that the filling step can be carried out under sterile air without compromising the chemical stability of a 300 mg/4 mL tobramycin formulation. It is surprising that the 300 mg/4 mL tobramycin formulation is chemically stable when prepared in this way. The process of the present invention therefore reduces the complexity and cost of manufacturing significantly compared to known processes for preparing a 300 mg/4 mL tobramycin formulation. Nitrogen has to be purchased or manufactured on site. In addition, use of nitrogen has safety implications as it is an asphyxiating agent and therefore requires oxygen-level monitoring and increased safety procedures for operators during manufacture.

The process of the present invention comprises step (i): providing a bulk solution consisting of tobramycin, water, sodium chloride, sodium citrate and a pH adjusting agent, wherein the bulk solution has a pH of 5.0 and an osmolality of 248 mOsmol/Kg.

The tobramycin formulation is initially provided as a bulk solution, i.e. a solution that contains multiple 4 mL/300 mg tobramycin doses.

The bulk solution consists of tobramycin, water, sodium chloride, sodium citrate and a pH adjusting agent. No other components are added to the bulk solution but trace impurities that do not affect the tobramycin dose may be present. The tobramycin formulation provided by the process of the present invention is preservative free. Examples of excluded preservatives are benzalkonium chloride, benzoic acid, ethanol and others as disclosed in US Pharmacopeia, USP 40 NF35. The absence of preservatives prevents the development of secondary undesirable side effects such as bronchospasm and a cough.

The bulk solution is a solution in that tobramycin, sodium chloride, sodium citrate and a pH adjusting agent are dissolved in water. The bulk solution is therefore homogenous throughout.

The active substance in the formulation is the aminoglycoside antibiotic, tobramycin. Tobramycin is specifically active against Pseudomonas aeruginosa. As used herein, the term tobramycin refers to tobramycin free base.

The tobramycin formulation is an aqueous formulation and further contains water. The water is preferably water for injection.

Sodium chloride is present in the bulk solution. The minimum amount of sodium chloride present in the formulation is defined by the osmolality. Preferably, sodium chloride is present in an amount of 3.375 mg/mL.

Sodium citrate is also present in the bulk solution. It is believed that sodium citrate in combination with sparging the bulk solution with nitrogen obviates the use of an oxygen-deficient atmosphere in the filling step when preparing a 300 mg/4 ml. tobramycin formulation.

Citric acid is trivalent and sodium citrate is preferably the trisodium salt. Unless indicated otherwise, reference to sodium citrate indicates the mono-, di- or trisodium salts or combinations thereof. Preferably, the sodium citrate used is trisodium citrate. Sodium citrate is preferably introduced to the bulk solution as trisodium citrate dihydrate.

Preferably, sodium citrate is present in an amount of 3.0 mg/mL, when sodium citrate is introduced to the bulk solution as trisodium citrate dihydrate.

The bulk solution has a pH of 5.0. This is achieved by adjusting the amount of the pH adjusting agent present. Preferably, the pH adjusting agent is sulfuric acid or a combination of sulfuric acid and sodium hydroxide. Most preferably, the pH adjusting agent is sulfuric acid. As concentrations, 10% sulfuric acid and 5M sodium hydroxide are suitable. The pH may be measured using a conventional pH meter at 20°C.

The bulk solution has an osmolality of 248 mOsmol/Kg. This is achieved by adjusting the amounts of the components present, in particular sodium chloride and sodium citrate. The osmolality may be measured using a freezing-point depression osmometer, following the procedure set out in the US Pharmacopeia, USP 34, National Formulary 29, 2011, chapter <785>. The controlled osmolality allows patients with compromised lungs such as CF patients to administer the tobramycin formulation provided by the process of the present invention.

Preferably, each mL of the tobramycin formulation prepared by the process of the present invention consists of 7.5% w/v of tobramycin, 0.3375% w/v of sodium chloride, 0.3% w/v of sodium citrate and an amount of sulfuric acid to reach a pH of 5.0.

Preferably, after its preparation, the bulk solution is filtered to remove any solid particles. A pre-use filter integrity test 0.2 pm sterilising filter may be used. The bulk solution may then be transferred into one or more holding tanks.

The process of the present invention comprises step (ii): sparging the bulk solution with nitrogen. The bulk formulation may be sparged with nitrogen in the one or more holding tanks. The nitrogen used is sterile and preferably has a purity of 99.998%. Preferably, the bulk solution is sparged until the bulk solution contains < 3 ppm dissolved oxygen. Preferably, the bulk solution is then held in the one or more holding tanks under a nitrogen atmosphere.

The process of the present invention comprises step (iii): filling, under sterile air, the bulk solution into ampoules, wherein each ampoule contains 4 ml. of the bulk solution and 300 mg tobramycin.

The bulk solution is filled into ampoules. Ampoules are well known in the art and are small, sealed vials, generally made up of glass, though plastic ampoules are also made. They are used to preserve and contain samples so that they can be protected from exposure to air and other contaminants.

Step (iii) is performed under sterile air. Sterile air is air that has been treated to remove microorganisms including bacteria. Microorganisms can be moved using conventional techniques such as filtration. Sterile air is therefore free from microorganisms.

In the present invention, filling is performed under sterile air instead of under sterile nitrogen as recommended in the literature. Step (iii) is therefore performed in the absence of an oxygen-deficient atmosphere. An oxygen-deficient atmosphere is one having less oxygen than atmospheric air. Typically, during the filling processes of the prior art, oxygen is replaced with inert gases, such as carbon dioxide, nitrogen or argon. However, in the process of the present invention, an oxygendeficient atmosphere is not required during the filling step to provide a chemically stable 300 mg/4 ml. tobramycin formulation. There is no additional stream of nitrogen used in the filling step. This reduces the complexity and cost of manufacturing significantly. Therefore, the term the absence of an oxygen-deficient atmosphere simply means that step (iii) of the process of the present invention does not require an oxygen-deficient atmosphere, unlike typical processes.

The bulk formulation may be transferred from the one or more holding tanks via an in-line pre-use filter integrity test filter (0.1 pm) to a Blow-Fill-Seal (BFS) machine. BFS technology is well known in the art. The basic concept of BFS is that a container is formed, filled, and sealed in a continuous process without human intervention, in a sterile enclosed area inside a machine. Thus this technology can be used to aseptically manufacture sterile pharmaceutical liquid dosage forms.

BFS brings together the two technologies of plastic container moulding and metered dose filling.

Preferably, low-density polyethylene (LDPE) is extruded by the BFS machine to form open topped ampoules which are presented to a filling head. The bulk solution is then filled into the formed ampoules via a time/pressure dosing unit which delivers a precise volume of 4 ml. of bulk solution via one or more filling needles. When the open ampoules have been filled, the one or more filling needles are withdrawn and the head section ofthe mould closes to seal the ampoule completely.

The maximum duration ofthe filling process is 168 hours.

Each ampoule contains 4 ml. ofthe bulk solution and 300 mg tobramycin. Therefore, each ampoule contains a single dose of a 4 ml. tobramycin for inhalation.

Preferably, each ampoule contains 13.5 mg sodium chloride.

Preferably, each ampoule contains 12.0 mg sodium citrate, when sodium citrate is introduced to the bulk solution as trisodium citrate dihydrate.

The 300 mg/4 ml. tobramycin formulation prepared by the process of the present invention is surprisingly chemically stable despite the bulk solution being transferred to the ampoules in the absence of an oxygen-deficient atmosphere.

The tobramycin formulation ofthe present invention is chemically stable in that it contains low levels of impurities. Such low levels of impurities are surprisingly maintained over time, even when the formulation is exposed to temperatures and relative humidity (RH) which are above those recommended for storage, such as a temperature of 25°C and a RH of 40%.

Surprisingly, the tobramycin formulation prepared using the process of the present invention was found to contain low levels of impurities which are maintained overtime, even though the tobramycin formulation has a pH limit of 5.0 and it was not filled with nitrogen prior to sealing each unit dose as recommended in the prior art for maintaining a longer shelf life.

In order to assess chemical stability, the tobramycin formulation prepared by the process of the present invention is stored at 5°C for 39 weeks and/or is stored at 25°C and 40% RH for 26 weeks, according to ICH Q1A guidelines, and the amounts of impurities and tobramycin present are assessed. The impurity profile is measured and the tobramycin assay is performed by HPLC according to the US Pharmacopeia Monograph for Tobramycin Inhalation Solution, USP 29, National Formulary 24. The following amounts of each impurity are presented as a percentage with respect to the amount of tobramycin present in a sample of the formulation and the following amounts of tobramycin in the assay are presented as a percentage relative to a labelled amount of tobramycin, i.e. 75 mg/1 ml_.

Preferably, the tobramycin formulation contains deoxystreptamine kanosaminide as an impurity in an amount of 0.3% by weight or less, more preferably 0.03% by weight or less.

In a preferred embodiment, the tobramycin formulation contains an impurity at a relative retention time (RRT) of 0.36 in an amount of 0.25% by weight or less, more preferably 0.1% by weight or less.

In another preferred embodiment, the tobramycin formulation contains one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.1% by weight or less, more preferably 0.04% by weight or less.

In a further preferred embodiment, the tobramycin formulation contains one or more impurities in a total amount of 1.0% by weight or less, more preferably 0.13% by weight and most preferably 0.03% by weight or less. This includes deoxystreptamine kanosaminide, the impurity at a RRT of 0.36 and the one or more impurities at a RRT of 0.70 and/or 0.74.

Preferably, the tobramycin formulation prepared according to the process of the present invention contains tobramycin in an amount of 90-110%, preferably 98-102%, relative to a labelled amount of 75 mg/1 ml. tobramycin using the HPLC method disclosed in the USP Monograph for Tobramycin Inhalation Solution, USP 29, National Formulary 24. This indicates that the extent of degradation of the tobramycin present in the formulation is very small or even negligible.

In a preferred embodiment, the tobramycin formulation contains: deoxystreptamine kanosaminide as an impurity in an amount of 0.3% by weight or less; an impurity at a RRT of 0.36 in an amount of 0.25% by weight or less; and one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.1% by weight or less, wherein one or more impurities are present in a total amount of 1.0% by weight or less.

In another preferred embodiment, the tobramycin formulation contains: deoxystreptamine kanosaminide as an impurity in an amount of 0.03% by weight or less; an impurity at a RRT of 0.36 in an amount of 0.1% by weight or less; and one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.04% by weight or less, wherein one or more impurities are present in a total amount of 0.13% by weight or less.

In a further preferred embodiment, the tobramycin formulation contains: deoxystreptamine kanosaminide as an impurity in an amount of 0.03% by weight or less; an impurity at a RRT of 0.36 in an amount of 0.1% by weight or less; and one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.04% by weight or less, wherein one or more impurities are present in a total amount of 0.03% by weight or less.

Preferably, the tobramycin formulation contains the above limitations relating to the amounts of the impurities in combination with tobramycin in an amount of 90-110%, preferably 98-102%, relative to a labelled amount of 75 mg/1 mL tobramycin using the HPLC method disclosed in the USP Monograph for Tobramycin Inhalation Solution, USP 29, National Formulary 24.

The formulation described herein may be used for the treatment of Pseudomonas aeruginosa infection, particularly in patients with CF. Specifically, it is presently indicated for the long-term management of chronic pulmonary infection due to Pseudomonas aeruginosa in CF patients aged 6 years and older. The antimicrobial kill rate test in Table 5 below demonstrates the effectiveness of the tobramycin formulation against microbial species, in particular against Pseudomonas aeruginosa, thus making it suitable for the treatment of Pseudomonas aeruginosa infection in CF patients.

The formulation is suitable for inhalation and preferably the treatment is by inhalation. This is apparent from the droplet size data in Table 6 below, and from the dose delivery data such as the active substance delivery rate (output rate) and the total active substance delivered (total drug output), as presented in Table 7 below.

Most preferably, the formulation is administered using various types of nebulisers, e.g. a jet, ultrasonic or vibrating mesh nebuliser, like the l-Neb. These nebulisers deliver the formulation as an aerosol. Typically, the aerosol has a particle size of 1-5 pm which is ideal for treatment by inhalation. Accordingly, the present invention may also provide a nebule containing the formulation.

In one embodiment, the tobramycin formulation prepared by the process of the present invention is characterised by one or more of the following active drug delivery features: output rate for an adult of 0.1-0.2 pg/second and fora child of 0.1-0.15 pg/second as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <1604> Products For Nebulization—Characterization Tests; and total drug output for an adult of 30-40 pg and for a child of 22-30 pg as measured by US Pharmacopeia, USP 40, National Formulary 35, 2017, chapter <1604> Products For Nebulization—Characterization Tests when using a Pari LC Plus connected to the Pari Vios compressor.

In another embodiment, the tobramycin formulation prepared by the process of the present invention is characterised by one or more of the following active drug delivery features: output rate for an adult of 0.1-0.2 pg/second and for a child of 0.1-0.15 pg/second as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <1604> Products For Nebulization—Characterization Tests; and total drug output for an adult of 30-40 pg and for a child of 22-30 pg as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <1604> Products For Nebulization— Characterization Tests when using a Pari LC Plus connected to the Pari Vios compressor, wherein the tobramycin formulation contains: deoxystreptamine kanosaminide as an impurity in an amount of 0.3% by weight or less; an impurity at a RRT of 0.36 in an amount of 0.25% by weight or less; and one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.1% by weight or less, wherein one or more impurities are present in a total amount of 1.0% by weight or less.

Preferably, the tobramycin formulation prepared by the process of the present invention is characterised by one or more of the following active drug delivery features: output rate for an adult of 0.1-0.2 pg/second and fora child of 0.1-0.15 pg/second as measured by USP 39, National Formulary 34, chapter <1604> Products For Nebulization—Characterization Tests; and total drug output for an adult of 30-40 pg and for a child of 22-30 pg as measured by US Pharmacopeia, USP 40, National Formulary 35, 2017, chapter <1604> Products For Nebulization—Characterization Tests when using a Pari LC Plus connected to the Pari Vios compressor, wherein the tobramycin formulation contains: deoxystreptamine kanosaminide as an impurity in an amount of 0.03% by weight or less; an impurity at a RRT of 0.36 in an amount of 0.1% by weight or less; and one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.04% by weight or less, wherein one or more impurities are present in a total amount of 0.13% by weight or less.

More preferably, the tobramycin formulation prepared by the process of the present invention is characterised by one or more of the following active drug delivery features: output rate for an adult of 0.1-0.2 pg/second and for a child of 0.1-0.15pg/second as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <1604> Products For Nebulization—Characterization Tests; and total drug output for an adult of 30-40 pg and for a child of 22-30 pg as measured by USP 39, National Formulary 34, chapter <1604> Products For Nebulization—Characterization Tests when using a Pari LC Plus connected to the Pari Vios compressor, wherein the tobramycin formulation contains: deoxystreptamine kanosaminide as an impurity in an amount of 0.03% by weight or less; an impurity at a RRT of 0.36 in an amount of 0.1% by weight or less; and one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.04% by weight or less, wherein one or more impurities are present in a total amount of 0.03% by weight or less.

Even more preferably, the tobramycin formulation contains the above limitations relating to the output rate, the total drug output and the amounts of the impurities in combination with tobramycin in an amount of 90-110%, preferably 98-102%, relative to a labelled amount of 75 mg/1 mL tobramycin using the HPLC method disclosed in the USP Monograph for Tobramycin Inhalation Solution, USP 29, National Formulary 24.

The tobramycin formulation prepared by the process of the present invention may have the following droplet size: d(v0.1) of 1-2 microns; d(v0.5) of 4-5.5 microns; and d(v0.9) of 10-12 microns, as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <429> Light Diffraction Measurements Of Particle Size. The tobramycin formulation prepared by the process of the present invention may also have the following droplet size: a span of 1-3 microns, as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <429> Light Diffraction Measurements Of Particle Size.

In one embodiment, the tobramycin formulation prepared by the process of the present invention has the following droplet size: d(v0.1) of 1-2 microns; d(v0.5) of 4-5.5 microns; d(v0.9) of 10-12 microns; and a span of 1-3 microns, as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <429> Light Diffraction Measurements Of Particle Size.

In another embodiment, the tobramycin formulation prepared by the process of the present invention is characterised by the following droplet size features: d(v0.1) of 1-2 microns; d(v0.5) of 4-5.5 microns; d(v0.9) of 10-12 microns; and a span of 1-3 microns, as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <429> Light Diffraction Measurements Of Particle Size, wherein the tobramycin formulation contains: deoxystreptamine kanosaminide as an impurity in an amount of 0.3% by weight or less; an impurity at a RRT of 0.36 in an amount of 0.25% by weight or less; and one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.1% by weight or less, wherein one or more impurities are present in a total amount of 1.0% by weight or less.

Preferably, the tobramycin formulation prepared by the process of the present invention is characterised by the following droplet size features: d(v0.1) of 1-2 microns; d(v0.5) of 4-5.5 microns, d(v0.9) of 10-12 microns; and a span of 1-3 microns, as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <429> Light Diffraction Measurements Of Particle Size, wherein the tobramycin formulation contains: deoxystreptamine kanosaminide as an impurity in an amount of 0.03% by weight or less; an impurity at a RRT of 0.36 in an amount of 0.1% by weight or less; and one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.04% by weight or less, wherein one or more impurities are present in a total amount of 0.13% by weight or less.

More preferably, the tobramycin formulation prepared by the process of the present invention is characterised by the following droplet size features: d(v0.1) of 1-2 microns; d(v0.5) of 4-5.5 microns; d(v0.9) of 10-12 microns; and a span of 1-3 microns, as measured US Pharmacopeia, USP 39, National Formulary 34, chapter <429> Light Diffraction Measurements Of Particle Size, wherein the tobramycin formulation contains: deoxystreptamine kanosaminide as an impurity in an amount of 0.03% by weight or less; an impurity at a RRT of 0.36 in an amount of 0.1% by weight or less; and one or more impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.04% by weight or less, wherein one or more impurities are present in a total amount of 0.03% by weight or less

Even more preferably, the tobramycin formulation contains the above limitations relating to the droplet size features and the amounts of the impurities in combination with tobramycin in an amount of 90110%, preferably 98-102%, relative to a labelled amount of 75 mg/1 mL tobramycin using the HPLC method disclosed in the USP Monograph for Tobramycin Inhalation Solution, USP 29, National Formulary 24.

The present invention also provides a 4 mL formulation for inhalation, the formulation consisting of:

300 mg tobramycin; water; 13.5 mg sodium chloride; 12.0 mg sodium citrate; and an amount of sulfuric acid to achieve a pH of 5.0, wherein the osmolality of the formulation is 248 mOsmol/Kg. The above limitations also apply to this formulation. The present invention further provides this formulation for use in the treatment of a Pseudomonas aeruginosa infection, preferably for use in patients with cystic fibrosis.

The invention will now be described with reference to the following examples, which are not intended to be limiting.

Examples

Example 1

A tobramycin formulation was prepared according to the process of the present invention using bulk solution 1 set out in Table 1.

Water for injection and a nominal amount of concentrated sulfuric acid were added slowly to a stainless steel compounding vessel and mixed to achieve an approximate 3% w/w solution of sulfuric acid. The excipient and tobramycin were then added to the compounding vessel via the charging port and the resultant bulk solution was mixed for a minimum of 30 minutes to allow complete dissolution of tobramycin.

A sample was then taken for pH adjustment. Following pH adjustment, the bulk solution was made up to volume with water for injection and then sparged with sterile nitrogen (99.998%) until the level of dissolved oxygen in the bulk was < 3 ppm. Samples of the bulk solution were taken for pH confirmation and TVAC (Total Viable Aerobic Count) and TYMC (Total Yeast and Mould Count) analysis according to USP <61 > & USP<62>. All compounding activities took place in an ISO Class 7 Area.

The bulk solution was then transferred to a holding tank via a pre-use filter integrity test 0.2 pm sterilising filter. Once in the holding tank, the solution was held under a sterile nitrogen (99.998%) blanket, which was maintained throughout the filling process. During the filling process, the bulk solution was transferred from the holding tank via an in-line pre-use filter integrity test filter (0.1 pm) to the Blow-Fill-Seal (BFS) machine. There was no additional stream of nitrogen that was used in the filling process. This means that the filling was done under sterile air instead of under sterile nitrogen as recommended in the literature.

Low-density polyethylene (LDPE) was extruded by the BFS machine to form open topped units which were presented to the filling head. The bulk solution was then filled into the formed ampoules via a time/ pressure dosing unit which delivered a precise volume, 4 mL, of the bulk solution via the filling needles. When the open ampoules had been filled, the filling needles were withdrawn and the head section of the mould closed to seal the ampoule completely.

Table 1. Bulk solution 1

Ingredients	Amount (mg/mL)	Amount (Kg)
Tobramycin, USP	75.0	20.0
Water for injection, USP	Qs	Qs
Sulfuric acid	Qs to pH 5	Qs to pH 5
Sodium chloride	3.375	0.957
Trisodium citrate dihydrate	3.0	0.851
Diluted sodium hydroxide	Qs to pH 5	Qs to pH 5
Total batch size (kg and units)	N/A	300 (66,666 Units)

Example 2

The chemical stability of the tobramycin formulations prepared using bulk solution 1 was assessed by placing 13 carton boxes containing 14 aluminum foils, each aluminium foil containing 4 LDPE ampoules in an incubator under the conditions specified in Tables 3 and 4. The testing was performed at time 0, 12, 26 and 39 weeks under the conditions specified in Tables 3 and 4. Each time, 1 box was removed from the incubator and 4 ampoules were checked for the parameters listed below. These boxes were then thrown away.

The impurity profile was measured and the tobramycin assay was performed using the HPLC method disclosed in the USP Monograph for Tobramycin Inhalation Solution, USP 29, National Formulary 24. The following amounts of each impurity are presented as a percentage with respect to the amount of tobramycin present in a sample of the formulation and the following amounts of tobramycin in the assay are presented as a percentage relative to a labelled amount of 75 mg/1 mL of tobramycin.

Table 3. Chemical stability data using bulk solution 1, stored at 5°C (± 3°C)

Parameter	Time point (weeks)
0	12	26	39
Deoxystreptamine kanosaminide [NMT⁽¹⁾ 0.3%]	ND	ND	<0.03%	<0.03%
RRT 0.36 [NMT⁽¹⁾ 0.25%]	ND	ND	ND	ND
RRT0.70 [ΝΜΤ^ω0.1%]	ND	ND	0.04%	0.03%
RRT 0.74 [ΝΜΤ^ω0.1%]	ND	ND	0.03%	ND
Total impurities [NMT⁽¹⁾ 1.0%]	0.00%	0.00%	0.07%	0.03%
Tobramycin Content	100.9%	98.6%	99.2%	100.2%
[90.0-110.0% (67.5-82.5 mg/mL)]	(75.7 mg/mL)	(73.9 mg/mL)	(74.4 mg/mL)	(75.2 mg/mL)

^lu Not more than

Table 4. Chemical stability data using bulk solution 1, stored at 25°C (± 3°C)/40% RH (±5% RH)

Parameter	Time point (weeks)
0	12	26
Deoxystreptamine kanosaminide [NMT⁽¹⁾ 0.3%]	ND	ND	<0.03%
RRT 0.36 [NMT⁽¹⁾ 0.25%]	ND	ND	ND
RRT0.70 [ΝΜΤ^ω0.1%]	ND	ND	0.04%
RRT 0.74 [ΝΜΤ^ω0.1%]	ND	ND	0.03%
Total impurities [NMT⁽¹⁾ 1.0%]	0.00%	0.00%	0.10%
Tobramycin Content	100.9%	99.4%	99.5%
[90.0-110.0% (67.5-82.5 mg/mL)]	(75.7 mg/mL)	(74.6 mg/mL)	(74.6 mg/mL)

Not more than

These data therefore demonstrate that the tobramycin formulation prepared by the process of the present invention is chemically stable.

Example 3

A study was conducted to demonstrate that a tobramycin formulation according to the present invention is equivalent to the commercial formulation, Bethkis®, in terms of in-vitro antimicrobial kill rate, droplet size distribution of the nebulised aerosol, active substance delivery rate and total active substance delivered.

Data generated within the study were evaluated statistically based upon the industry guidance. The results demonstrated that for all parameters tested the products were statistically equivalent. The methodology and results are detailed hereinbelow.

(i) In-vitro antimicrobial kill rate

The in-vitro antimicrobial kill rate was determined in accordance with the Wickham Laboratory method “In Vitro Antimicrobial Kill Rate Test by Membrane Filtration” as applied to the 300 mg/4 mL tobramycin formulation of the present invention and to Chiesi's Tobramycin Inhalation Solution 300mg/4mL reference product, Bethkis®. The results are shown in Table 5.

This test involved inoculating samples at 0 hours and enumerating the number of surviving microorganisms at various time points throughout the test period (0, 7.5, 15, 30 and 60 minutes). Twelve replicates of both the test and reference samples were taken at each time point. The organisms selected for this test are those which are stated in the United States Pharmacopoeia 40 <51 > and those which are listed in the reference product labelling.

The test was performed against five organisms; Aspergillus brasiliensis, Candida albicans, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The media selected for this test are those which are stated in the United States Pharmacopoeia 40 <51 >.

The population for each test organism/product, for designated time intervals, was determined by counting surviving colonies after incubation. Kill rates were determined for each product and equivalence declared if both the test and reference products produced the same in vitro kill rates on all organisms tested.

Table 5

Bacterium	Measure	Ratio' ’’	Ratio 90% Cl’
		(T/R)%	Lower	Upper
Aspergillus brasiliensis	60 mins	96.4	89.7	103.6
Candida albicans	60 mins	96.6	82.4	113.2
Escherichia coli	60 mins	100.5	99.6	101.4
Staphylococcus aureus	60 mins	100.3	99.5	101.1
Pseudomonas aeruginosa	60 mins	N/A^w	n/a'²⁾	n/a'²⁾

^ιυ Ratio and Ratio 90% Cl calculated using Fieller's method (8.3).

⁽²⁾ The total kill of Pseudomonas aeruginosa within the first test period.

(ii) Droplet size distribution ofthe nebulised aerosol

The droplet size distribution ofthe nebulised aerosol was assessed using the US Pharmacopeia, USP 39, National Formulary 34, chapter <429> Light Diffraction Measurements Of Particle Size. The apparatus used was the Malvern Spraytec, with an inhalation cell and lens type of 300 mm. The refractive index was 1.00 and the refractive index (real) was 1.33. Ten measurements were made for each batch of the reference product, Bethkis®, and the tobramycin formulation of the present invention (n=10). The results for the Dv[10], Dv[50], Dv[90] and Span are summarized in Table 6.

Table 6

	Geometric Mean Ratio	95% Upper confidence bound for linearised criteria
Parameter	(T/R)	Upper 95% CL
Dv[10] (pm)	1.01	-0.0223
Dv[50] (pm)	1.00	-0.0185
Dv[90] (pm)	1.00	-0.0191
Span	1.00	-0.0208

(iii) Active substance delivery rate and total active substance delivered

The drug delivery rate and the total drug delivered was assessed. A breathing simulator was used to determine active substance delivery rate and total active substance delivered using the methodology set out in USP <1601 > Products for nebulization: Characterization or Ph. Eur 2.9.44 Preparations for nebulisation: Characterisation. Ten measurements were made for each batch of the reference product, Bethkis®, and the tobramycin formulation of the present invention (n=10). The results are 10 shown in Table 7.

Table 7

		Geometric Mean Ratio	95% Upper confidence bound for linearised criteria
Breathing pattern	Parameter/Measure	(T/R)	Upper 95% CL
Child	Drug delivery rate (pg/second)	1.02	-0.0181
Total active delivered (%LC)	1.08	-0.0126
Adult	Drug delivery rate (pg/second)	1.00	-0.0151
Total active delivered (%LC)	1.04	-0.0167

These data show that the 300 mg/4 ml. tobramycin formulation of the present invention is comparable to Chiesi's Bethkis® reference product in terms in-vitro antimicrobial kill rate, droplet size distribution of the nebulised aerosol, active substance delivery rate and total active substance delivered

Claims

1. A process for preparing a tobramycin formulation, the process comprising the following steps, in order:

(i) providing a bulk solution consisting of tobramycin, water, sodium chloride, sodium citrate and a pH adjusting agent, wherein the bulk solution has a pH of 5.0 and an osmolality of 248 mOsmol/Kg;

(ii) sparging the bulk solution with nitrogen;

(iii) filling, under sterile air, the bulk solution into ampoules, wherein each ampoule contains 4 ml. of the bulk solution and 300 mg tobramycin.

2. A process as claimed in claim 1, wherein the pH adjusting agent is sulfuric acid or a combination of sulfuric acid and sodium hydroxide.

3. A process as claimed in claim 2, wherein the pH adjusting agent is sulfuric acid.

4. A process as claimed in any preceding claim, wherein sodium citrate is introduced to the bulk solution as trisodium citrate dihydrate.

5. A process as claimed in any preceding claim, wherein the bulk solution is sparged until the bulk solution contains < 3 ppm dissolved oxygen.

6. A process as claimed in any preceding claim, wherein each ampoule contains 13.5 mg sodium chloride.

7. A process as claimed in claim any preceding claim, wherein each ampoule contains 12.0 mg sodium citrate.

8. A 4 mL formulation for inhalation, the formulation consisting of: 300 mg tobramycin; water; 13.5 mg sodium chloride; 12.0 mg sodium citrate; and an amount of sulfuric acid to achieve a pH of 5.0, wherein the osmolality of the formulation is 248 mOsmol/Kg.

9. A formulation as claimed in claim 8, wherein the formulation has the following droplet size: d(v0.1) of 1-2 microns; d(v0.5) of 4-5.5 microns; and d(v0.9) of 10-12 microns, as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <429> Light Diffraction Measurements Of Particle Size.

10. A formulation as claimed in claim 8 or 9, wherein the formulation has the following droplet size: a span of 1-3 microns, as measured by US Pharmacopeia, USP 39, National Formulary 34, chapter <429> Light Diffraction Measurements Of Particle Size.

11. A formulation as claimed in any of claims 8-10, wherein the formulation contains deoxystreptamine kanosaminide as an impurity in an amount of 0.3% by weight or less, as measured by HPLC according to the US Pharmacopeia Monograph for Tobramycin Inhalation Solution, USP 29, National Formulary 24.

5

12. A formulation as claimed in any of claims 8-11, wherein the formulation contains an impurity at a relative retention time (RRT) of 0.36 in an amount of 0.25% by weight or less, as measured by HPLC according to the US Pharmacopeia Monograph for Tobramycin Inhalation Solution, USP 29, National Formulary 24.

13. A formulation as claimed in any of claims 8-12, wherein the formulation contains one or more 10 impurities at a RRT of 0.70 and/or 0.74, each in an amount of 0.1% by weight or less, as measured by

HPLC according to the US Pharmacopeia Monograph for Tobramycin Inhalation Solution, USP 29, National Formulary 24.

14. A formulation as claimed in any of claims 8-13 for use in the treatment of a Pseudomonas aeruginosa infection.

15

15. A formulation as claimed in claim 14 for use in patients with cystic fibrosis.