WO2023006565A1

WO2023006565A1 - Process for preparing formulations of edaravone

Info

Publication number: WO2023006565A1
Application number: PCT/EP2022/070466
Authority: WO
Inventors: José Andrés LOZANO VELILLA; Montserrat GARCÍA ROVIRA; Ernesto DURAN LÓPEZ
Original assignee: Medichem SA
Current assignee: Medichem SA
Priority date: 2021-07-26
Filing date: 2022-07-21
Publication date: 2023-02-02
Anticipated expiration: 2024-01-26

Abstract

This invention relates to a process for preparing a formulation of edaravone or a pharmaceutically acceptable salt thereof, concretely an aqueous injectable formulation, which contains low amounts of a certain impurity. This process comprises heat-sterilization of an aqueous formulation of edaravone or pharmaceutically acceptable salt thereof, which comprises bisulfite anions (HSO3-) and contains less than 1 ppm of oxygen.

Description

PROCESS FOR PREPARING FORMULATIONS OF EDARAVONE

This invention relates to a process for preparing an aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof with low contents of impurities that is stable during storage, transportation, and use.

BACKGROUND OF THE INVENTION

Edaravone (Compound I) is the international commonly accepted non-propietary name (INN) for 3-methyl-1-phenyl-2-pyrazolin-5-one, and has an empirical formula of C10H10N2O and a molecular weight of 174.20 g/mol.

Compound 1

Edaravone, sold as under the brand names Radicava or Radicut, is a medication used to help with recovery following a stroke and to treat amyotrophic lateral sclerosis (ALS).

Edaravone when given to patients intravenously it is supplied in the form of ampoules or bags containing the injectable aqueous solution of edaravone. For example, the US commercial injectable formulation Radicava is an aqueous solution of edaravone, which is administered intravenously by infusion or drip. It is supplied in a polypropylene bag, which is further overwrapped with polyvinyl alcohol overpouch. The US commercial injectable formulation Radicava contains the following excipients: L-cysteine hydrochloride hydrate, sodium bisulfite, sodium chloride and phosphoric acid and sodium hydroxide to adjust the pH to about 4.

Edaravone is extremely stable in a solid state, but aqueous solutions of edaravone have been found to be unstable. Several impurities have been disclosed to be present in aqueous solutions of edaravone. China Licensed Pharmacist Aug 2014, Vol. 11 No.8, discloses several impurities present in Radicut, aqueous solution of edaravone, being Impurity I, 2-(3- methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)-2-sulfopropanoic acid, one of the disclosed impurities:

Impurity I CN106316957A and CN110090225A disclose the Impurity I as an impurity present in aqueous solutions of edaravone which contain sodium bisulfite as excipient. CN106316957A describes a preparation method for the Impurity I. CN102336710A also discloses the preparation of the Impurity I.

This impurity is supposed to be formed due to the presence of sodium bisulfite, which is used as stabilizer in the aqueous pharmaceutical formulations of edaravone.

There is the need of providing processes for preparing aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof, comprising bisulfite anions (HSOT), while reducing the formation of impurities such as Impurity I.

BRIEF SUMMARY OF THE INVENTION It is an object of the present invention to provide a simple and industrially scalable process for preparing aqueous formulations, preferably aqueous injectable formulations, of edaravone or a pharmaceutically acceptable salt thereof, comprising bisulfite anions (HSOT), which contain low amounts of impurities, concretely low amounts of the Impurity I. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process for preparing an aqueous formulation comprising edaravone or a pharmaceutically acceptable salt thereof and bisulfite anions (HSOT), wherein the process comprises: a) providing an aqueous formulation comprising i) edaravone or a pharmaceutically acceptable salt thereof and ii) bisulfite anions, and controlling the oxygen content of said formulation to be less than 1 ppm of oxygen, and b) heat sterilizing the formulation resulting from step a).

The aqueous formulations prepared according to the process of the present invention are preferably pharmaceutical formulations, namely the aqueous formulations prepared according to the process of the present invention are safe, non-toxic and acceptable for human therapeutic use. The aqueous formulations prepared according to the process of the present invention are preferably injectable pharmaceutical formulations.

In a preferred embodiment of the present invention, the oxygen content of the formulation is controlled to be less than 0.5 ppm of oxygen.

The objective of the above-described process is to have an aqueous formulation comprising edaravone or a pharmaceutically acceptable salt thereof, bisulfite anions and less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen, (step a), which is then subjected to heat sterilization (step b).

Thus, the invention involves ensuring that the oxygen content of the aqueous formulation just before the sterilization step b) is less than 1 ppm, preferably less than 0.5 ppm of oxygen. This may be achieved through the following steps:

1) providing an aqueous formulation comprising i) edaravone or a pharmaceutically acceptable salt thereof and ii) bisulfite anions having an oxygen content of less than 1 ppm, preferably less than 0.5 ppm of oxygen.

2) optionally further controlling the oxygen content of the environment during the handling of the aqueous formulation of step 1). The provision of the aqueous formulation of step 1) may be achieved by one or more of the following steps: i) controlling the oxygen content of the water used to prepare the aqueous formulation, ii) controlling the oxygen content of the environment in which the aqueous formulation is prepared, iii) taking active measures to reduce the oxygen content of the aqueous formulation if said formulation has an oxygen content of 1 ppm or more.

Optionally, in an embodiment of the present invention, the oxygen content is also controlled during the heat-sterilization.

In a preferred embodiment of the present invention, the aqueous formulation comprising edaravone or a pharmaceutically acceptable salt thereof, bisulfite anions and less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen, (step a)), which is then subjected to heat sterilization (step b) is an aqueous solution.

In an embodiment of the invention the above-mentioned formulation (resulting from step a)) is prepared using water which has a content of less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen, (i.e. deoxygenated water) while controlling the oxygen of the environment to prevent that oxygen is absorbed by the aqueous formulation (which could yield to the aqueous formulation having 1 or more ppm of oxygen, for example from 1 to 9 ppm of oxygen). In this embodiment, the control of oxygen content takes place before the actual preparation of the aqueous formulation (by using deoxygenated water), during the preparation of the aqueous formulation, during the handling of the aqueous formulation and during sterilization (by using an environment poor in, or free of oxygen). In another embodiment, water having a content of 1 or more ppm of oxygen is used to prepare the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof and bisulfite anions. Then, after obtaining said aqueous formulation, the content of oxygen is reduced to obtain an aqueous formulation having less than 1 ppm of oxygen, more preferably less than 0.5 ppm of oxygen, before heat-sterilization. In yet another embodiment a combination of the embodiments described in the two former paragraphs is used. In this embodiment, deoxygenated water (having a content of less than 1 ppm of oxygen, preferably of less than 0.5 ppm of oxygen) is used but no measure or not all the measures to control the oxygen content of the environment are taken. Consequently, the resulting aqueous formulation will very likely have an oxygen content of 1 ppm or more, for example from 1 to 9 ppm of oxygen, due to the absortion of oxygen from the environment. For this reason, after obtaining the aqueous formulation, the content of oxygen is reduced to obtain an aqueous formulation having less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen, before heat- sterilization.

Any method of reducing oxygen of an aqueous formulation can be used to control the oxygen content of the aqueous formulation to be less than 1 ppm of oxygen, preferably to be less than 0.5 ppm of oxygen.

In one embodiment, the method of reducing oxygen comprises placing the aqueous formulation together with an oxygen scavenger in a closed environment for a duration of time sufficient to reduce the oxygen content of the aqueous formulation to the desired levels. The aqueous formulation may be in direct contact with the oxygen scavenger or, even preferably, separated from the scavenger by an oxygen-permeable material.

An alternative embodiment of reducing oxygen is placing the aqueous formulation in an oxygen-free (or oxygen-poor) environment and bubbling an inert gas (such as nitrogen or argon) through the aqueous formulation.

Another alternative embodiment of reducing oxygen is applying vacuum to the aqueous formulation, preferably in an oxygen-free (or oxygen-poor) environment. In another embodiment of the present invention, the container, preferably a plastic bag, more preferably a multi-layered plastic bag, which contains the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof having more than 1 ppm of oxygen, for example from 1 to 9 ppm of oxygen, is placed, preferably under an inert atmosphere of argon or nitrogen, preferably of nitrogen, in an overpouch, preferably an aluminum overpouch, provided with an oxygen scavenger (e.g. Mitsubishi Gas Chemical Company, Inc, “Ageless” or Dry Pak oxygen scavangers) and this packaging is kept until the oxygen content in the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof is less than 1 ppm before heat-sterilization, preferably less than 0.5 ppm before heat-sterilization. The handling of the aqueous formulation while controlling its oxygen content may include, for example, transporting the aqueous formulation and/or filling the aqueous formulation in a container, for example a plastic bag. It is possible to control the oxygen content of the formulation during the handling steps by working on an environment poor in, or free of oxygen and/or by flushing with argon or nitrogen the headspace of the container filled with the aqueous formulation.

The authors of the present invention have found that the formation of impurities, concretely of the Impurity I, is surprisingly and drastically reduced when the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof comprising bisulfite anions, preferably a bisulfite salt, which is subjected to the heat-sterilization, contains less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen,

The authors of the present invention have also found that the formation of phenylhydrazine, which is a genotoxic impurity, is also reduced when the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof comprising bisulfite anions which is subjected to the heat-sterilization, contains less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen. The amount of oxygen in the aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof as defined in the present invention refers to the amount of dissolved oxygen in said formulations.

The bisulfite anions (HSOT) present in the formulations obtainable by the process of the present invention may be added in the form of any compound or mixture of compounds yielding in water solution said bisulfite anions (HSOT)· Non-limiting examples of compounds which yield bisulfite anions (HSO₃) in aqueous solutions are a salt of bisulfite and a cation, preferably a salt selected from the group consisting of sodium bisulfite, potassium bisulfite and mixtures thereof; a salt of metabisulfite and a cation, preferably a salt selected from sodium metabisulfite, potassium metabisulfite and mixtures thereof; a salt of sulfite and a cation, preferably a salt selected from sodium sulfite, potassium sulfite and mixtures thereof; and mixtures of any one of these salts. In a preferred embodiment of the present invention, the source of bisulfite ion is sodium bisulfite, sodium metabisulfite or mixtures thereof. In a preferred embodiment of the present invention, the bisulfite anions (HSO3) are present in the form of sodium bisulfite.

Bisulfite anions (HSO₃) have been used as excipient, concretely as stabilizer in aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof. However, its presence entails the formation of Impurity I, mainly during the heat- sterilization step of the aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof.

The process of the present invention allows to use bisulfite anions (HSOT) in the aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof, thus protecting edaravone from degradation, while reducing the formation of Impurity I whose formation is directly linked to the presence of bisulfite anions (HSOT) when the aqueous formulation is subjected to heat-sterilization.

The aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof obtained by the process of the present invention after step b) is a sterilized aqueous formulation, preferably an injectable formulation, namely a pharmaceutical injectable formulation.

The excipients used in the present invention must be suitable for use in contact with tissues or organs of humans and/or animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications consistent with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” as used herein refers to any salt that is useful for preparing a pharmaceutical formulation and it is tolerated physiologically, i.e. , it is non- toxic, when used for therapeutic purposes in humans and/or animals. As examples of pharmaceutically acceptable salts, mention may be made of salts with mineral acids such as hydrochloric acid, sulfuric acid, hydrobromide, phosphoric acid, and the like; salts with organic acids such as methanesulfonic acid, p-toluenesulfonic acid, acetic acid, oxalic acid, citric acid, malic acid, fumaric acid, and the like; salts with alkali metals such as sodium, potassium, and the like; salts with alkaline earth metals such as magnesium, and the like; and salts with amines such as ammonia, ethanolamine, 2- amino-2-methyl-1 -propanol, and the like. The aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, comprising bisulfite anions (HSOT) obtained according to the process of the present invention after step b) contains less than 0.10 % (area) of the Impurity I, preferably less than 0.05 % (area) of the Impurity I, more preferably less than 0.03 % (area) of the Impurity I, when analyzed by a column chromatography method for purity, for example a HPLC or UHPLC method.

The aqueous formulation of edaravone or a pharmaceutically salt thereof, comprising bisulfite anions (HSOT) obtained according to the process of the present invention after step b) is stable for at least six months at 40° C ± 2° C and a relative humidity of 75% and for at least six months, preferably for at least nine months, more preferably for at least twelve months, more preferably for at least twenty-four months, even more preferably for at least thirty-six months, at 25° C ± 2° C and a relative humidity of 60%.

The term “stable” as used herein means that the amount of each impurity, concretely of the Impurity I, is maintained in amounts less than 0.25% (area), preferably less than 0.20% (area), more preferably less than 0.15% (area), even more preferably less than 0.10 (area), when analyzed by a column chromatography method for purity, for example a HPLC or UHPLC method. In one embodiment, the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, comprising bisulfite anions (HSOT) obtained according to the process of the present invention after step b) contains less than 300 ppm, preferably less than 200 ppm, more preferably less than 150 ppm, even more preferably less than 100 ppm of phenylhydrazine, when analyzed by a column chromatography method, for example a HPLC or UHPLC method. In an embodiment, the aqueous formulation of edaravone or a pharmaceutically salt thereof, comprising bisulfite anions (HSO₃) obtained according to the process of the present invention after step b) and subsequently maintained for at least six months at 40° C ± 2° C and a relative humidity of 75% or for at least six months, preferably for at least nine months, more preferably for at least twelve months, more preferably for at least twenty-four months, even more preferably for at least thirty-six months, at 25° C ± 2° C and a relative humidity of 60%, contains less than 600 ppm, preferably less than 500 ppm, more preferably less than 400 ppm of phenylhydrazine, when analyzed by a column chromatography method, for example a HPLC or UHPLC method.

The aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, which comprises bisulfite anions (HSOT), preferably in the form of sodium bisulfite, which is subjected to the heat-sterilization is preferably contained in a container which can be a glass or a plastic ampoule or a plastic bag. In a preferred embodiment, the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, which comprises bisulfite anions (HSOT), preferably in the form of sodium bisulfite, and which is subjected to the heat-sterilization step is contained in a plastic bag, preferably in a multi-layered plastic bag, more preferably in a three, four, five or six-layer plastic bag. Any multi-layered plastic bag known in the art to be suitable for storing aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof can be used in the process for preparing aqueous formulations of edaravone or a pharmaceutically salt thereof of the present invention. Non-limiting examples of the plastic bags used in the present invention are bags equivalent or equal to those disclosed in JP6863845B2, W02009066752A1 , W02010016404A1, JP4596124B2, JP2018192643A1,

W02008001496A 1 , WO201079693A1, WO2019151532A1 or WO2019159967A1.

In a preferred embodiment of the present invention, the multi-layered plastic bag comprises at least one layer, which comprises a cycloolefin polymer. Cycloolefin polymers are materials that contain, or are made from, at least one cyclic monomer. Cycloolefin polymers include cyclic olefin polymers (COP) and cyclic olefin copolymers (COC). In a preferred embodiment of the present invention, the multi-layered plastic bag comprises at least one layer, which comprises at least one cyclic olefin polymer (COP). In an embodiment of the present invention the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof which is subjected to the heat-sterilization is contained in a multi-layered plastic bag which is a three-layer plastic bag which comprises: an innermost layer which comprises at least one cyclic olefin polymer (COP); a resin layer disposed on the innermost layer and an outer layer disposed on the resin layer which might be made of polypropylene and/or polyethylene. The adhesive resin layer may be composed of linear low-density polyethylene, styrene- based elastomer and polypropylene-based resin. For example, in an embodiment of the present invention the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof which is subjected to the heat-sterilization is contained in Fujimori’s/Zacros Nip^® - å Bag or Fujimori’s/Zacros MediTect^® IV Bag.

In another embodiment of the present invention the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof which is subjected to the heat-sterilization is contained in a multi-layered plastic which is a five-layer plastic bag which comprises: an innermost layer made of polypropylene and/or polyethylene; an adhesive layer disposed on the innermost layer; a layer made of at least one cyclic olefin polymer (COP) disposed on the adhesive layer; an adhesive layer disposed on the cyclic olefin polymer (COP) layer and an outermost layer disposed on the adhesive layer made of polypropylene and/or polyethylene. The adhesive resin layer may be composed of linear low-density polyethylene and polypropylene-based resin.

Any means to control the oxygen content of the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof comprising bisulfite anions, preferably in the form of sodium bisulfite, which is subjected to the heat-sterilization contains less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen, are encompassed in the process of the present invention.

The term “deoxygenated water”, for example “deoxygenated water for injection” as used herein means that the water, for example the water for injection, contains less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen.

Deoxygenated water, preferably deoxygenated water for injection, can be obtained by bubbling an inert gas such as argon or nitrogen, preferably nitrogen, at room temperarute, i.e. , at 25 ± 5°C, into water, preferably water for injection, until obtaining the desired levels of oxygen, i.e., less than 1 ppm, preferably less than 0.5 ppm of oxygen.

In a preferred embodiment of the present invention, after providing the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, the aqueous formulation is filled in a container, preferably a plastic bag, more preferably a multi layered plastic bag, under an inert atmosphere of argon or nitrogen, preferably of nitrogen and the headspace is flushed with an inert gas such as argon or nitrogen, preferably nitrogen. In a preferred embodiment of the present invention, the oxygen content in the headspace atmosphere after flushing with an inert gas is less than 10% (w/w), preferably less than 5% (w/w), for example 2%-3% (w/w).

In an embodiment of the present invention, the control of oxygen is carried out by placing the container, preferably a plastic bag, more preferably a multi-layered plastic bag, which contains the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof having more than 1 ppm of oxygen, for example from 1 to 9 ppm of oxygen, preferably under an inert atmosphere of argon or nitrogen, preferably of nitrogen, in an overpouch, preferably an aluminum overpouch, provided with an oxygen scavenger (e.g. Mitsubishi Gas Chemical Company, Inc, “Ageless” or Dry Pak oxygen scavangers) and keeping this packaging until the oxygen content in the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof is less than 1 ppm, preferably less than 0.5 ppm, before heat-sterilization.

In a preferred embodiment of the present invention, the packaging formed by the container, preferably the plastic bag, more preferably the multi-layered plastic bag, which contains the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, having more than 1 ppm of oxygen, for example from 1 to 9 ppm of oxygen, which is placed in the overpouch, preferably an aluminum overpouch, is kept, preferably at 25 ± 5 °C, for a time from 5 to 20 days, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 days, more preferably fora time from 7 to

15 days, in order to reduce the amount of oxygen in the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof to less than 1 ppm of oxygen, preferably to less than 0.5 ppm of oxygen, before heat-sterilization. Any overpouch, i.e., any bag, which is non-permeable to oxygen and suitable for protecting formulations, preferably aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof, can be used in the process of the present invention. Non-limiting examples of materials which may form part of the overpouch, include silica- or alumina-vapor-deposited polyethylene terephthalate film, silica- or alumina-vapor-deposited nylon film, aluminum-vapor-deposited polyethylene terephthalate, aluminum-vapor-deposited nylon, aluminum foil, ethylene-vinyl-alcohol, poly(vinyl alcohol), polyvinylidene chloride and mixtures thereof.

In an embodiment of the present invention, once the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof contained in a container, preferably a plastic bag, more preferably a multi-layered plastic bag, which is placed in an overpouch, preferably an aluminum overpouch, provided with an oxygen scavenger (e.g. Mitsubishi Gas Chemical Company, Inc, “Ageless” or Dry Pak oxygen scavangers), has a content of oxygen of less than 1 ppm, preferably less than 0.5 ppm, the overpouch, preferably the aluminum overpouch, and the oxygen scavenger (e.g. Mitsubishi Gas Chemical Company, Inc, “Ageless” or Dry Pak oxygen scavangers) are removed, and the container, preferably the plastic bag, more preferably the multi layered plastic bag, which contains the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, having a content of oxygen of less than 1 ppm, preferably less than 0.5 ppm, is placed in a second overpouch, preferably a second aluminum overpouch, and the headspace between the container and the second overpouch is totally or partially removed under vacuum before heat- sterilization.

In an embodiment of the present invention, the heat-sterilization of the aqueous formulation resulting from step a) having less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen, which is contained in a container which is a plastic bag, preferably a multi-layered plastic bag, can be performed under vacuum or under an inert atmosphere, for example an argon or nitrogen atmopshere, preferably under a nitrogen atmosphere.

The term vacuum as used herein means under reduced pressure, i.e., a pressure lower than 760 mmHg or 101.3 kPa.

In an embodiment of the present invention, the heat-sterilization of the aqueous formulation resulting from step a) having less than 1 ppm of oxygen, preferably less than 0.5 ppm of oxygen, which is contained in a container which is a plastic bag, preferably a multi-layered plastic bag, being this container placed in an overpouch, preferably an aluminum overpouch, is performed under vacuum or under an inert atmosphere, for example an argon or nitrogen atmosphere, preferably under a nitrogen atmosphere, or under atmospheric air.

Any heat-sterilization cycle known in the art can be performed in the process of the present invention.

In a preferred embodiment, the heat-sterilization is for 5 minutes to 30 minutes at 115°C to 125°C. For example, for 5 minutes at 123°C, for 10 minutes at 123°C or for 15 minutes at 121°C.

In an embodiment of the present invention, the overpouch, preferably an aluminum overpouch, is removed after the heat-sterilization step and the container, preferably a plastic bag, more preferably a multi-layered plastic bag, is placed in an overpouch, preferably a transparent plastic overpouch, preferably a transparent multi-layered overpouch or bag, provided with an oxygen scavenger, for example Dry Pak^® oxygen absorber, and an oxygen indicator, for example, Dry Pak^® oxygen indicator. Any overpouch which is non-permeable to oxygen and suitable for protecting formulations, preferably aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof, can be used after heat-sterilization in the process of the present invention. For example, the transparent multi-layered plastic ovepouch might be a three-layer, four-layer, five-layer or six-layer overpouch. Non-limiting examples of materials which may form part of this transparent overpouch, include polypropylene, polyethylene, poly(vinyl alcohol), nylon and silicified vaporized polyethylene terephthalate.

The aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof prepared according to the process of the present invention may contain other excipients apart from the source of bisulfite, preferably in the form of sodium bisulfite. Non-limiting examples of these excipients are stabilizers, surfactants, buffers, solubilizers, antioxidants, antifoamers, tonicity agents, emulsifiers, suspending agents, preservatives, soothing agents, solubilizers, solubilizing adjuvants and mixtures thereof. In a preferred embodiment of the present invention, the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof which is obtained according to the process of the present invention is an aqueous injectable formulation of edaravone or a pharmaceutically salt thereof comprising as excipients sodium bisulfite, sodium chloride, L-cysteine hydrochloride hydrate and sodium hydroxide and/or phosphoric acid to adjust the pH to about 4.

In a preferred embodiment, the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, preferably an aqueous injectable formulation of edaravone or a pharmaceutically acceptable salt thereof, which is obtained according to the process of the present invention has an edaravone concentration preferably between 0.06 mg/ml_ to 1.0 mg/ml_, for example 0.3 mg/ml_ and 0.6 mg/ml_, and the liquid volume of the aqueous pharmaceutical formulations is preferably 50 ml_ to 500 ml_, for example 100 mL.

In a preferred embodiment, the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, preferably an aqueous injectable formulation of edaravone or a pharmaceutically acceptable salt thereof, which is obtained according to the process of the present invention contains bisulfite anions, preferably in the form of sodium bisulfite, in an amount from 0.1 mmol/L to 10.0 mmol/L of bisulfite anions, preferably from 0.5 mmol/L to 5 mmol/L, more preferably between about 1.9 mmol/L and about 2 mmol/L.

In a preferred embodiment, the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, preferably an aqueous injectable formulation of edaravone or a pharmaceutically acceptable salt thereof, which is obtained according to the process of the present invention contains L-cysteine hydrochloride hydrate in an amount from 0.01 mg/mL to 1.0 mg/mL, preferably from 0.05 mg/mL to 0.5 mg/mL, more preferably about 0.1 mg/mL.

In a preferred embodiment of the present invention, the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, preferably an aqueous injectable formulation of edaravone or a pharmaceutically acceptable salt thereof, which is obtained according to the process of the present invention contains sodium chloride in an amount from 1.0 mg/mL to 20.0 mg/mL, preferably from 5.0 mg/mL to 10.0 mg/mL, more preferably about 8.7 mg/mL. In a preferred embodiment of the present invention, the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, preferably an aqueous injectable formulation of edaravone or a pharmaceutically acceptable salt thereof, which is obtained according to the process of the present invention contains: i) sodium bisulfite, in an amount from 0.1 mmol/L to 10.0 mmol/L of bisulfite anions, preferably from 0.5 mmol/L to 5 mmol/L, more preferably about 1.9 mmol/L and about 2 mmol/L; ii) L-cysteine hydrochloride hydrate in an amount from 0.01 mg/mL to 1.0 mg/mL, preferably from 0.05 mg/mL to 0.5 mg/mL, more preferably about 0.1 mg/mL; iii) sodium chloride in an amount from 1.0 mg/mL to 20.0 mg/mL, preferably from 5.0 mg/mL to 10.0 mg/mL, more preferably about 8.7 mg/mL and iv) sodium hydroxide and/or phosphoric acid to adjust the pH to about 4.

All the oxygen contents, for example in the deoxygenated water, preferably deoxygenated water for injection, in the aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof or in the the headspace of the container, preferably a plastic bag, more preferably a multi-layered plastic bag, containing the aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof, can be measured with an oximeter, for example the Mettler Toledo oximeter SevenGo pro.

Another aspect of the present invention provides an aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, more preferably an aqueous injectable formulation of edaravone or a pharmaceutically acceptable salt thereof, obtainable according to the process of the present invention.

The term "obtainable” is used herein for defining aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, more preferably an aqueous injectable formulation of edaravone or a pharmaceutically acceptable salt thereof, by its preparation process. For the purposes of the invention, the expressions “obtainable”, “obtained” and similar equivalent expressions are used interchangeably and, in any case, the expression “obtainable” encompasses the expression “obtained”.

The aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, more preferably an aqueous injectable formulation of edaravone or a pharmaceutically acceptable salt thereof, which is obtained according to the process of the present invention can be used for the treatment of stroke and motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), or arthrogryposis multiplex congenita (AMC).

The term “about” when used in the present invention preceding a number and referring to it, is meant to designate any value which lies within the range defined by the number ±10% of its value, preferably a range defined by the number ±5%, more preferably range defined by the number ±2%, still more preferably a range defined by the number ±1%. For example, “about 10” should be construed as meaning within the range of 9 to 11 , preferably within the range of 9.5 to 10.5, more preferably within the range of 9.8 to 10.2, and still more preferably within the range of 9.9 to 10.1.

The method for determining the purity (i.e. assay) and the impurities (for exemple Impurity I) comprises any column chromatography method, for example an HPLC or UHPLC method, used to determine the purity and the impurities (for exemple Impurity I) of aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof. Preferably, the chromatographic method for determining the purity and the impurities (for examples Impurity I) comprises the UHPLC method used in the present invention.

The % area of a specific compound, for exemple of Impurity I, as used in the present invention is calculated as follows: the area of the specific compound, for exemple of Impurity I, of the chromatograpm of the column chromatography method for purity, preferably the UHPLC chromatogram obtained as defined in the UHPLC method for purity of the present invention, is divided by the total area of the peaks in the chromatogram of the column chromatography method for purity, preferably of the UHPLC chromatogram obtained as defined in the UHPLC method for purity of the present invention. The value obtained is then multiplied by 100.

The method for determining the phenylhydrazine comprises any column chromatography method, for example an HPLC or UHPLC method, used to determine the phenylhydrazine of aqueous formulations of edaravone or a pharmaceutically acceptable salt thereof. Preferably, the chromatographic method for determining the phenylhydrazine comprises the HPLC method used in the present invention.

In another aspect the present invention relates to the use of a formulation of edaravone or a pharmaceutically salt thereof obtainable or obtained by the process described above for the treatment of stroke and motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), or arthrogryposis multiplex congenita (AMC).

In another aspect the present invention relates to a formulation of edaravone or a pharmaceutically salt thereof obtainable or obtained by the process described above for use in the treatment of stroke and motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), or arthrogryposis multiplex congenita (AMC). In another aspect the present invention relates to the use of a formulation of edaravone or a pharmaceutically salt thereof obtainable or obtained by the process described above for the manufacture of a medicament for the treatment of stroke and motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), or arthrogryposis multiplex congenita (AMC).

In another aspect the present invention relates to the a method for the treatment of stroke and motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), or arthrogryposis multiplex congenita (AMC) by administration to a subject in need thereof of a formulation of edaravone or a pharmaceutically salt thereof obtainable or obtained by the process described above.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES

General experimental conditions:

UHPLC method used for analysing the injectable aqueous pharmaceutical formulations of edaravone or a pharmaceutically acceptable salt thereof

The chromatographic separation was carried out in UHPLC apparatus with an UV- Visible detector (244 nm).

Column: YMC Triart C18150 x 2.0 mm, 1.9 pm column or equivalent Column temperature: 45°C The mobile phase A was:

- 90% Buffer Di-ammonium hydrogen phosphate salt, 10 mM, pH 6.2 - 10% HPLC-grade Methanol.

The mobile phase B was: - 90% HPLC-grade Methanol

- 10% HPLC-grade Acetonitrile

The chromatograph was programmed as follows: initial 5.0 min isocratic 100% mobile phase A; 5.0-9.4 min linear gradient to 95% mobile phase A; 9.4-37.5 min linear gradient to 80% mobile phase A; 37.5-50.0 min linear gradient to 50% mobile phase A; 50.0-62.5 min isocratic 50% mobile phase A; 62.5-64.0 min linear gradient to 100% mobile phase A; 64.0 to 75.0 min isocratic 100% mobile phase A.

The flow rate was 0.2 mL/min. Injection volume: 2.0 pL. Solutions

- Diluent A: HPLC-grade acetonitrile: HPLC-grade water (50:50 v/v)

- Diluent B: HPLC-grade water

Standard Solution 100 % (about 300 mg/mL Edaravone): 50.0 mg of edaravone working standard were weighted in a 50.0 mL volumetric flask, diluted with diluent A and sonicated for 5 minutes. 6.0 mL of this solution was diluted to 20.0 mL with diluent B. This solution was filtered through 0.22 pm PVDF filter.

Test solution: the sample from the plastic bag was withdrawn and directly injected without the need of filtration.

Calculations for the assay 100

ru: peak response of edaravone from test solution rs: peak response of edaravone from the standard solution 100%

Cu: concentration of edaravone in the test solution (30 mg/100mL dose: 300 pg/mL) Cs: concentration of edaravone in the standard solution 100%

Limit of detection for Impurity I: 0.03% (area) Limit of quantification for Impurity I: 0.05% (area)

HPLC method used for analysing the amount of phenylhydrazine in the injectable aqueous pharmaceutical formulations of edaravone or a pharmaceutically acceptable salt thereof

The chromatographic separation was carried out in HPLC apparatus with PDA detector (Wavelength: 232 nm).

Column: Symmetry C18 (250 x 4.6 mm) 5.0 pm or equivalent Column temperature: 25°C The mobile phase A: Ammonium acetate buffer, 10 mM, pH 6.6 The mobile phase B: HPLC-grade Acetonitrile

The chromatograph was programmed as follows: initial 18.5 min isocratic 90% mobile phase A; 18.5-21.0 min linear gradient to 60% mobile phase A; 21.0-26.0 min isocratic 60% mobile phase A; 26.0-28.0 min linear gradient to 90% mobile phase A; 28.0-35.0 min isocratic 90% mobile phase A.

The flow rate was 1.0 mL/min.

Injection volume: 1000 pl_ (extended sample loop)

Solutions Diluent A: HPLC-grade Methanol Diluent B: HPLC-grade water

Stock Standard Solution (approximately 75 pg/mL phenylhydrazine): 20.0 mg of phenylhydrazine hydrochloride working standard were weighted in a 200.0 mL volumetric flask, diluted up with diluent A and sonicated for 2 minutes or more until dissolution was achieved.

A calibration curve was calculated from the analysis of different Standard solutions of phenylhydrazine concentration from 16 to 1500 ppm of phenylhydrazine with respect to edaravone which were prepared from the stock Standard solution.

Test solution: 30 mg/100mL Dose (preparation for approximately 300 pg/mL of Edaravone): 1.0 mL of the sample from the plastic bag was taken into a 5.0 mL volumetric flask and diluted up with diluent B.

Example 1: Preparation of the aqueous formulation of edaravone 0.3 mg/mL of the following formula:

In a stainless-steel vessel, edaravone, a source of sodium bisulfite (consisting of a mixture of < 10% (w/w) sodium bisulfite + > 90% (w/w) sodium metabisulfite yielding 20.00 mg of sodium bisulfite), sodium chloride and L-cysteine hydrochloride hydrate were dissolved in deoxygenated water for injection containing less than 1 ppm of oxygen.

The pH of the aqueous solution was adjusted to about 4 with the addition of sodium hydroxide 1N and phosphoric acid 85%.

The aqueous solution was filtered through a single PVDF 0.22 pm sterilizing filter and the filtered solution was filled inside a grade C isolator and into a non-sterile 100 mL three-layer plastic bag (Fujimori’s Nip^® solution Bags for Pharmaceuticals). The headspace of the filled bags was flushed with nitrogen and then were sealed with a stopper and cap that was welded into place by using a Branson ultrasonic welder.

The aqueous edaravone solution was analyzed before sterilization (see table 1). Samples were collected for the bulk sample, i.e., before the filling of the bags, and at end of the filling.

Table 1

The results above show that Impurity I cannot be detected before the heat-sterilization. Each 100 mL bag was placed into an aluminum bag provided with an oxygen absorber (300cc Dry Pak^®) and nitrogen was flushed in the space between the plastic bag and the aluminum overpouch. Two set of samples were measured for oxygen content. In the first set of samples (samples 1-14) the oxygen content of the aqueous solutions of edaravone contained in the bags were analysed after being prepared, i.e., at time = 0, whereas in the second set of samples (samples 15-28) the oxygen content of the aqueous solutions of edaravone contained in the bags were analyzed seven days after, i.e., time = 7 days after being stored at 20-25°C in the aluminum overpouch provided with the oxygen scavenger. The results of the oxygen content of the aqueous edaravone solutions are depicted in Table 2:

Table 2

Impurity I content after heat-sterilization Heat-sterilization was performed under atmospheric air by an autoclave cycle for 10 minutes at 123°C.

The aqueous edaravone solution was analyzed after heat-sterilization. Samples of three bags were analyzed by duplicate.

Table 3 shows the average results after heat-sterilization of the aqueous formulation of edaravone having about 1.78 ppm of oxygen.

Table 3

As it can be observed around 0.12-0.14% (area) of Impurity I is formed when the aqueous formulation of edaravone containing about 1.78 ppm of oxygen is autoclaved.

Table 4 shows the average results after heat-sterilization of the aqueous formulation of edaravone having about 0.39 ppm of oxygen.

Table 4

As it can be observed no Impurity I can be detected when the aqueous formulation of edaravone having about 0.39 ppm of oxygen is heat-sterilized. Example 2: Preparation of aqueous formulation of edaravone 0.3 mg/mL (90 L batch size) according to the process of the present invention (comprising the step of heat- sterilizing an aqueous formulation of edaravone containing less than 1 ppm of oxygen):

In a stainless-steel vessel, edaravone, a source of sodium bisulfite (sodium metabisulfite yielding 20.00 mg of sodium bisulfite), sodium chloride and L-cysteine hydrochloride hydrate were dissolved in deoxygenated water for injection containing less than 1 ppm of oxygen.

The pH of the aqueous solution was adjusted to about 4 with the addition of sodium hydroxide 1N and phosphoric acid 85%. The aqueous solution was filtered through a single PVDF 0.22 pm sterilizing filter and the filtered solution was filled inside a grade C isolator and into a non-sterile 100 ml three-layer plastic bag (Fujimori’s/Zacros Nip^® - å Bag, also called MediTect^® IV Bag).

The headspace of the filled bags was flushed with nitrogen and then were sealed with a stopper and cap that was welded into place by using a Branson ultrasonic welder.

The aqueous edaravone solution was analyzed before sterilization (see table 5). Samples were collected for the bulk sample, i.e. , before the filling of the bags:

Table 5

The result above shows that Impurity I can not be detected before the heat-sterilization. Each 100 mL bag was placed into an aluminum bag provided with an oxygen absorber (300cc Dry Pak^®) and nitrogen was flushed in the space between the plastic bag and the aluminum overpouch. The plastic bags in the aluminum overpouch together with an oxygen scavenger were kept for 11 days at 25 ± 5 °C. During this period, some bags were measured for oxygen content at different times. The results of the average amount of oxygen content are depicted in Table 6:

Table 6

After these 11 days, the aluminum bag and the oxygen absorber (300cc Dry Pak^®) were removed and the bags were placed in a second aluminum bag.

Heat-sterilization was then performed under atmospheric air by an autoclave cycle for 10 minutes at 123°C.

Impurity I content after heat-sterilization (t = 0) and after stability under different conditions:

The aqueous edaravone solution was analyzed after heat-sterilization. The samples were analyzed by duplicate. Table 7 shows the average values of assay, Impurity I and phenylhydrazine:

Table 7

As it can be observed Impurity I can be detected in low amounts (0.03%) when the aqueous formulation of edaravone having about 0.40 ppm of oxygen is autoclaved. The impurity content remains below 0.25% during stability and the amount of phenylhydrazine also remains below 300 ppm.

Claims

Claim 1: A process for preparing an aqueous formulation comprising edaravone or a pharmaceutically acceptable salt thereof and bisulfite anions (HSOT), wherein the process comprises: a) providing an aqueous formulation comprising i) edaravone or a pharmaceutically acceptable salt thereof and ii) bisulfite anions, and controlling the oxygen content of said formulation to be less than 1 ppm of oxygen, and b) heat sterilizing the formulation resulting from step a).

Claim 2: The process according to claim 1 wherein the oxygen content of the formulation is controlled to be less than 0.5 ppm of oxygen.

Claim 3: The process according to anyone of claims 1 or 2, wherein the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof resulting from step b) contains less than 0.10% (area) of the Impurity I, when analyzed by a HPLC or UHPLC method for chromatographic purity.

Claim 4: The process according to anyone of claim 1 to 3, wherein the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, resulting from step b) contains less than 300 ppm, preferably less than 200 ppm, more preferably less than 150 ppm, even more preferably less than 100 ppm of phenylhydrazine, when analyzed by a HPLC or UHPLC method.

Claim 5: The process according to any one of claims 1 to 4 wherein step a) comprises: 1) providing an aqueous formulation comprising i) edaravone or a pharmaceutically acceptable salt thereof and ii) bisulfite anions having an oxygen content of less than 1 ppm, preferably less than 0.5 ppm of oxygen.

2) optionally further controlling the oxygen content of the environment during the handling of the aqueous formulation of step 1).

Claim 6: The process according to claim 5 wherein step 1) is performed by one or more of the following steps: i) controlling the oxygen content of the water used to prepare the aqueous formulation, ii) controlling the oxygen content of the environment in which the aqueous formulation is prepared, iii) taking active measures to reduce the oxygen content of the aqueous formulation if said formulation has an oxygen content of 1 ppm or more.

Claim 7: The process according to anyone of claims 1 to 6, wherein bisulfite anions (HSOT) are present in the form of sodium bisulfite.

Claim 8: The process according to any one of claims 1 to 7 wherein the aqueous formulation of edaravone resulting from step b) is contained in a plastic ampoule or a plastic bag. Claim 9: The process according to claim 8 wherein the plastic bag is a multi-layered plastic bag comprising at least one layer, which comprises a cycloolefin polymer.

Claim 10: The process according to any one of claims 6 to 9, wherein step iii) is carried out by: (I) placing the plastic ampoule or plastic bag in an aluminum overpouch together with an oxygen scavenger and (II) keeping the product of step (I) until the oxygen contained in the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof has been reduced to less than 1 ppm, preferably to less than 0.5 ppm, before b) heat-sterilization. Claim 11: The process according to claim 10, wherein step (2) is carried out by keeping the product of step (1) at 25 ± 5 °C for a time from 5 to 20 days. Claim 12: The process according to anyone of claims 1 to 11 further comprising controlling the oxygen content of the environment during the heat-sterilization. Claim 13: The process according to claim 12, wherein the heat-sterilization is performed under vacuum or under nitrogen or argon atmosphere.

Claim 14: The process according to any one of claims 1 to 11, wherein the heat- sterilization is performed under atmospheric air.

Claim 15: The process according to anyone of claims 1 to 14, wherein the heat- sterilization is carried out for 5 minutes to 30 minutes at 115°C to 125°C.

Claim 16: The process according to claim 15, wherein the heat-sterilization is carried out for 5 minutes to 10 minutes at 123°C.

Claim 17: The process according to claim 15, wherein the heat-sterilization is carried out for 5 minutes at 123°C. Claim 18: The process according to claim 15, wherein the heat-sterilization is carried out for 15 minutes at 121°C.

Claim 19: The process according to any one of claims 1 to 18, wherein the aqueous formulation of edaravone or a pharmaceutically acceptable salt thereof, which is subjected to step b) further comprises L-cysteine hydrochloride hydrate, sodium chloride and sodium hydroxide and phosphoric acid to adjust the pH to about 4.

Claim 20: The process according to any one of claims 1 to 19, wherein the obtained formulation of edaravone or a pharmaceutically acceptable salt thereof is an injectable formulation.

Claim 21: A formulation of edaravone or a pharmaceutically salt thereof obtained according to any one of claims 1 to 20. Claim 22: Use of a formulation of edaravone or a pharmaceutically salt thereof as defined in claim 21 for the treatment of stroke and motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), or arthrogryposis multiplex congenita (AMC).

Claim 23: Formulation of edaravone or a pharmaceutically salt thereof as defined in claim 21 for use in the treatment of stroke and motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), or arthrogryposis multiplex congenita (AMC).

Claim 24: Use of a formulation of edaravone or a pharmaceutically salt thereof as defined in claim 21 for the manufacture of a medicament for the treatment of stroke and motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), or arthrogryposis multiplex congenita (AMC). Claim 25: A method for the treatment of stroke and motor neuron diseases such as amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), or arthrogryposis multiplex congenita (AMC) by administration to a subject in need thereof of a formulation of edaravone or a pharmaceutically salt thereof as defined in claim 21.