FIELD

The disclosure relates to liquid pharmaceutical compositions comprising ceftriaxone.

BACKGROUND

Ceftriaxone is a third-generation cephalosporin antibiotic and may be represented by Formula I:

Ceftriaxone has a broad antibacterial activity against a variety of gram-negative and grampositive bacteria. The antibacterial activity of ceftriaxone is due to its inhibition of bacterial cell wall synthesis. It binds to transpeptidases thereby inhibiting these which leads to damage and destruction of the bacterial cell wall and eventually to cell lysis.

A challenge with ceftriaxone compositions prepared for patient administration is relatively short stability at room temperature and under refrigerated conditions.

Considering its instability in solution, ceftriaxone is currently commercially available only in the form of powder that needs to be reconstituted and further diluted prior administration to a patient. Depending on the diluent used and resulting ceftriaxone concentration, according to the label, ceftriaxone intramuscular and intravenous preparations are stable for maximum 2 days if stored at room temperature and for maximum 10 days if stored refrigerated. Accordingly, there remains a need for liquid compositions of ceftriaxone suitable for administration to a subject and which possess prolonged stability when refrigerated and at room temperature, in respect of degradation of ceftriaxone and/or formation of impurities.

SUMMARY

The inventors have found that liquid pharmaceutical compositions comprising ceftriaxone, a derivative thereof, or a pharmaceutically acceptable salt of ceftriaxone or a derivative thereof and at least one magnesium salt, magnesium oxide, and/or magnesium hydroxide possess surprisingly enhanced storage stability.

It was found that when ceftriaxone is formulated in compositions according to the present disclosure, degradation is retarded, and accordingly, such compositions exhibit prolonged stability when stored under refrigerated conditions, i.e., at a temperature of from 2°C to 8°C.

Because of their enhanced storage stability, the compositions disclosed can be advantageously stored for relatively long periods.

Accordingly, the disclosure relates to liquid pharmaceutical compositions comprising ceftriaxone, a derivative thereof, or a pharmaceutically acceptable salt of ceftriaxone or a derivative thereof and at least one magnesium salt, magnesium oxide, and/or magnesium hydroxide. The disclosure also relates to compositions in which dissolved oxygen is removed or significantly reduced in the compositions.

Furthermore, the disclosure relates to the use of compositions according to the invention as a medicament, and to a method for treating bacterial infections in a subject by administering a liquid pharmaceutical composition to the subject.

DETAILED DISCLOSURE

The disclosure relates to ceftriaxone in a liquid pharmaceutical composition together with at least one magnesium salt, magnesium oxide, and/or magnesium hydroxide, wherein the ceftriaxone possesses surprisingly enhanced storage stability. Accordingly, the invention relates to stable liquid pharmaceutical compositions comprising ceftriaxone together with at least one magnesium salt, magnesium oxide, and/or magnesium hydroxide. The stability of the liquid compositions is unexpected in view of teachings that ceftriaxone has limited stability in liquid solutions. The term “ceftriaxone” as used herein means (1) ceftriaxone free acid known under the CAS No. 74484-59-5; (2) a derivative thereof; and/or (3) a pharmaceutically acceptable salt of ceftriaxone or a derivative thereof.

In one embodiment, ceftriaxone is ceftriaxone sodium.

The compositions disclosed comprise at least one magnesium salt, magnesium oxide, and/or magnesium hydroxide. This includes any form of magnesium suitable to provide magnesium ions in a liquid composition (i.e., a source of magnesium ions). In some aspects, the composition has a neutral charge; the positive charge of the magnesium ions is balanced by a negative charged counterion.

In one embodiment, the source of magnesium (which may be in the form of magnesium ions when in solution in the liquid composition) is a magnesium salt, magnesium oxide, or magnesium hydroxide. In one embodiment the magnesium salt, magnesium oxide, or magnesium hydroxide may be pharmaceutically acceptable. The term "pharmaceutically acceptable salt" when used in respect to this embodiment refers to a salt, oxide, or hydroxide containing a magnesium cation in which the associated acid-derived anion is generally considered suitable for human consumption. The salt may be derived from inorganic or organic acids.

In some embodiments, the magnesium is not elemental magnesium. In some embodiments, the magnesium is soluble in the liquid composition (meaning that the liquid composition has acceptable clarity, and no visible particles present in the liquid solution at the concentration of magnesium employed). In some embodiments, the magnesium is at least one magnesium salt or inorganic magnesium compound (such as a hydroxide or oxide).

Suitable organic acids generally include but are not limited to aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids.

Specific examples of suitable organic acids include but are not limited to acetic acid, trifluoroacetic acid, formic acid, propionic acid, succinic acid, benzoic acid, citric acid, maleic acid, tartaric acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glycolic acid, gluconic acid, digluconic acid, lactic acid, ascorbic acid, glucuronic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, stearic acid, salicylic acid, p-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid, ethanesulfonic acid, pantothenic acid, 2-hydroxyethanesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, beta-hydroxybutyric acid, galactaric acid, galacturonic acid, adipic acid, alginic acid, butyric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, dodecylsulfic acid, glycoheptanoic acid, glycerophosphoric acid, heptanoic acid, hexanoic acid, nicotinic acid, 2- naphthalenesulfonic acid, oxalic acid, palmoic acid, pectinic acid, 3-phenylpropionic acid, picratic acid, pivalic acid, thiocyanic acid, and undecanoic acid.

Suitable magnesium salts also include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, boric acid, fluoroboric acid, phosphoric acid, metaphosphoric acid, nitric acid, carbonic acid, sulfonic acid, and sulfuric acid.

In one embodiment, the magnesium salt is selected from at least one of magnesium sulfate, magnesium trifluoromethanesulfonate, magnesium acetylacetonate, magnesium fumarate, magnesium succinate, magnesium maleate, magnesium citrate, magnesium perchlorate, and magnesium nitrate.

In one embodiment, the magnesium (i.e., magnesium in a form suitable to provide magnesium ions in a liquid composition) may be magnesium oxide or magnesium hydroxide.

The amount of ceftriaxone and at least one magnesium salt, magnesium oxide, and/or magnesium hydroxide relative to each other in the composition may be defined by the molar ratio of the individual components. The molar ratios are calculated using the atomic masses of magnesium and ceftriaxone, and not the atomic masses of the salts of magnesium and ceftriaxone. The molar ratio of ceftriaxone and magnesium may be from 1 :0.05 to 1 :2, such as e.g. from 1 :0.05 to 1 :2, from 1 :0.06 to 1 :2, from 1 :0.07 to 1 :2, from 1 :0.08 to 1 :2, from 1 :0.09 to 1 :2, from 1 :0.1 to 1 :2, 1 :0.2 to 1 :2, from 1 :0.3 to 1 .9, from 1 :0.4 to 1 :1.9, from 1 :0.4 to 1 :1.5, from 1 :0.5 to 1 :1.8, from 1 :0.6 to 1 :1.7, from 1 :0.7 to 1 :1.6, from 1 :0.8 to 1 :1.5, from 1 :0.9 to 1 :1.4, from 1 :1 to 1 :1.3, and from 1 :1 to 1 :1.2.

In one embodiment, the molar ratio of ceftriaxone and at least one magnesium salt, magnesium oxide, and/or magnesium hydroxide, calculated using the atomic mases of magnesium and ceftriaxone, respectively, may be 1 :0.05; 1 :0.06, 1 :0.07, 1 :0.08, 1 :0.09, 1 :0.1 , 1 :0.2, 1 :0.3, 1 :0.4, 1 :0.5, 1 :0.6, 1 :0.7, 1 :0.8, 1 :0.9, 1 :1 , 1 :1.1 , 1 :1.2, 1 :1.3, 1 :1.4, 1 :1.5, 1 :1.6, 1 :1.7, 1 :1.8, 1 :1.9 or 1 :2.

The term “stable” means that the liquid pharmaceutical compositions meets one or more of the following criteria:

(1 ) the liquid pharmaceutical composition exhibits an acceptable amount of ceftriaxone having degraded after a certain period compared to the amount of ceftriaxone present at the beginning of the period; and/or

(2) the liquid pharmaceutical composition exhibits an acceptable amount of impurities being formed after a certain period compared to the amount of impurities present at the beginning of the period; and/or

(3) the liquid pharmaceutical composition retains a pharmaceutically desirable appearance such as clarity, no significant change of color, and no visible particles. Visual inspection for visible particles may be performed as follows: the container under inspection is gently swirled and inverted, ensuring that no air bubbles are produced, and inspected for approximately 5 sec with naked eye. Visual inspection for change of color may be performed as follows: The container is inspected by eye and a color is assigned to the composition. The color may also be determined by a UV/VIS spectrometer and the difference in color between two samples may be expressed as delta E.

Stability of a liquid pharmaceutical composition may also be defined as the improvement of a parameter, such as, e.g., less degradation of ceftriaxone, less formation of impurities, and/or less change of clarity, less change of color, and less formation of visible particles, in comparison to a suitable control. As an example, a stable liquid pharmaceutical composition according to the disclosure may be one in which degradation of ceftriaxone is reduced in comparison to a control composition stored under same conditions, such as, e.g., a liquid pharmaceutical composition comprising ceftriaxone and a magnesium salt, magnesium oxide and/or magnesium hydroxide, in which the degradation of ceftriaxone is reduced in comparison to a control composition comprising ceftriaxone without magnesium salt, magnesium oxide and/or magnesium hydroxide stored for a predetermined period at predetermined conditions.

Stability may be determined in various ways, for instance by one of the following methods:

(1 ) determination of stability includes measuring the concentration or absolute amount of ceftriaxone remaining in the composition after a predetermined time period at predetermined storage conditions by determining the concentration or absolute amount of ceftriaxone free base in the composition by using an external standard; and/or

(2) measuring the purity of ceftriaxone in a composition after a predetermined period at predetermined storage conditions and comparing any reduction in purity to the purities present at an initial time point, such as by measuring chromatographic purity (e.g., by measuring peak-area percentage of a chromatogram, such as an HPLC or UHPLC chromatogram, over time and expressing stability of the compositions in terms a percentage), and/or

(3) measuring the amount of impurities in a composition after a predetermined period at predetermined storage conditions and comparing any increase in impurities to impurities at an initial time point (such as by using peak-area percentage of a HPLC chromatogram, as well as by evaluating the concentration or absolute amount of an impurity or combinations of impurities or total impurities present).

As it appears from the above the term “stable” encompasses both chemical and physical stability of a composition. Chemical stability relates for example to the degradation of ceftriaxone and formation of impurities while physical stability relates for example to the formation of visible particles and color change.

Generally, for pharmaceutical compositions according to the disclosure, as little degradation of the active ingredient as possible is desired.

A liquid pharmaceutical composition in which an acceptable amount of ceftriaxone has degraded after a certain period compared to the amount of ceftriaxone present at the beginning of the period is one which has no more than 10 %, such as, e.g., no more than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% drop or decrease of ceftriaxone after a predetermined period at predetermined storage conditions.

Furthermore, the formation of impurities may be unwanted. In some cases, the formation of specific impurities may be unwanted.

A liquid pharmaceutical composition which exhibits an acceptable amount of impurities being formed after a certain period compared to the amount of impurities present at the beginning of the period is one, in which no more than 10%, such as, e.g., no more than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% impurities have formed after a predetermined period at predetermined storage conditions. In one aspect the impurity measured is Impurity C (Cas No. 58909-39-0).

The liquid pharmaceutical composition of ceftriaxone described herein may be stable over time periods of 5 days, 6 days, 7 days (1 week), 14 days (2 weeks), 30 days (1 month), 60 days (2 months), 3 months, 4 months, 5 months, 6 months (180 days), 9 months, 12 months (1 year) and longer.

The compositions disclosed are surprisingly stable when stored at a temperature of 25°C or below, such as at a temperature of 24°C, 23°C, 22°C, 21 °C, 20°C, 19°C, 18°C, 17°C,

16°C, 15°C, 14°C, 13°C, 12°C, 11 °C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C or 2°C .

In one embodiment, the compositions are stored at a temperature of from 2°C to 8°C, such as, e.g., at a temperature of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C.

In one embodiment, the compositions may be stable for at least 5 days at 25°C, wherein the amount of ceftriaxone that has degraded in the composition at 5 days is less than 10%, such as, e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%.

In one embodiment, the compositions may be stable for at least 14 days at 15°C, wherein the amount of ceftriaxone that has degraded in the composition at 14 days is less than 7%, such as, e.g., less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%.

In one embodiment, the compositions may be stable for at least 21 days at 15°C, wherein the amount of ceftriaxone that has degraded in the composition at 21 days is less than 8%, such as, e.g., less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%.

In one embodiment, the compositions may be stable for at least 30 days at 2-8°C, wherein the amount of ceftriaxone that has degraded in the composition at 30 days is less than 3%, such as, e.g., less than 2% or less than 1%.

In one embodiment, the compositions may be stable for at least three months at 2-8°C, wherein the amount of ceftriaxone that has degraded in the composition at three months is less than 4%, such as, e.g., less than 3% or less than 2% or less than 1%. In one embodiment, the compositions may be stable for at least six months at 2-8°C, wherein the amount of ceftriaxone that has degraded in the composition at six months is less than 8%, such as, e.g., less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%.

In one embodiment, the compositions may be stable for at least nine months at 2-8°C, wherein the amount of ceftriaxone that has degraded in the composition at nine months is less than 10%, such as, e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%.

In one embodiment, the compositions may be stable for at least six months at 2-8°C, wherein the amount of Impurity C that has formed in the composition at six months is less than 1.5%, such as, e.g., less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1% less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6% or less than 0.5%.

In some compositions comprising ceftriaxone and magnesium formation of visible particles may appear after storage for more than 60 (2 months), 70, 80 or 90 (three months) days. The inventors have surprisingly found that removing dissolved oxygen or significantly reducing the amount of dissolved oxygen in the compositions according to the disclosure to obtain a substantially oxygen-free composition, may have an impact on the formation of visible particles in the composition during storage. By removing or significantly reducing the oxygen from the compositions to obtain a substantially oxygen- free composition, the formation of visible particles during storage is prevented. Accordingly, in one embodiment the compositions are free from visible particles after storage for at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12 months at 2-8°C.

By the term “substantially oxygen-free” is understood a composition in which the amount of oxygen in the composition is kept at a very low level or where the composition is free of oxygen. It may be a composition having an oxygen content equal to or lower than 2.0 ppm, such as, e.g., lower than 1.5 ppm. 1.0 ppm, 0.5 ppm, 0.4 ppm. 0.3 ppm, 0.2 ppm,

0.1 ppm, 0.09 ppm, 0.08 ppm, 0.07 ppm, 0.06 ppm, 0.05 ppm, 0.04 ppm, 0.03 ppm, 0.02 ppm or lower than 0.01 ppm. Compositions in which the amount of oxygen is significantly reduced are understood to be compositions having an oxygen content equal to or lower than 2.0 ppm, such as, e.g., lower than 1.5 ppm. 1.0 ppm, 0.5 ppm, 0.4 ppm. 0.3 ppm, 0.2 ppm, 0.1 ppm, 0.09 ppm, 0.08 ppm, 0.07 ppm, 0.06 ppm, 0.05 ppm, 0.04 ppm, 0.03 ppm, 0.02 ppm or lower than 0.01 ppm.

When stored, it has been observed that the liquid ceftriaxone composition may change color from colorless or slightly yellow to light orange, orange or dark orange. The inventors have surprisingly found that removing oxygen or significantly reducing the amount of oxygen in the compositions according to the invention to obtain a substantially oxygen- free composition, will lead to a composition wherein no significant change of color of the composition takes place during storage. Accordingly, colorless, or slightly or light-colored compositions are obtained after storage.

By the term “no significant change of color” is understood that the color of the composition is changing to a color with no significance to the useability of the composition. The compositions may for example obtain a color from colorless to slightly yellow, slightly orange to light orange after storage for predetermined period under predetermined conditions. By the term may also be understood that the degree of color change of the composition is without significance to the usability of the compositions. The composition may for instance be changing from colorless or slightly yellow or any color in between to slightly yellow, slightly orange or light orange or any color in between. The term may also cover situations where the color of the compositions according to the disclosure are compared to the color of a suitable control, and where the color change of the composition is less than that of the control composition. As an example, a stable liquid pharmaceutical composition according to the disclosure may be one in which color change is reduced in comparison to a control composition stored under same conditions, such as, e.g., a liquid pharmaceutical composition comprising ceftriaxone and optionally a magnesium salt, magnesium oxide and/or magnesium hydroxide and wherein oxygen is removed or significantly reduced in the composition, and wherein color change is reduced as compared to a control composition comprising ceftriaxone and optionally a magnesium salt, magnesium oxide and/or magnesium hydroxide, and wherein oxygen has not been removed or significantly reduced, after storage for a predetermined period at predetermined conditions.

Accordingly, in one embodiment there is no significant change of the color of the composition after storage for at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11 , at least 12 months at 2-8°C.

In one embodiment, the disclosure relates to a substantially oxygen-free liquid pharmaceutical composition comprising ceftriaxone.

In one embodiment, the disclosure relates to a substantially oxygen free liquid pharmaceutical composition, as described herein, comprising ceftriaxone and a magnesium salt, magnesium oxide, and/or magnesium hydroxide.

In one embodiment the liquid pharmaceutical compositions as described herein may be aqueous pharmaceutical composition.

By the term “aqueous pharmaceutical composition” is understood any composition in which water is present in or above 50% v/v, such as, e.g., a composition comprising from 50% v/v to 99.5% v/v water, from 50 % v/v to 90% v/v, from 60% v/v to 85% v/v, from 70% v/v to 80 % v/v water. Accordingly, aqueous compositions include compositions comprising 50% v/v or more, 60% v/v or more, 70% v/v or more, 75% v/v or more, 80% v/v or more, 85% v/v or more, 90% v/v or more, 95% v/v or more or 99% v/v water or more.

The compositions may further comprise one or more additional excipients.

The composition may also comprise a tonicity agent in order to obtain an osmolarity of the composition suitable for parenteral administration, i.e., to render the pharmaceutical compositions according to the invention isotonic with physiological fluids. Exemplary tonicity agents include sodium chloride, glycine, sucrose, mannitol, dextrose, amino acids, and combinations thereof.

The compositions according to the invention may also comprise one or more pH adjusting agents, which may be selected from mineral acids, organic acids, weak and strong bases, and salts and derivatives thereof. Examples of agents include hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, succinic acid, lactic acid, citric acid, phenolic acids, sodium hydroxide, ammonium hydroxide, sodium bicarbonate, or similar. The pH of the composition is adjusted to 6.0 to 8.5 before storage of the composition. Accordingly, the compositions according to the invention may have a pH before storage of from 6.0 to 8.5, such as, e.g., from 6.1 to 8.4, from 6.2 to 8.2, from 6.3 to 8.0, from 6.4 to 7.8, from 6.4 to 7.6, from 6.4 to 7.4, from 6.4 to 7.2, from 6.4 to 7.0, from 6.5 to 7.0, from 6.5 to 6.9, from 6.6 to 6.9, from 6.7 to 6.9, i.e. a pH of 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,

7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,0, 8.1 , 8.2, 8.3, 8.4 or 8.5.

The compositions may further comprise a buffer, such as, e.g., a buffer selected from citrate, maleate, ACES, MES, MOPS or PIPES. If the compositions comprise a buffer the pH may be adjusted to 6.5 to 7.3 before storage, such as, e.g., to a pH from 6.6 to 7.2, from 6.7 to 7.1 , from 6.7 to 7.0 and from 6.7 to 6.9. The compositions may have a pH of

6.5, 6.6., 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2 or 7.3.

In one embodiment, the compositions according to the disclosure does not comprise a source of calcium, carbonate or phosphate.

The compositions may be in form of a ready-to-administer or a ready-to-use composition. The term “ready-to-use” includes liquid preconcentrates which require a single step of dilution with a liquid diluent fluid such as water for injection or saline before administration. A "ready-to-administer" composition is synonymous with "ready-to-infuse" or “ready-to- inject” and is not to be read as the term "ready-to-use” composition. A “ready-to- administer” composition is suitable for administration directly to the patient and does not require any dilution steps.

The term "ready-to-administer” is also distinguished from lyophilized products that require two steps, a first step of reconstitution to form a preconcentrate and then a second step where the preconcentrate is subjected to dilution with a liquid infusion fluid.

Suitable fluids for diluting the ready-to-use composition include some which are pharmaceutically safe and non-toxic for administration to a human and are compatible for the preparation of a diluted formulation. Example of such diluent include water, e.g., sterile water for injection, sodium chloride solutions, dextrose solution solutions and combinations thereof. In one embodiment, the composition may be prepared by aseptically withdrawing an appropriate volume of the liquid composition of ceftriaxone and transferring it into an infusion bag comprising a suitable diluent.

The concentration of ceftriaxone (i.e., ceftriaxone, a derivative thereof, or a pharmaceutically acceptable salt of ceftriaxone) in compositions may be from 2 mg/ml to 350 mg/ml, such as, e.g. from 10 mg/ml to 325 mg/ml from 12 mg/ml to 325 mg/ml, from

14 mg/ml to 300 mg/ml, from 16 mg/ml to 275 mg/ml, from 18 mg/ml to 250 mg/ml, from

20 mg/ml to 225 mg/ml, from 20 mg/ml to 200 mg/ml, from 20 mg/ml to 175 mg/ml, from

20 mg/ml to 150 mg/ml, from 20 mg/ml to 125 mg/ml, from 20 mg/ml to 100 mg/ml and from 10 mg/ml to 100 mg/ml.

Accordingly, the concentration of ceftriaxone may be 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml,

15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 36 mg/ml, 37mg/ml, 38 mg/ml, 39 mg/ml, 40 mg/ml, 41 mg/ml, 42 mg/ml, 43 mg/ml, 44 mg/ml, 45 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 95 mg/ml, 96 mg/ml, 97 mg/ml, 98 mg/ml, 99 mg/ml, 100 mg/ml, 101 mg/ml, 102 mg/ml, 103 mg/ml, 104 mg/ml and 105 mg/ml.

In one embodiment the concentration of ceftriaxone is 10 mg/ml, 20 mg/ml, 40 mg/ml or 100 mg/ml.

The amount of ceftriaxone in the liquid pharmaceutical composition disclosed herein depends on whether the composition is intended to be used directly or after dilution as defined above.

Manufacturing

The disclosure also provides processes for manufacturing or preparing a liquid composition comprising ceftriaxone, a derivative thereof, or a pharmaceutically acceptable salt of ceftriaxone or a derivative thereof comprises a) providing ceftriaxone, a derivative thereof, or a pharmaceutically acceptable salt of ceftriaxone or a derivative thereof; b) providing at least one magnesium salt, magnesium oxide, and/or magnesium hydroxide (in other words, any form of magnesium suitable to provide magnesium ions in a liquid composition); c) optionally, providing one or more additional components into the liquid, d) combining a) and b) and optionally c) with a liquid. Any of the compositions described herein may be prepared using this method.

In one embodiment the liquid pharmaceutical compositions may be an aqueous pharmaceutical composition, such as, e.g., water for injection.

A method of stabilizing ceftriaxone in a liquid pharmaceutical composition comprises mixing ceftriaxone and at least one magnesium salt, magnesium oxide, and/or magnesium hydroxide (in other words, any form of magnesium suitable to provide magnesium ions in a liquid composition) and optionally one or more additional components. Any of the compositions described herein may be prepared using this method.

As mentioned above removing or significantly reducing the amount of dissolved oxygen in the composition is a part of the disclosure. Accordingly, in one embodiment the compositions are produced under conditions in which dissolved oxygen in the composition is removed or significantly reduced.

The dissolved oxygen in the composition may be removed prior to filling the composition into a suitable container. This removal of oxygen may be performed by deaerating or degassing the composition by bubbling or blowing the composition with a modified atmosphere, such as, e.g., an inert gas. Examples of inert gasses may be nitrogen or argon or mixtures thereof. The removal or reduction of oxygen by deaeration or degassing may also be performed on the liquid before the ceftriaxone, magnesium and optionally additional components are added to the liquid.

In another embodiment, the oxygen may be removed from the composition by applying a vacuum to the composition prior to filling it into a suitable container.

If a filtration step is applied to the composition before filling it into a suitable container this step may be performed by passing the composition through a sterilizing grade filter. Passage may be performed by pressurizing the solution with inert gas.

Before the composition is filled into a container, the container may be flushed with an inert gas such as, e.g., nitrogen or argon or a mixture thereof, to remove oxygen, or a vacuum may be applied to the container to remove oxygen.

After filing the composition into a suitable container, overlay with a modified atmosphere may be performed, in order to minimize the volume of residual oxygen in the headspace of the container, before closure of the container.

In another embodiment, the atmosphere of the container is not exchanged with an inert atmosphere prior to filling the composition into the container. The steps of preparing and filtering the composition and filling it into a suitable container may all be performed in a modified atmosphere, such as, e.g., in nitrogen, argon or mixtures thereof.

Packaging and kits

As described above, the compositions disclosed herein may be prepared by dissolving and mixing the ingredients, filtering the obtained mixture and transferring it to a suitable container.

The container may be suitable for maintaining the sterility or preventing the contamination of the composition. The container may be a sealed container. Examples of such containers include, but is not limited to, glass vials, ampoules, plastic flexible containers, for examples, but not limited to, polyvinyl chloride containers, CR3 elastomer copolyester ether containers, CZ resin containers, polypropylene containers and syringes.

In one embodiment the container comprising a composition may comprise a modified atmosphere.

The modified atmosphere may be an inert atmosphere. In one embodiment the atmosphere may be an atmosphere comprising no or a significantly reduced amount of oxygen. Examples of suitable atmospheres comprising no oxygen, or a significantly reduced amount of oxygen are atmospheres based on e.g. nitrogen or argon. In one embodiment the modified atmosphere is a mixture of gasses, such as, e.g., a mixture of nitrogen and argon.

In one embodiment the first container is overwrapped with a secondary packaging. The material may be one that has a low oxygen permeability. In one embodiment the overwrapping is an aluminum pouch in which the container is placed, and the pouch is subsequently sealed. Prior to sealing the amount of oxygen in the secondary packaging may be removed or significantly reduced by applying a vacuum to the secondary packaging. Alternatively, an oxygen absorber or scavenger may be placed in the secondary packaging to help remove or decrease the level of dissolved oxygen in the package. There are many types of oxygen absorbers available on the market and often the oxygen absorber or scavenger is enclosed in a porous sachet or packet, but it may also be part of the secondary packaging, in which case the placing of the oxygen absorber in the secondary packaging is of course not needed. In one embodiment the first container is a flexible plastic bag comprising a degassed/deaerated composition according to the invention. In one embodiment, the flexible plastic bag is overwrapped with an overwrap in which an oxygen absorber or scavenger is placed.

An embodiment relates to a kit comprising a composition as disclosed herein and instructions for storage and/or use, e.g., written instructions of how to administer a composition. The kit may comprise compositions that are ready to use, such they can be administered parenterally without any further compounding or processing. However, it is also contemplated that the composition may be diluted with a pharmaceutically acceptable diluent before administration to a patient or subject. The kit may further comprise such diluents, and devices for diluting the composition. It may also comprise devices for dispensing or delivering the compositions. An example of such a diluent and device is e.g., an infusion bag of a sodium chloride or dextrose solution, or of sterile water for injection. The composition and the instructions may be provided in a suitable container or package.

Use

The compositions disclosed herein may be used as a medicament.

The compositions disclosed may be used to treat infections in a patient or subject, such as by administering any of the compositions described herein to the patient or subject.

The terms "patient” and “subject” are used interchangeably and refer to any human or animal individual who is receiving a composition.

The compositions may be administered to a patient or subject in need thereof by a variety of parenteral delivery methods. Examples of such methods include, but is not limited to, administration via injection, such as, e.g., intravenously, subcutaneously, intracutaneously, intramuscularly, intraarticularly, intrasynovially, intrasternally, intralesionally, intracranially injection or infusion, or via pulmonary administration.

In one embodiment the administration method is intravenous injection or infusion.

The infections may include those caused by susceptible aerobic gram-negative, aerobic gram-positive, and anaerobic bacteria. Ceftriaxone can, in some instances, treat bacterial infections resistant to other antibiotics, including some multiply resistant to other antibiotics. Some bacterial infections that can be treated include, but are not limited to, infections caused by Acinetobacter calcoaceticus, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Haemophilus influenzae (including ampicillin-resistant and beta- lactamase producing strains), Haemophilus parainfluenzae, Klebsiella oxytoca, Klebsiella pneumoniae, Moraxella catarrhalis (including beta-lactamase producing strains), Morganella morganii, Neisseria gonorrhoeae (including penicillinase- and nonpenicillinase-producing strains), Neisseria meningitidis, Proteus mirabilis, Proteus vulgaris, Serratia marcescens, Staphylococcus aureus (including penicillinase-producing strains), Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Viridans group streptococci, Bacteroides fragilis, Clostridium species and/or Peptostreptococcus species

Conditions for treatment may include, but are not limited to, acute epididymitis, acute uncomplicated pyelonephritis, bacteremia, bone and joint infections (including arthritis related to infection) chancroid (genital sores caused by bacteria), ear infection (including acute bacterial otitis media), endocarditis (infection of the heart lining and valves), epiglottitis, gonococcal infections (such as those of the pharynx, cervix, vulva, vagina, urethra, rectum, gonococcal conjunctivitis, gonococcal endocarditis, gonococcal meningitis, gonococcal epididymitis, disseminated gonococcal infection, opthalmia neonatorum, gonococcal scalp abscesses; prophylaxis for infants of mothers with gonococcal infection), intra-abdominal infections, lung and lower respiratory tract infection, Lyme disease, meningitis, pelvic inflammatory disease, prophylaxis in close contacts of someone who is sick with meningitis, prophylaxis related to surgical procedure(s), prophylaxis and/or treatment in patients who have fever and are at high risk for infection because they have very few white blood cells, prophylaxis in certain penicillin-allergic patients who have a heart condition and are having a dental or upper respiratory tract (nose, mouth, throat, voice box) procedure, prophylaxis in people who have been bitten by humans or animals, prophylaxis of sexually transmitted diseases, prosthetic joint infection, relapsing fever (an infection that is transmitted by tick bites that causes repeated episodes of fever), salmonella (an infection that causes severe diarrhea), sepsis (blood infection) and septic shock, serious infections, shigella (an infection that causes severe diarrhea), sinus infections (including severe acute bacterial rhinosinusitis), skin infection (including skin and soft necrotizing infection and skin/skin structure infection), typhoid fever, urinary tract infection, and/or Whipple's disease. One embodiment relates to a method for treating a bacterial infection in a patient or subject, comprising administering a therapeutically effective amount of a composition to the patient or subject. Methods of treatment also include methods of prevention, such as when a patient or subject has been exposed to a bacteria that may cause an infection, has incurred an injury or wound, or is undergoing surgery or other medical procedure that could inadvertently lead to an infection.

A “therapeutically effective amount” is an amount which is sufficient to treat a patient’s bacterial infection.

All numbers in the specification and claims are modified by the term “about”. This means that each number includes minor variations as defined ±10% of the numerical value or range in question.

FIGURES

Figure 1 illustrates the color of ceftriaxone compositions having no oxygen or significantly reduced oxygen content compared to the color of ceftriaxone compositions comprising oxygen after storage for 120 days at 2-8°C.

EXAMPLES

Experimental methods

Preparation of ceftriaxone sodium formulations

During the manufacturing process and filling of the compositions of ceftriaxone sodium into vials the compositions were protected from natural light (exposed to regular laboratory lightning). The preparation of the formulations is conducted within the temperature range of 15°C-25°C.

The preparation of the compositons is described for compostions having a ceftriaxone content of 20 mg/ml, however compostions having other concentrations of ceftriaoxne, such as, e.g., 10 mg/ml, 40 mg/ml or 100 mg/ml ceftriaxone are prepared in a similar manner.

Control formulation in water for injection prepared under regular atmosphere A predefined volume of Water for Injection (WFI) at the temperature of 15°C-25°C was collected in a laboratory glass. The dispensed quantity of Ceftriaxone sodium API was added into the WFI to achieve the final ceftriaxone concentration of 20 mg/mL and solution was mixed until API was dissolved (clear solution without visible particles was obtained). WFI at the temperature in the range of 15°C-25°C was added to make the solution up to the prescribed volume to give the concentration of 20 mg/ml of ceftriaxone, and the solution was stirred for no less than 1 min.

The solution was then filtered through a 0.2 pm filter and filled into glass vials, stoppered with rubber stoppers and sealed with aluminum seals. Vials were either analyzed at the start for predefined parameters or loaded at stress/stability testing and analyzed as per defined schedule for agreed parameters.

Control formulation in water for injection prepared in inert atmosphere A predefined volume of Water for Injection (WFI) at the temperature of 15°C-25°C was collected in a laboratory glass. WFI was degassed until dissolved oxygen was below 2.0 ppm. Then the dispensed quantity of Ceftriaxone sodium API was added into the WFI to achieve the final ceftriaxone concentration of 20 mg/ml_ and solution was mixed until API was dissolved (clear solution without visible particles was obtained). Dissolved oxygen (DO) level in solution was maintained below 2.0 ppm during Ceftriaxone Sodium dissolution. Degassed WFI (with DO below 2.0 ppm) at the temperature in the range of 15°C-25°C was added to make the solution up to the prescribed volume to give the concentration of 20 mg/ml of ceftriaxone, and the solution was stirred for no less than 1 min.

The solution was then filtered through a 0.2 pm filter and filled into glass vials. Solution in the vials was overlaid with nitrogen prior to stoppering with rubber stoppers and sealing with aluminum seals. Vials were either analyzed at the start for predefined parameters or loaded at stress/stability testing and analyzed as per defined schedule for agreed parameters.

Alternative to the above described process, filtration and filling of the solution in vials, as well as vials stoppering, was perfomed in a nitrogen cabinet where the level of oxygen was maintained below 0.5 ppm. WFI used for solution preparation and volume make up was degassed prior to use to assure DO content below 2.0 ppm.

Formulation in water for injection containing magnesium prepared under regular atmosphere A predefined volume of Water for Injection (WFI) at the temperature of 15°C-25°C was collected in the laboratory glass. The dispensed quantity of magnesium salt in an amount to give a concentration of between 8.6 mg/ml to 86 mg/ml was added and mixed until dissolved (clear solution without visible particles is obtained). If needed, the pH was adjusted in the range from 6.5 to 9 using a sodium hydroxide solution. Ceftriaxone sodium API was then added in an amount to achieve the desired concentration of 20 mg/ml, and the solution was mixed until API was dissolved (clear solution without visible particles is obtained). WFI at the temperature in the range of 15°C-25°C was added to make the solution up to the prescribed volume to give the desired concentrations, and the solution was stirred for no less than 1 min.

The solution was then filtered through a 0.2 pm filter and filled into glass vials, stoppered with rubber stoppers and sealed with aluminum seals. Vials were either analyzed at the start for predefined parameters or loaded at stress/stability testing and analyzed as per defined schedule for agreed parameters.

Formulation in water for injection containing magnesium prepared in inert atmosphere A predefined volume of Water for Injection (WFI) at the temperature of 15°C-25°C was collected in the laboratory glass. WFI was degassed until dissolved oxygen was below 2.0 ppm. Then the dispensed quantity of magnesium salt in an amount to give a concentration of between 8.6 mg/ml to 86 mg/ml was added and mixed until dissolved (clear solution without visible particles is obtained). If needed, the pH was adjusted in the range from 6.5 to 6.9 using a sodium hydroxide solution. Ceftriaxone sodium API was then added in an amount to achieve the desired concentration of 20 mg/ml, and the solution was mixed until API was dissolved (clear solution without visible particles is obtained). DO level in solution was maintained below 2.0 ppm during magnesium salt and Ceftriaxone Sodium dissolution. Degassed WFI (with DO below 2.0 ppm) at the temperature in the range of 15°C-25°C was added to make the solution up to the prescribed volume to give the desired concentrations, and the solution was stirred for no less than 1 min.

The solution was then filtered through a 0.2 pm filter and filled into glass vials. The solution in the vials was overlaid with nitrogen prior to stoppering with rubber stoppers and sealing with aluminum seals. Vials were either analyzed at the start for predefined parameters or loaded at stress/stability testing and analyzed as per defined schedule for agreed parameters. Alternative to the above described process in laboratory scale preparation, filtration and filling of the solution in vials, as well as vials stoppering, was perfomed in a nitrogen cabinet where the level of oxygen was maintained below 0.5 ppm. WFI used for solution preparation and volume make up was degassed prior to use to assure DO content below 2.0 ppm.

Analytical methods

1. RP Liquid Chromatography Assay and impurities method Mobile phase A: 10 mM phosphate buffer, pH 2.3 adjusted with phosphoric acid Mobile phase B: 50% Buffer + 50% Acetonitrile (% by volume)

Column: C18 Supelco Titan 100*2.1 mm; 1.9 pm Column temperature: 35 °C Flow: 0.400 mL/min Gradient:

Detection: UV@ 254 nm Injection volume: 3 pL

2. Sample and standard Concentration of Ceftriaxone (free base) in Sample and Standard solution is about 0.08 mg/ml_ in MiliQ water.

Standard solution was prepared by weighing 4.000-5.000 mg of Ceftriaxone Sodium reference standard in 50 ml_ volumetric flask, dissolve and dilute to volume with MilliQ water. Sample preparation for 20 mg/ml_ formulations:

About 0.4 ml. of sample was weighed into 100 ml. volumetric flask and diluted to volume with MilliQ water. The density of the composition was determined. Sample and standard formulations were prepared in duplicate.

3. Calculation

Response factor (RF) is calculated as means of calibration of the system and then used to calculate ceftriaxone in sample.

Calculation of the detector response factor (RF) of ceftriaxone in Standard solutions:

RF = Area of ceftriaxone peak / concentration of ceftriaxone free base in standard solution

Ceftriaxone assay calculation:

Area of Cef peak in sample solution * Density of formulation ^) * 100 (mL) * 100% Average RF of standard solution * weight of sample ( g ) * 20

Relative standard deviation (RSD) of standard response factors should be less than 1% Recovery of Assay results of two sample preparation should be between 0.985-1.015

Impurities calculation:

Impurities are calculated as Area% (percent ratio of sigle impurity area and total chromatogram area)

Relative retention time of Imp C is about 0.2 and relative response factor at 254 nm is 0.8. Impurity C is calculated as Area % * 0.8

Stability analysis

Stability of Ceftriaxone molecule in the compositions is expressed as drop of ceftriaxone assay measured at specific time point to Ceftriaxone assay measured at the time of preparation.

Ceftriaxone assay as percentage of concentration of ceftriaxone at start relative to targeted concentration - Ceftriaxone assay as percentage of concentration of ceftriaxone at specific time point relative to targeted concentration = Assay drop % Example 1: Stability of ceftriaxone compositions prepared under regular atmosphere

In order to illustrate the effect of magnesium in liquid compositions of ceftriaxone compositions comprising various magnesium salt and various concentrations were prepared as described above.

Table 1: Stability of 20 mg/ml ceftriaxone with magnesium salts after 30 days at 2-

8°C represented as assay drop of ceftriaxone

Formulation Molar ratio Assay drop

Ceftriaxone : Mg %

Ceftriaxone sodium with magnesium chloride 1 : 1 1.3

Ceftriaxone sodium with magnesium chloride 1 : 0.8 1.9

Ceftriaxone sodium with magnesium chloride 1 : 0.5 2.1

Ceftriaxone sodium with magnesium chloride 1 : 0.4 2.9

Ceftriaxone sodium with magnesium chloride 1 : 2 1.6

Ceftriaxone sodium with magnesium 1 : 1 1.2 trifluoromethanesulfonate

Ceftriaxone sodium with magnesium sulfate 1 : 1 1.3

Ceftriaxone sodium with magnesium acetylacetonate 1 : 1 1.9

Ceftriaxone sodium with magnesium tri-magnesium 1 : 0.95 2.5 dicitrate

Ceftriaxone sodium with magnesium fumarate 1 : 1 1.0

Ceftriaxone sodium with magnesium maleate 1 : 1 1.0

Ceftriaxone sodium with magnesium succinate 1 : 1 1.3

Ceftriaxone sodium with magnesium perchlorate 1 : 1.5 1.6

Ceftriaxone sodium with magnesium chloride 1 : 0.6 1.7 Ceftriaxone sodium with magnesium perchlorate 1 : 1 1.8

Control 3.2

Ceftriaxone sodium in WFI

As clearly seen from the results all compositions comprising magnesium showed less drop of ceftriaxone after 30 days at 2-8°C than the control without magnesium.

Table 2: Stability of 20 mg/ml ceftriaxone with magnesium salts after 60 days at 2-

8°C represented as assay drop of ceftriaxone

Formulation Molar ratio Assay drop

Ceftriaxone : Mg

Ceftriaxone sodium with magnesium chloride 1 : 1 2.1

Ceftriaxone sodium with magnesium chloride 1 : 0.5 5.0

Ceftriaxone sodium with magnesium chloride 1 : 0.2 6.6

Ceftriaxone sodium with magnesium sulfate 1 : 1 1.2

Ceftriaxone sodium with magnesium 1 : 1 1.6 trifluoromethansulfonate

Ceftriaxone sodium with magnesium 1 : 1 2.3 acetylacetonate

Ceftriaxone sodium with tri-magnesium dicitrate 1 : 0.95 3.7

Control 7.0

Ceftriaxone sodium in WFI

As clearly seen from the results all compositions comprising magnesium showed less drop of ceftriaxone after 60 days at 2-8°C than the control without magnesium. Table 3: Stability of 20 mg/ml ceftriaxone with magnesium salts after 90 days at 2- 8°C represented as assay drop of ceftriaxone

Formulation Molar ratio Assay drop Ceftriaxone : Mg

Ceftriaxone sodium with magnesium chloride 1 : 0.5 6.1

Control Solution

Ceftriaxone sodium in WFI precipitated

The control composition had precipitated after 90 days. Because the precipitation of the control composition prevented comparison of the magnesium chloride with a control composition after 90 days, a comparison was made to a control composition after 60 days in a different experiment. While from different experiments, the results in Table 2 and 3 that a composition comprising ceftriaxone to magnesium in a molar ratio of 1:0.5 showed less assay drop of ceftriaxone after 90 days at 2-8°C than the control without magnesium after 60 days at 2-8°C.

Table 4: Stability of 20 mg/ml ceftriaxone with magnesium chloride after 180 days at 2-8°C represented as assay drop of ceftriaxone and increase of Impurity C

Formulation Molar ratio EP impurity C (increase Assay drop

Ceftriaxone: Mg from start) % %

Ceftriaxone sodium with 1 : 1 0.78 3.9

MgCI2

Control 2.94 16.1

Ceftriaxone sodium in WFI As clearly seen from the results the composition comprising magnesium showed less drop of ceftriaxone and less formation of Impurity C after 180 days at 2-8°C than the control without magnesium.

Table 5: Stability of 20 mg/ml ceftriaxone formulated with magnesium after 270 days at 2-8°C represented as assay drop of ceftriaxone and increase of Impurity C

Formulation Molar ratio EP impurity C (increase Assay drop

Ceftriaxone: Mg from start) % %

Ceftriaxone sodium with 1 : 1 1.47 7.9

MgCI2 initially adjusted to pH

7.5 Ceftriaxone sodium with 1 : 1 1.61 8.4

MgCI2 initially adjusted to pH

8.5

As seen from the results the compositions comprising magnesium showed a drop of ceftriaxone of about 8% after 270 days at 2-8°C. Table 6: Stability of 20 mg/ml ceftriaxone with magnesium salt after 14 days at 15°C represented as assay drop of ceftriaxone

Formulation Molar ratio Assay drop

Ceftriaxone : Mg

Ceftriaxone sodium with magnesium chloride 1 : 1 5.5

Ceftriaxone sodium with magnesium chloride 1 : 0.8 6.3

Ceftriaxone sodium with magnesium 1 : 1 4.2 trifluoromethanesulfonate

Ceftriaxone sodium with magnesium sulfate 1 : 1 4.3

Ceftriaxone sodium with magnesium acetylacetonate 1 : 1 4.4

Ceftriaxone sodium with magnesium tri-magnesium 1 : 0.95 6.8 dicitrate

Ceftriaxone sodium with magnesium fumarate 1 : 1 4.7

Ceftriaxone sodium with magnesium succinate 1 : 1 5.2

Ceftriaxone sodium with magnesium maleate 1 : 1 5.0

Ceftriaxone with magnesium perchlorate 1 : 1.5 5.2

Ceftriaxone with magnesium perchlorate 1 : 1 5.6

Ceftriaxone with magnesium nitrate 1 : 1 6.2

Control 7.1

Ceftriaxone sodium in WFI As clearly seen from the results all compositions comprising a magnesium salt showed less drop of ceftriaxone after 14 days at 15°C than the control without magnesium.

Table 7: Stability of 20 mg/ml ceftriaxone with magnesium salt after 7 days at 25°C represented as assay drop of ceftriaxone

Formulation Molar ratio Assay drop

Ceftriaxone : Mg

Ceftriaxone sodium with magnesium chloride 1 : 1 10.3

Ceftriaxone sodium with magnesium chloride 1 : 2 10.0

Control 12.2

Ceftriaxone sodium in WFI

As clearly seen from the results all compositions comprising a magnesium salt showed less drop of ceftriaxone after 7 days at 25°C than the control without magnesium. Example 2: Stability of ceftriaxone compositions under inert atmosphere

To demonstrate the impact of manufacturing the compositions in inert atmosphere (keeping the dissolved oxygen level below 2.0 ppm by the aid of one or more inert gasses) on chemical and physical stability of ceftriaxone, compositions were produced in inert and regular atmosphere according to the Experimental section above. By regular atmosphere is understood the natural occurring atmosphere having an oxygen content of approximately 21% and a nitrogen content of approximately 78%. Obtained results are presented in Figure 1 and Tables 8 and 9 below.

Figure 1 shows a comparison of 20 mg/ml ceftriaxone in Water for Injection under regular and inert atmosphere, respectively, after storage for 120 days at 2-8°C and clearly illustrates the effect of no or significantly reduced oxygen in the ceftriaxone composition on color and particle formation. Vials 1 -4 were manufactured under inert atmosphere (as described above and in additional preparation was performed in nitrogen cabinet leading to a very low level of dissolved oxygen in the compostions) while vials 5-10 were manufactured under regular atmosphere. The color of the compositions in vials 1-4 were slightly orange and free of visible particles, while the color of the compositions in vials 5- 10 were orange and with a precipitate (visible particles). Table 8 shows a comparison of 20 mg/ml ceftriaxone manufactured with an inert and regular atmosphere with and without magnesium. The compositions manufactured with an inert atmosphere were not prepared in a nitrogen cabinet. Table 8: Stability of 20 mg/ml ceftriaxone with/without magnesium and stored in inert or regular atmosphere after 60 days at 2-8°C represented as assay drop of ceftriaxone, increase of Impurity C and color

Formulation Molar ratio EP impurity C Assay Color

Ceftriaxone: Mg (increase from drop start) % %

Ceftriaxone sodium with 1 : 0.6 0.28 1.9 Slightly orange MgS04 x 7H20 @ pH 6.8 (inert atmosphere)

Control 1 : 0.6 0.36 2.9 Light orange

Ceftriaxone sodium with MgCI2 (regular atmosphere)

Control 1.14 6.5 Light orange

Ceftriaxone sodium in WFI in inert atmosphere (average of 4)

Control 1.06 6.1 Orange

Ceftriaxone sodium in WFI average of 3

(regular atmosphere) _

* All compositions were almost colorless before storage. The results show that the manufacturing under inert atmosphere has an impact on color and accordingly, has an impact on the physical stability of the composition. It also shows that magnesium has a positive effect on the chemical stability of ceftriaxone as the assay drop and the formation of Impurity C are both reduced in the compositions comprising magnesium as compared to the compositions comprising only ceftriaxone. Furthermore, it also illustrates that removing or significantly reducing oxygen in a composition comprising ceftriaxone without magnesium will lead to a reduction in color change, but will not impact the chemical stability as represented by assay drop or increase in formation of impurities. The results also illustrates that the assay drop of ceftriaxone and formation of Impurity C is reduced in the composition comprising ceftriaxone and magnesium and manufactured under inert atmosphere, as compared to a similar composition produced under regular atmosphere. Accordingly, a composition of ceftriaxone comprising magnesium having no or a significantly reduced amount of oxygen are having both a better chemical and physical stability, as compared to a composition with magnesium in which oxygen has not been removed or significantly reduced.

The results in Table 9 shows a comparison of 20 mg/ml ceftriaxone composition with magnesium produced under inert atmosphere to a 20 mg/ml ceftriaxone composition produced under regular atmosphere at 120 days:

Table 9: Stability of 20 mg/ml ceftriaxone formulated with magnesium under regular atmosphere versus inert atmosphere after 120 days at 2-8°C represented as assay drop of ceftriaxone, increase of Impurity C, color and visible particles

Formulation Molar ratio EP impurity C Assay Visible Color

Ceftriaxone: (increase from drop particles

Mg start) % %

Ceftriaxone sodium with 1 : 1 0.46 2.5 None Slightly

MgCI2 (inert atm.) observed orange

Ceftriaxone sodium with 1 : 1 0.62 3.8 After Orange

MgCI2 (regular atm.) app. 90 days

The results confirm that the color change of a ceftriaxone composition comprising magnesium reduced when manufactured in a way, so the amount of oxygen is removed or significantly reduced. It also illustrates that manufacturing under substantially oxygen-free conditions will have a positive effect on the formation of visible particles as no particles are formed after 120 days. Furthermore, the results show that the assay drop, and formation of Impurity C is reduced in the composition comprising ceftriaxone and magnesium and manufactured under inert atmosphere, as compared to the similar composition produced under regular atmosphere. Accordingly, a composition of ceftriaxone comprising magnesium having no or a significantly reduced amount of oxygen are having both a better chemical and physical stability, as compared to a composition with magnesium in which oxygen has not been removed or significantly reduced.

Table 10: Stability of 20 mg/ml and 100 mg/ml ceftriaxone with magnesium under inert atmosphere after 21 days at 15°C represented as assay drop of ceftriaxone and increase of Impurity C

Formulation Concentration Molar ratio EP impurity C Assay drop

(mg/ml) Ceftriaxone: (increase from start) %

Mg %

Ceftriaxone sodium with 20 1 : 0.6 1.26 7.5

MgS04 x 7H20 Ceftriaxone sodium with 100 1 : 0.6 0.78 4.9

MgS04 x 7H20

The result of Table 10 illustrates the stability of 20 mg/ml and 100 mg/ml ceftriaxone compositions respectively.

Example 3: Antimicrobial susceptibility testing

In order to determine whether the addition of magnesium to a composition comprising ceftriaxone would have any impact on the activity of ceftriaxone, antimicrobial susceptibility testing was performed.

The activity of ceftriaxone compositions against a panel of four representative bacterial strains including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae was measured.

Test was performed using broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Output of the method is minimal inhibitory concentration (MIC), defined as the lowest concentration of the tested compound which inhibits visible growth after an overnight incubation.

Inoculum was prepared by resuspending several colonies from 18-24 h old Mueller Hinton agar plate (for S. pneumoniae Muller Hinton agar with 5% blood sheep was used) in sterile 0.9% saline in order to obtain suspension with turbidity equivalent to 0.5 McFarland. The bacterial suspension prepared in this manner was then diluted 100x in appropriate test medium and added to 96-well plate with diluted compounds.

Test items were dissolved in water to obtain initial stock solution and were further diluted in test medium, cation adjusted Mueller Hinton broth. For Streptococcus pneumoniae cation adjusted Mueller Hinton broth with 5% lysed horse blood was used. Testing was performed in 10 consecutive 2-fold dilutions (from 32 to 0.0625 pg/mL) in duplicates, in parallel with Ceftriaxone reference standard as pharmacological control.

Minimum inhibitory concentration (MIC) was determined as the lowest concentration of compound that inhibits the visible growth of a microorganism after 18-24h incubation at 37°C. Table 11: Minimum inhibitory concentration (MIC) of various ceftriaxone compositions As clearly seen from Table 11 , activity of the tested ceftriaxone formulations against representative bacterial strains was found to be equivalent to ceftriaxone USP reference standard which was used as pharmacological control. Results clearly indicate that magnesium added to composition do not have any negative impact on ceftriaxone efficacy.