# Patent

## Title
Ceftolozane antibiotic compositions

## Abstract

This disclosure provides pharmaceutical compositions comprising ceftolozane, pharmaceutical compositions comprising ceftolozane and tazobactam, methods of preparing those compositions, and related methods and uses of these compositions.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 62/051,859, filed Sep. 17, 2014 and is a continuation-in-part application of U.S. application Ser. No. 14/251,381, filed Apr. 11, 2014, which is a continuation application of International Application No. PCT/US2014/028642, filed Mar. 14, 2014, which claims priority to U.S. Provisional Application No. 61/792,092, filed Mar. 15, 2013; U.S. Provisional Application No. 61/793,007, filed Mar. 15, 2013; U.S. Provisional Application No. 61/882,936, filed Sep. 26, 2013; and U.S. Provisional Application No. 61/893,436, filed Oct. 21, 2013. The contents of these applications are incorporated hereby by reference in their entirety.


TECHNICAL FIELD
This disclosure relates to pharmaceutical compositions comprising ceftolozane, pharmaceutical compositions comprising tazobactam and ceftolozane, methods of preparing those compositions, and related methods and uses thereof.
BACKGROUND
Ceftolozane is a cephalosporin antibacterial agent. The antibacterial activity of ceftolozane is believed to result from its interaction with penicillin binding proteins (PBPs) to inhibit the biosynthesis of the bacterial cell wall which acts to stop bacterial replication. Ceftolozane is also referred to as “CXA-101”, FR264205, (6R,7R)-3-[(5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-1-methyl-1H-pyrazol-2-ium-2-yl)methyl]-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, or (6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureido]-1-methyl-1H-pyrazol-2-ium-2-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(Z)-1-carboxy-1-methylethoxyimino]acetamido]-3-cephem-4-carboxylic acid). As used herein, the term “ceftolozane” means (6R,7R)-3-[(5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-1-methyl-1H-pyrazol-2-ium-2-yl)methyl]-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate or (6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureido]-1-methyl-1H-pyrazol-2-ium-2-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(Z)-1-carboxy-1-methylethoxyimino]acetamido]-3-cephem-4-carboxylic acid in its free-base or salt form, including a sulfate form. Unless otherwise indicated, the term “CXA-101” as used herein can refer to ceftolozane in any pharmaceutically acceptable form, e.g., ceftolozane in its free-base or salt form, including a ceftolozane sulfate salt form. Ceftolozane sulfate is a pharmaceutically acceptable salt of ceftolozane that can be combined with sodium chloride and other components to obtain an antibiotic composition suitable for administration by injection or infusion.
Antibacterial pharmaceutical compositions can include ceftolozane as a pharmaceutically acceptable salt formulated for intravenous administration. Ceftolozane sulfate is a pharmaceutically acceptable ceftolozane salt of formula (I) that can be formulated for intravenous administration or infusion.




U.S. Pat. No. 7,129,232 discloses ceftolozane and various ceftolozane salts. For example, a ceftolozane hydrogen sulfate salt is disclosed among ceftolozane salts that can be formed “with a base or an acid addition salt such as a salt with an inorganic base, for example, an alkali metal salt [e.g., sodium salt, potassium salt, etc.], an alkaline earth metal salt [e.g., calcium salt, magnesium salt, etc.], an ammonium salt; a salt with an organic base, for example, an organic amine salt [e.g., trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N′-dibenzylethylenediamine salt, etc.]; an inorganic acid addition salt [e.g., hydrochloride, hydrobromide, sulfate, hydrogen sulfate, phosphate, etc.]; an organic carboxylic or sulfonic acid addition salt [e.g., formate, acetate, trifluoroacetate, maleate, tartrate, citrate, fumarate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.]; and a salt with a basic or acidic amino acid [e.g., arginine, aspartic acid, glutamic acid, etc.].”
Antibiotic pharmaceutical compositions comprising a beta-lactam antibiotic compound (e.g., a cephalosporin) (i.e., an antibiotic compound possessing one or more beta-lactam moieties) can be administered with a beta-lactamase inhibitor (BLI) compound. For example, beta-lactam antibiotic compounds such as ceftolozane or other cephalosporin antibiotic compounds can be formulated with, and/or administered in combination with beta-lactamase inhibiting compounds (e.g., tazobactam and salts thereof) in order to mitigate the effects of bacterial beta-lactamase enzymes that can lead to bacterial resistance to antibiotic therapy. Tazobactam is a BLI compound approved for use in fixed dose combination with piperacillin in an injectable antibacterial product available under commercial names ZOSYN (U.S.) and TAZOCIN (e.g., in Canada, and the United Kingdom). Tazobactam sodium, a derivative of the penicillin nucleus, is a penicillanic acid sulfone having the chemical name sodium (2S,3S,5R)-3-methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylate-4,4-dioxide. The chemical formula is C10H11N4NaO5S and the molecular weight is 322.3. The chemical structure of tazobactam sodium is:




Ceftolozane can be formulated with tazobactam in antibiotic compositions called CXA-201 (ceftolozane/tazobactam for injection), comprising ceftolozane and tazobactam in a 2:1 weight ratio between the amount of ceftolozane active and the amount of tazobactam acid, regardless of the salt forms of these compositions (e.g., 1,000 mg of ceftolozane active can be included in about 1,147 mg of ceftolozane sulfate). CXA-201 compositions include an amount of tazobactam in a pharmaceutically acceptable form providing 500 mg of tazobactam acid per 1,000 mg of ceftolozane active as a composition formulated for injection, or for reconstitution prior to parenteral administration. In one product presentation, CXA-201 can be provided in a single container comprising ceftolozane sulfate and tazobactam sodium, administered by reconstituting a container-unit dosage form container of solid CXA-201 to form a reconstituted injectable formulation. In one presentation (e.g., for treatment of certain urinary tract infections and/or certain intra-abdominal infections), each unit dosage form container of CXA-201 can contain 1000 mg of ceftolozane active (free base equivalent weight, e.g., provided as a pharmaceutically acceptable salt such as ceftolozane sulfate) and sterile tazobactam sodium at a quantity equivalent of 500 mg of tazobactam free acid, in a solid form. In another presentation (e.g., for treatment of hospital acquired/ventilator-associated bacterial pneumonia (HABP/VABP)), a CXA-201 product can include a unit dosage form container providing 2,000 mg of ceftolozane active (e.g., as an equivalent amount of ceftolozane sulfate) and 1,000 mg of tazobactam acid (e.g., as an equivalent amount of tazobactam sodium). CXA-201 compositions display potent antibacterial activity against various gram-negative infections such as, for example, complicated intra-abdominal infection (cIAI), complicated urinary tract infection (cUTI), or hospital acquired/ventilator-associated bacterial pneumonia (HABP/VABP).
As disclosed herein, ceftolozane was initially found to be chemically unstable in certain lyophilized compositions evaluated during the development of CXA-101 and CXA-201 pharmaceutical compositions. For example, ceftolozane had a residual rate of about 51% in the absence of a stabilizing agent during both a 3 day stability test at 70 degrees C., indicating loss of almost half of the ceftolozane during the test (Example 2, Table 2A control sample), and a 5.88% reduction in ceftolozane purity during a 7 day stability test at 60 degrees C. in the absence of a stabilizing agent (Example 2, Table 2B control sample). Second, the formation of a number of additional ceftolozane degradation products formed during the preparation of initial compositions was observed by additional peaks using high performance liquid chromatography (HPLC) during stability tests of ceftolozane alone (e.g., Peak P12 in Table 4 of Example 3, and the RT63′ peak in Table 15 of Example 8), and testing of compositions with tazobactam and ceftolozane formed by co-lyophilization of ceftolozane and tazobactam (e.g., RRT 1.22 peak in Tables 12 and 13 of Example 7a). Accordingly, there remains an unmet need to identify formulations and manufacturing methods that effectively stabilize ceftolozane both in a solid and liquid form to provide suitably stable pharmaceutical compositions comprising ceftolozane and tazobactam (both in a powder form for reconstitution and in a reconstituted form for parenteral delivery). These formulations should address the need to provide pharmaceutical compositions having desired levels of ceftolozane and tazobactam potency, as well as levels of impurities that are therapeutically acceptable for parenteral administration.
SUMMARY
As provided herein, ceftolozane can be stabilized in pharmaceutical composition comprising ceftolozane and a stabilizing effective amount of a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose, maltose, trehalose and sucrose. The pharmaceutical compositions provided herein are based in part on the surprising discovery that ceftolozane pharmaceutical compositions comprising these stabilizing agents demonstrate improved ceftolozane residual rates (e.g., % ceftolozane remaining after 3 days at 70° C. as measured by HPLC) and/or chemical stability (e.g., lower reduction in ceftolozane purity measured by HPLC after 7 days at 60° C. in a stability test) compared control samples comprising ceftolozane without a stabilizing agent.
Accordingly, preferred pharmaceutical antibiotic compositions can include ceftolozane sulfate and a stabilizing agent (e.g., 300 to 500 mg of a stabilizing agent per 1,000 mg ceftolozane active) in a lyophilized unit dosage form (e.g., powder in a container). The unit dosage form can be dissolved with a pharmaceutically acceptable carrier (e.g., 0.9% sodium chloride aqueous isotonic saline and/or water for injection), and then intravenously administered. In certain ceftolozane compositions, the stabilizing agent can be selected from the group consisting of: sodium chloride, lactose, maltose and dextran 40, and/or selected from the group consisting of: sodium chloride, trehalose and sucrose.
In addition, the present disclosure provides ceftolozane pharmaceutical compositions based in part on the surprising discovery that ceftolozane pharmaceutical compositions comprising about 1000 mg of ceftolozane active per 189 mg sodium from sodium chloride demonstrate improved chemical stability and purity compared with pharmaceutical compositions comprising ceftolozane with comparatively less sodium chloride. For example, the invention is based in part on the discovery of the absence of the “RT63 Impurity” (also referred to herein as “Formula III”) in HPLC analysis of pharmaceutical compositions comprising about 1,000 mg of ceftolozane and 189 mg sodium from sodium chloride. By comparison, reducing the amount of sodium chloride relative to ceftolozane in tested compositions resulted in at least 1.5-fold greater impurity at RT=63 minutes (observed by HPLC using the method described in Example 1). The ceftolozane formulations with reduced levels of sodium were not as stable as the ceftolozane formulation containing about 1,000 mg of ceftolozane per 189 mg sodium from sodium chloride per. Ceftolozane formulations containing about 1,000 mg of ceftolozane effective per stabilizing-effective amount of sodium from sodium chloride maintained the level of RT63 Impurity below the detection limit (e.g., 0.03%) measured by HPLC using the method described in Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric Acid:



Acetonitrile, 4:1 (v/v)













Gradient Profile
Time (min.)
% A
% B






0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









In a further embodiment provided herein, ceftolozane sulfate is stabilized in pharmaceutical compositions by incorporation of an effective amount of an inorganic salt stabilizing agent, in particular 125 to 500 mg (e.g., 480 to 500 mg) of sodium chloride per gram of ceftolozane active. This is based in part on the surprising discovery that ceftolozane pharmaceutical compositions comprising 125 to 500 mg (e.g., 480 to 500 mg) of sodium chloride per 1000 mg of ceftolozane active demonstrate improved ceftolozane purity and chemical stability compared to pharmaceutical compositions comprising ceftolozane with comparatively less sodium chloride. For example, the disclosed pharmaceutical compositions have an improved stability as a decrease in the rate of ceftolozane purity and/or a decrease in the rate of formation of substances characterized by HPLC peaks 1 and 7 identified during a 7-day stability study in Example 5. The disclosed ceftolozane pharmaceutical compositions comprise a stabilizing amount of sodium chloride (e.g., 125 to 500 mg of sodium chloride [more specifically, 480 to 500 mg] per 1000 mg of ceftolozane active). Certain preferred compositions demonstrate improved ceftolozane purity (e.g., Table 6) and chemical stability (e.g., with respect to the composition of HPLC peak 1 in Table 7) compared with pharmaceutical compositions comprising ceftolozane with comparatively less sodium chloride. For example, the disclosed pharmaceutical compositions typically comprise less than about 4% total impurity after being stored for seven days at 60° C., as determined by HPLC using the method described in Example 1. Alternatively, the disclosed pharmaceutical compositions comprise less than about 2% of the impurity represented by Peak 1 after being stored for seven days at 60° C., as determined by HPLC using the method described in Example 1, where Peak 1 has a retention time relative to ceftolozane of 0.1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









In an embodiment, pharmaceutical antibiotic compositions provided herein can include ceftolozane sulfate and stabilizing amount of sodium chloride (e.g., 125 to 500 mg more specifically 480 to 500 mg of sodium chloride and 1,000 mg ceftolozane active) in a unit dosage form (e.g., powder in a container). The unit dosage form can be dissolved with a pharmaceutically acceptable carrier, and then intravenously administered.
In another aspect, provided herein is a pharmaceutical composition comprising 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozane active, wherein the decrease in ceftolozane total purity is not greater than about 4% after storing the pharmaceutical composition for seven days in a sealed container at 60° C., as determined by HPLC using the method described in Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









In another aspect, provided herein is a pharmaceutical composition comprising 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozane active, wherein the increase in the amount of the impurity represented by Peak 1 is not greater than about 2% after storing the pharmaceutical composition for seven days at 60° C., as determined by HPLC using the method described in Example 1, where Peak 1 has a retention time relative to ceftolozane of about 0.1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









In embodiments of these aspects, the pharmaceutical composition further comprises L-arginine, or citric acid. In other embodiments, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the compositions can be in a unit dosage form comprising 125 mg to 500 mg sodium chloride, 1,000 mg of ceftolozane in the form of ceftolozane sulfate, L-arginine and citric acid.
In other embodiments of these aspects, the pharmaceutical composition is lyophilized. In another embodiment, the ceftolozane is ceftolozane sulfate.
In another aspect, provided herein is a unit dosage form injectable pharmaceutical composition comprising 125 mg to 500 mg sodium chloride and 1,000 mg of ceftolozane active present as a composition of formula (I)




In another aspect, provided herein is a pharmaceutical composition comprising 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozane active present as ceftolozane sulfate, wherein the ceftolozane total purity is at least about 94% after storing the pharmaceutical composition for three days at 60° C., as determined by HPLC using the method described in Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









Applicants have further discovered pharmaceutical compositions comprising ceftolozane and tazobactam with reduced and even undetectable amounts of the compound RRT 1.22, and methods of manufacturing these compositions. This is based in part on the discovery that the formation of RRT 1.22 can be reduced if not completely suppressed by lyophilizing ceftolozane in the absence of tazobactam and then blending the lyophilized ceftolozane with a dry tazobactam composition, such as a tazobactam composition lyophilized in the absence of ceftolozane (See Example 10 and the results reported in Tables 23 and 24). Based on these results, pharmaceutical compositions comprising ceftolozane and tazobactam, and pharmaceutical compositions prepared using ceftolozane and tazobactam are provided herein. In particular, these pharmaceutical compositions can include ceftolozane and/or tazobactam with reduced or even undetectable amounts of the compound RRT 1.22:




In one embodiment, a pharmaceutical composition can include ceftolozane and tazobactam with less than 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC or even undetectable amounts of RRT 1.22 (e.g., less than about 0.03% of the compound RRT 1.22 measured by HPLC). These pharmaceutical compositions can be obtained by a process comprising the steps of (a) lyophilizing ceftolozane in the absence of tazobactam to obtain a lyophilized ceftolozane composition; and (b) combining the lyophilized ceftolozane with tazobactam under conditions suitable to obtain said pharmaceutical composition with the aforementioned purity levels. The combination of the lyophilized ceftolozane composition with tazobactam can include blending the lyophilized ceftolozane composition with lyophilized or crystalline tazobactam material.
Also provided herein is a pharmaceutical composition comprising a blend of separately lyophilized tazobactam and ceftolozane sulfate in an amount providing 1,000 mg of ceftolozane active per 500 mg of tazobactam active, further comprising less than 0.15%, 0.10%, 0.05% or 0.03% by weight; from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC; or even undetectable amounts (e.g., less than about 0.03% by HPLC) of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography (HPLC) using the method of Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









CXA-201 compositions comprising less than about 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC of the compound of formula (III) can be obtained by a process comprising the steps of: (a) forming a first aqueous solution comprising ceftolozane (e.g., in a pharmaceutically acceptable salt such as formula (I)), (b) lyophilizing the first aqueous solution to obtain a lyophilized ceftolozane composition, and (c) blending the lyophilized ceftolozane composition with a tazobactam composition (e.g., tazobactam acid lyophilized in the absence of ceftolozane) in an amount that provides a 2:1 weight ratio between the amount of ceftolozane active and tazobactam active.
In yet another aspect, provided herein is a method for the treatment of a bacterial infection in a mammal, comprising administering to said mammal a therapeutically effective amount of any one of the pharmaceutical compositions provided herein. In an embodiment, the bacterial infection is caused by the bacterial infection is caused by bacteria selected from the group consisting of: Staphylococcus aureus, Escherichia coli, Acinetobacter baumanii, Haemophilus influenzae, Klebsiella pneumonia, and Pseudomonas aeruginosa. In another embodiment, the bacterial infection is selected from the group consisting of nosocomial pneumonia, complicated intra-abdominal infection and complicated urinary tract infection.
In yet another aspect, any of the pharmaceutical compositions provided herein may be used for the manufacture of a medicament for the treatment of complicated intra-abdominal infection (cIAI), complicated urinary tract infection (cUTI), or hospital acquired/ventilator-associated bacterial pneumonia (HABP/VABP).
In still another aspect provided herein, an antibiotic pharmaceutical composition comprises ceftolozane (or a pharmaceutically acceptable salt thereof) and tazobactam (or a pharmaceutically acceptable salt thereof) in a fixed dose combination of 1,000 mg of ceftolozane active per 500 mg of tazobactam active, and a ceftolozane-stabilizing amount of 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozane active.
In a further aspect disclosed herein, a pharmaceutical composition comprising stabilized ceftolozane sulfate is obtained by a process comprising lyophilizing an aqueous solution comprising 125 mg to 500 mg sodium chloride with an amount of ceftolozane sulfate providing 1,000 mg of ceftolozane active, to obtain the lyophilized stabilized ceftolozane sulfate composition.
Yet another aspect provided herein discloses an antibacterial pharmaceutical composition comprising ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of:
a) lyophilizing a first aqueous solution in the absence of tazobactam, the first aqueous solution comprising ceftolozane sulfate prior to lyophilization to obtain a first lyophilized ceftolozane composition; and
b) blending the first lyophilized ceftolozane composition with tazobactam to obtain an antibacterial composition comprising less than 0.13% by HPLC of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using the method described in Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









to detect:








BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a flowchart showing the steps for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a blending process, wherein the ceftolozane and tazobactam are lyophilized separately prior to blending as described herein.
FIG. 2 is a flowchart showing the steps for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a co-lyophilization process, as described herein.
FIG. 3 is a plot of the data points from Table 6, showing the purity of the ceftolozane in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.
FIG. 4 is a plot of the data points from Table 7, showing the peak area of the composition peak 1 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.
FIG. 5 is a plot of the data points from Table 8A, showing the total peak area of the composition with a RRT of 0.43 and the composition peak 3 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.
FIG. 6 is a plot of the data points from Table 8B, showing the peak area of the composition peak 7 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.
FIG. 7 is a plot of the data points from Table 17, showing the purity of ceftolozane in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.
FIG. 8 is a plot of the data points from Table 18, showing the peak area of the composition peak 1 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.
FIG. 9 is a plot of the data points from Table 19, showing the total peak area of the composition with a RRT of 0.43 and the composition peak 3 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.
FIG. 10 is a plot of the data points from Table 20, showing the peak area of the composition peak 7 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.
FIG. 11 is a flowchart showing the manufacturing process for a ceftolozane/tazobactam composition via co-filling.
FIG. 12 is a flowchart showing the process for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a blending process in a dedicated production area according to FDA Guidance.
FIG. 13 is a flowchart showing the process for preparing a ceftolozane/tazobactam composition via co-filling in a dedicated production area according to FDA Guidance.
FIG. 14 shows the mass spectra obtained for the RRT 1.22 compound.
FIG. 15 shows the chemical structures for certain peaks in the spectra in FIG. 14.



DETAILED DESCRIPTION
I. Stabilizing Ceftolozane
Ceftolozane can be stabilized in a pharmaceutical composition comprising ceftolozane and a stabilizing effective amount of a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose, maltose, trehalose and sucrose. The stabilizing agent and the stabilizing effective amount of the stabilizing agent for combination with ceftolozane were determined by high performance liquid chromatography (HPLC) analysis, for example by detecting the ratio of peak areas obtained for ceftolozane compared to peaks for other substances.
Preferred stabilized ceftolozane compositions have a ceftolozane residual rate of greater than the residual rate measured for a comparable ceftolozane composition without the stabilizing agent. Unless otherwise indicated, the residual rate is measured by detecting the amount of ceftolozane in a sample before and after a stability test using HPLC, and determining the percentage of ceftolozane last during the stability test.
Referring to Example 2 (including Table 2A), the residual rate of ceftolozane in the control sample without a stabilizing agent (i.e., 100 mg of ceftolozane) after 3 days at 70 degrees C. was 51.2%, meaning that the HPLC peak area after the stability test for ceftolozane was about 51.2% of the HPLC peak area for ceftolozane at the start of the stability test (i.e., 3 days at 70 degrees C.). Sodium chloride, dextran 40, lactose and maltose all showed higher ceftolozane residual rates than the control in Example 2, while ceftolozane was less stable than the control when combined with fructose, xylitol, sorbitol and glucose (e.g., as evidenced by a residual rate lower than that of the control). In one embodiment, stabilized ceftolozane compositions comprise ceftolozane (e.g., ceftolozane sulfate) and a stabilizing effective amount of a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose and maltose, where the stabilizing effective amount provides a residual rate of at least 51.2% for the ceftolozane in the stabilized ceftolozane composition after 3 days at 70 degrees C. Preferably, the stabilized ceftolozane pharmaceutical compositions after 3 days at 70 degrees C. can comprise at least about 70% of an initial amount of the stabilized ceftolozane in the pharmaceutical composition (i.e., a residual rate of about 70% or greater, as shown in Example 2), where the % of ceftolozane is measured by high performance liquid chromatography (HPLC) according to Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









Referring to Example 2 (Table 2A), stabilized ceftolozane compositions are characterized by a reduction in ceftolozane of less than about 5% after 7 days at 60 degrees C., where the % reduction of ceftolozane is measured by HPLC according to Example 1. The stabilized ceftolozane pharmaceutical composition comprising ceftolozane and a stabilizing agent selected from the group consisting of: sodium chloride, trehalose and sucrose can lose less than 5% of the amount of ceftolozane after 7 days at 60 degrees C., where the % loss of ceftolozane is measured by HPLC according to Example 1. Sodium chloride, trehalose and sucrose all showed reduced reductions in ceftolozane purity after a 7 day stability test at 60 degrees C. (as measured by the % HPLC peak corresponding to ceftolozane before and after the stability test). In one embodiment, stabilized ceftolozane compositions comprise ceftolozane (e.g., ceftolozane sulfate) and a stabilizing effective amount of a stabilizing agent selected from the group consisting of: sodium chloride, trehalose and sucrose, where the stabilizing effective amount provides a reduction in ceftolozane purity of not more than about 5% (e.g., not more than about 4%) for the ceftolozane in the stabilized ceftolozane composition after 3 days at 70 degrees C.
Accordingly, in one aspect, provided herein is a pharmaceutical composition comprising stabilized ceftolozane obtained by a process comprising lyophilizing a composition including ceftolozane and a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose, maltose, trehalose and sucrose, to obtain a lyophilized stabilized ceftolozane pharmaceutical composition. In an embodiment, the stabilizing agent is selected from the group consisting of: sodium chloride, trehalose and sucrose. In another aspect, provided herein is a pharmaceutical composition comprising stabilized ceftolozane and a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose, maltose, trehalose and sucrose, wherein the pharmaceutical composition after 3 days at 70 degrees C. comprises at least about 70% of an initial amount of the stabilized ceftolozane in the pharmaceutical composition.
In another aspect, provided herein is a container containing a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising 1,000 mg of ceftolozane active, L-arginine, citric acid and about 300-500 mg of a stabilizing agent selected from the group consisting of: sodium chloride, trehalose, and sucrose, wherein the pharmaceutical composition after 3 days at 70 degrees C. comprises at least about 70% of an initial amount of the ceftolozane active in the pharmaceutical composition.
Various ceftolozane compositions are described herein. One stabilized ceftolozane composition comprises ceftolozane (e.g., ceftolozane sulfate), L-arginine, citric acid, and a stabilizing agent. Preferably, the stabilized ceftolozane composition comprises 1,000 mg of ceftolozane active, L-arginine and stabilizing-effective amount of the stabilizing agent. The stabilizing effective amount can be readily determined using HPLC and a stability test as disclosed herein. The stabilizing-effective amount can be effective to provide: (1) a residual rate measured by HPLC of ceftolozane of at least about 51.2% (including, e.g., at least about 70%, and at least about 80%) after 3 days at 70 degrees C. and/or (2) a reduction in ceftolozane purity measured by HPLC of not more than about 5.11% (including, e.g., reductions of not more than about 5%, or 4%) after 7 days at 60 degrees C. Examples of stabilizing effective amounts include 100 mg-500 mg of the stabilizing agent per 1,000 mg of the ceftolozane active, more preferably about 300-500 mg of the stabilizing agent per 1,000 mg of the ceftolozane active.
In the screening of ceftolozane stabilizing agents, it has been found that, surprisingly, a preferred amount of sodium chloride can improve the stability of ceftolozane, including ceftolozane in the ceftolozane sulfate form. For example, in one experiment, a ceftolozane composition comprising about 100 mg (about 1.71 mmol) sodium chloride per 100 mg (about 0.15 mmol) of ceftolozane was more stable compared to many ceftolozane compositions comprising known stabilizing sugars, such as fructose, xylitol, sorbitol, glucose, and D-mannitol, and as stable as other ceftolozane compositions comprising the same amount of certain sugars, such as dextran 40, lactose, and maltose (see Example 2). Interestingly, additional experiments demonstrated that the use of maltose in a ceftolozane composition resulted in a significant amount of additional compounds (see Example 3).
Surprisingly, pharmaceutical compositions comprising ceftolozane and 125 to 1000 mg sodium chloride per 1000 mg of ceftolozane have been observed to exhibit better chemical stability over the course of time and/or in the presence of heat, and fewer additional compounds than those pharmaceutical compositions comprising ceftolozane and less sodium chloride (i.e., less than 125 mg sodium chloride per 1000 mg of ceftolozane) (see, e.g., Example 5). In particular embodiments described herein, the pharmaceutical compositions comprising ceftolozane and 125 to 500 mg sodium chloride per 1000 mg of ceftolozane have been found to be more stable than the compositions comprising ceftolozane and less than 125 mg sodium chloride per 1000 mg of ceftolozane.
Ceftolozane compositions having 50-481 mg of sodium chloride per 1,000 mg ceftolozane active were prepared as described in Table 5 and tested for stability as described in Example 5. Ceftolozane was more stable in compositions containing at least 125 mg of sodium chloride per 1,000 mg of ceftolozane active, as measured by high performance liquid chromatography (HPLC) analysis by detecting the ratio of peak areas obtained for ceftolozane compared to peaks for other substances. Unless otherwise indicated, HPLC measurements reported herein are obtained using the conditions described in Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









During the stability test of Example 5, ceftolozane samples containing 125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozane active showed a decrease in ceftolozane total purity measured by HPLC that was at least about 35% less than reductions in ceftolozane total purity observed for formulations containing 50 mg or 75 mg sodium chloride per 1,000 mg ceftolozane active. Thus, ceftolozane compositions having at least 125 mg or more sodium chloride relative to the fixed amount of ceftolozane were about 35-90% more stable than comparable ceftolozane compositions having less than 125 mg sodium chloride (e.g., the % decrease in ceftolozane for the sample containing 75 mg sodium chloride was about 35% greater than the comparable % decrease in ceftolozane for the sample containing 190 mg sodium chloride). In addition, samples obtained from ceftolozane compositions containing 125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozane active showed a decrease in ceftolozane that was up to about 90% less than reductions in ceftolozane observed for formulations containing 50 mg or 75 mg sodium chloride per (e.g., the % decrease in ceftolozane for the sample containing 50 mg sodium chloride was about 90% greater than the comparable % decrease in ceftolozane for the sample containing 481 mg sodium chloride).
The ceftolozane sodium-stabilized compositions having 125 mg or more sodium chloride relative to the fixed amount of 1,000 mg ceftolozane active also had lower quantities of additional substances identified by peaks 1 and 7 having characteristic retention times measured by HPLC (see Table 1, indicating retention times of about 0.1 for peak 1 and about 1.3 for peak 7 relative to ceftolozane measured according to the HPLC method of Example 1). In particular, these sodium chloride stabilized ceftolozane compositions were characterized by about 37-94% less of the material of peak 1 and about 38-306% less of the material of peak 7 (measured by corresponding HPLC peak areas) than comparable ceftolozane compositions having less than 125 mg sodium chloride (e.g., see 7-day stability study in Example 5). Referring to the data in Table 7 (FIG. 4), the amount of the composition of peak 1 (measured by HPLC according to Example 1) was measured by the % increase in the peak 1 HPLC peak during the 7-day stability test of Example 5.
In particular, samples containing 125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozane active showed at least a 37% reduction in the amount of the peak 1 composition observed for these formulations containing at least 125 mg sodium chloride per 1,000 mg ceftolozane active, compared to the compositions with 50 mg or 75 mg sodium chloride per 1,000 mg of ceftolozane active (e.g., the % increase in peak 1 for the sample containing 75 mg sodium chloride was about 37% greater than the comparable % decrease in ceftolozane for the sample containing 190 mg sodium chloride). In addition, compositions containing 125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozane active showed up to a 94% reduction in the amount of the peak 1 composition observed for these formulations containing at least 125 mg sodium chloride per 1,000 mg ceftolozane active, compared to the compositions with 50 mg or 75 mg sodium chloride per 1,000 mg of ceftolozane active (e.g., the % increase in peak 1 for the sample containing 50 mg sodium chloride was about 94% greater than the comparable % decrease in ceftolozane for the sample containing 481 mg sodium chloride).
The formulation of pharmaceutical compositions can be selected to minimize decomposition of the constituent drug substances and to produce a composition that is stable under a variety of storage conditions.
Provided herein are pharmaceutical compositions useful for the treatment of bacterial infections comprising ceftolozane and sodium chloride, wherein the sodium chloride is present in an amount sufficient to stabilize the ceftolozane. Also provided herein are pharmaceutical compositions comprising ceftolozane, tazobactam, and sodium chloride, wherein the sodium chloride is present in an amount sufficient to stabilize the ceftolozane. Advantageously, these pharmaceutical compositions have fewer additional compounds and are more chemically stable, and can therefore be stored for longer periods of time.
In one embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and 125 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 75% or greater after 3 days at 70° C. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and about 487 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 75% or greater after 3 days at 70° C. In certain embodiments, the purity of the ceftolozane in the composition is 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or 99% or greater after 3 days at 70° C.
In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and 125 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 94.8% or greater after 3 days at 60° C. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and about 487 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 94.8% or greater after 3 days at 60° C. In certain embodiments, the purity of the ceftolozane in the composition is 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater after 3 days at 60° C.
In still another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and 125 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition decreases by 3.1% or less after 3 days at 60° C. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and about 487 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition decreases by 3.1% or less after 3 days at 60° C. In certain embodiments, the purity of the ceftolozane in the composition decreases by 3.0% or less, 2.5% or less, 2.0% or less, 1.5% or less, or 1% or less after 3 days at 60° C.
In another aspect, provided herein is a pharmaceutical composition comprising about 1,000 mg of ceftolozane active per 189 mg sodium from sodium chloride, and not more than 0.03% by high performance liquid chromatography (HPLC) of a RT63 Impurity at a retention time of about 63 minutes observed by HPLC using the method described in Example 1, the pharmaceutical composition obtained by a process comprising the step of lyophilizing an aqueous solution comprising 189 mg sodium from sodium chloride per 1,000 mg of ceftolozane active in the form of ceftolozane sulfate to obtain a lyophilized ceftolozane composition, and formulating the pharmaceutical composition from the lyophilized ceftolozane composition.
In one embodiment, the pharmaceutical composition comprises a total of 1,000 mg of ceftolozane active. In another aspect, provided herein is a pharmaceutical composition obtained by a process comprising the step of lyophilizing an aqueous solution comprising 189 mg sodium from sodium chloride per 1,000 mg of ceftolozane in the form of ceftolozane sulfate to obtain a lyophilized ceftolozane composition.
In one embodiment, the pH of the aqueous solution is 5.0 to 7.0, e.g., 6.0 to 7.0, and the aqueous solution further comprises L-arginine. In another embodiment, the pharmaceutical composition is formulated for parenteral administration and further comprises citric acid. In another embodiment, the composition is a unit dosage form in a container comprising tazobactam and 189 mg sodium from sodium chloride per 1,000 mg of ceftolozane active in the form of ceftolozane sulfate.
In another embodiment, the aqueous solution further comprises L-arginine and citric acid; the pH of the aqueous solution is 6.0 to 7.0 prior to lyophilization; and the pharmaceutical composition further comprises tazobactam blended with the lyophilized ceftolozane composition.
In still another aspect, provided herein is a container containing a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising 189 mg sodium from sodium chloride, and 1,000 mg ceftolozane active in the form of ceftolozane sulfate.
In one embodiment, the container comprises the ceftolozane sulfate, tazobactam and the sodium chloride and not more than 0.03% by high performance liquid chromatography (HPLC) of a RT63 Impurity at a retention time of about 63 minutes observed by HPLC using the method described in Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









Typically, antibiotic compositions do not contain sodium chloride or contain only a small amount of sodium chloride. For example, MAXIPIME®, which is approved for pneumonia, empiric therapy for febrile neutropenic patients, uncomplicated and complicated urinary tract infections, uncomplicated skin and skin structure infections, and complicated intra-abdominal infections, is a dry mixture of cefepime hydrochloride and L-arginine, wherein the mixture does not contain sodium chloride. CEFAZOLIN® for injection, which is approved for respiratory tract infections, urinary tract infections, skin and skin structure infections, biliary tract infections, bone and joint infections, genital infections, septicemia, and endocarditis and perioperative prophylaxis, comprises lyophilized cefazolin sodium that does not contain additional sodium salt. Furthermore, ROCEPHIN®, which is approved for lower respiratory tract infections, acute bacterial otitis media, skin and skin structure infections, urinary tract infections, uncomplicated gonorrhea, pelvic inflammatory disease, bacterial septicemia, bone and joint infections, intra-abdominal infections, meningitis, and surgical prophylaxis, comprises ceftriaxone sodium that only comprises 13.5 mg of free sodium per 1000 mg of ceftriaxone sodium, which equals about 34 mg sodium chloride per 1000 mg of ceftriaxone sodium if the free sodium is in sodium chloride form. In contrast, the pharmaceutical compositions provided herein (compositions comprising ceftolozane and sodium chloride, and compositions comprising ceftolozane, tazobactam, and sodium chloride), have high amounts of sodium chloride, e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane.
Ceftolozane
The compound 5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-2-{[(6R,7R)-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl}-1-methyl-1H-pyrazolium monosulfate (also known also as ceftolozane sulfate, FR264205, “CXA-101”) is a cephalosporin compound (shown below), the synthesis of which is described in U.S. Pat. No. 7,129,232, wherein the compound is also named 7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(1-carboxy-1-methylethoxyimino)acetamido]-3-{3-amino-4-[3-(2-aminoethyl)ureido]-2-methyl-1-pyrazolio}methyl-3-cephem-4-carboxylate. Ceftolozane has the chemical formula below and CAS registry number 689293-68-3. “Ceftolozane” can be provided as the salt, ceftolozane sulfate.




Unless otherwise indicated herein, the phrase “1000 mg ceftolozane” or “1 g ceftolozane” refers to an amount of ceftolozane containing the free base equivalent weight of ceftolozane provided in the free base form or any suitable salt form, as appropriate. For example, a composition containing 1000 mg of ceftolozane in the ceftolozane sulfate solid form will include greater than 1000 mg of material (e.g., due to at least the additional weight of the sulfate counter ion). Preferably, the ceftolozane is present as ceftolozane sulfate. If a ceftolozane sulfate composition contains “1000 mg of ceftolozane” then it includes an amount of ceftolozane sulfate comprising 1000 mg of the ceftolozane molecule in free base equivalent form. For example, as shown in Table 29, 1147 mg ceftolozane sulfate corresponds to 1000 mg of ceftolozane free base.
In another embodiment, “1000 mg ceftolozane” refers to an amount of ceftolozane that is considered a bioequivalent by the United States Food and Drug Administration (FDA), i.e. for which 90% CI of the relative mean Cmax, AUC(0-t) and AUC(0-∞) is within 80.00% to 125.00% of the reference formulation in the fasting state (see: “Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally Administered Drug Products—General Considerations”. Center for Drug Evaluation and Research, United States Food and Drug Administration, 2003).
“Ceftolozane active” refers to the active portion of a salt form of ceftolozane, i.e., the free base form of ceftolozane.
As used herein, “125 to 1000 mg sodium chloride per 1000 mg of ceftolozane” refers to a ratio of sodium chloride to ceftolozane free base equivalent. For example, “125 to 1000 mg sodium chloride per 1000 mg of ceftolozane” includes, for example, 62.5 to 500 mg sodium chloride per 500 mg of ceftolozane, as well as, for example, 25 to 200 mg sodium chloride per 200 mg ceftolozane, etc.
In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane as described herein.
II. Ceftolozane in the Presence of Tazobactam
It has also been observed that pharmaceutical compositions comprising ceftolozane, tazobactam, and 125 to 1000 mg sodium chloride per gram of ceftolozane exhibit better chemical stability and fewer additional compounds than those pharmaceutical compositions comprising ceftolozane and tazobactam, but less sodium chloride (see, e.g., Example 8). In particular embodiments described herein, the pharmaceutical compositions comprising ceftolozane, tazobactam, and 125 to 500 mg sodium chloride per 1000 mg of ceftolozane have been found to be more stable than the compositions comprising ceftolozane, tazobactam, and less than 125 mg sodium chloride per gram of ceftolozane.
Adding high amounts of sodium chloride to CXA-201 compositions (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane, 125-500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane) also inhibits the formation of certain additional compounds. Adding about 487 mg sodium chloride per 1000 mg of ceftolozane to CXA-201 composition can also inhibit the formation of certain additional compounds. For example, in one experiment, CXA-201 compositions comprising 125-481 mg sodium chloride per 1000 mg ceftolozane developed a reduced amount of a composition having a retention time of 63 minutes (“RT 63′”) after three months at 25° C. (see the HPLC measurements shown in Example 8A).
Accordingly, in one aspect, provided herein is a pharmaceutical composition comprising ceftolozane, tazobactam, and 125-1000 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125-500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane, tazobactam, and about 487 mg sodium chloride per 1000 mg of ceftolozane.
In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane, tazobactam, and 125-1000 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125-500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 94.9% or greater after 3 days at 60° C. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane, tazobactam, and about 487 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 94.9% or greater after 3 days at 60° C. In certain embodiments, the purity of the ceftolozane in the composition is 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater after 3 days at 60° C.
Tazobactam
The compound (2S,3S,5R)-3-methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide (also known as tazobactam) is a β-lactamase inhibitor of the following structure:




As used herein, tazobactam can be a free acid, a sodium salt, an arginine salt, or a hydrate or solvate thereof. The phrases “250-750 mg tazobactam”, “250-700 mg tazobactam,” “300-700 mg tazobactam”, “300-650 mg tazobactam”, “350-650 mg tazobactam”, “350-600 mg tazobactam”, “400-600 mg tazobactam”, “400-550 mg tazobactam”, “450-550 mg tazobactam” or “about 500 mg tazobactam” refer to an amount of tazobactam containing the free acid equivalent weight of tazobactam provided in the free acid form or any suitable salt form. For example, a composition containing 500 mg of tazobactam in the tazobactam sodium solid form will include greater than 500 mg of material (e.g., due to at least the additional weight of the sodium counter ion). For example, as shown in Table 29, 537 mg tazobactam sodium corresponds to 500 mg of tazobactam free acid. Preferably, the tazobactam is present as tazobactam sodium. If a tazobactam sodium composition contains “500 mg of tazobactam” then it includes an amount of tazobactam sodium comprising 500 mg of the tazobactam molecule in free acid equivalent form.
In an embodiment, the tazobactam is tazobactam sodium sterile powder. In yet a further embodiment, the tazobactam sodium sterile powder is generated by neutralizing tazobactam acid with sodium bicarbonate followed by lyophilization.
As used herein, the term “tazobactam active” refers to the active portion of a salt form of tazobactam, i.e., tazobactam free acid.
In certain embodiments, the pharmaceutical compositions further comprise tazobactam sodium at a quantity equivalent of 500 mg of tazobactam free acid in a lyophilized powder form per 1000 mg of ceftolozane (anhydrous, free base equivalent).
III. Liquid Pharmaceutical Compositions or Formulations
In another aspect, provided herein is a liquid pharmaceutical composition (e.g., an intravenous infusion solution) comprising ceftolozane and tazobactam, wherein the composition is suitable for intravenous administration. In one embodiment, the composition further comprises 125-1000 mg sodium chloride per 1000 mg of ceftolozane. In another embodiment, the composition further comprises 125-500 mg sodium chloride per 1000 mg of ceftolozane. In an embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) is prepared by reconstitution of a ceftolozane and tazobactam composition with sterile water and/or normal sterile saline, followed by dilution with sterile water and/or normal sterile saline. In an embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) is prepared by reconstitution of a ceftolozane and tazobactam composition with normal sterile saline, followed by dilution with normal sterile saline. In another embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) has an osmolality between about 300 mOsm/kg and 900 mOsm/kg, including injectable formulations with an osmolality of 350-900 mOsm/kg to 350-800 mOsm/kg, 400-500 mOsm/kg and 500-600 mOsm/kg. In a further embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) comprising 1,000 mg ceftolozane active and 500 mg of tazobactam active (as pharmaceutically acceptable salts thereof) has an osmolality that is between about 400 mOsm/kg and 500 mOsm/kg (e.g, 446-478 mOsm/kg, 440-480 mOsm/kg, 420-490 mOsm/kg). In a further embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) comprising 2,000 mg ceftolozane active and 1000 mg of tazobactam active (as pharmaceutically acceptable salts thereof) has an osmolality that is between about 500 mOsm/kg and 650 mOsm/kg. In yet a further embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) has an osmolality that is less than about 600 mOsm/kg (e.g, 290-610 mOsm/kg, 350-605 mOsm/kg, 550-605 mOsm/kg, 589-604 mOsm/kg). In another embodiment, the ceftolozane and tazobactam of the liquid pharmaceutical composition (e.g., an intravenous infusion solution) are controlled to pH 5 to 7. In a further embodiment, the ceftolozane and tazobactam of the liquid pharmaceutical composition (e.g., an intravenous infusion solution) are controlled to about pH 6.
In one embodiment, the methods further comprise reconstituting the lyophilized mixture in an aqueous solvent, such that the resulting solution is suitable for infusion. The mixture can be reconstituted in saline and/or sterile water for injection.
Methods of Preparing Pharmaceutical Compositions Comprising Ceftolozane and Sodium Chloride
Pharmaceutical compositions comprising ceftolozane and stabilizing-effective amount of a stabilizing agent can be obtained by lyophilization. As is known to those skilled in the art, lyophilization is a process of freeze-drying in which water is sublimed from a frozen solution of one or more solutes. Specific methods of lyophilization are described in Remington's Pharmaceutical Sciences, Chapter 84, page 1565, Eighteenth Edition, A. R. Gennaro, (Mack Publishing Co., Easton, Pa., 1990). A pharmaceutical composition comprising ceftolozane can be prepared by adding a stabilizing amount of sodium chloride in a fixed ratio to ceftolozane in an aqueous solution prior to lyophilization, then lyophilizing the solution to obtain a lyophilized composition comprising sodium chloride and ceftolozane.
In particular, the pharmaceutical antibiotic compositions can include stabilized ceftolozane sulfate obtained by a process comprising the steps of lyophilizing an aqueous solution containing ceftolozane and a stabilizing-effective amount of a stabilizing agent, where the stabilizing-effective amount of the stabilizing agent is about 100 to 500 mg (preferably 300-500 mg) of the stabilizing agent per 1,000 mg ceftolozane active in the aqueous solution prior to lyophilization. A therapeutically effective amount of ceftolozane (e.g., ceftolozane sulfate) and a stabilizing-effective amount of the stabilizing agent can dissolved in an aqueous solution that can be lyophilized to obtain a stabilized ceftolozane pharmaceutical composition.
The method can further comprise the steps of: (1) forming a solution comprising sodium chloride and ceftolozane or a salt thereof followed by lyophilizing the solution; and (2) combining the lyophilized ceftolozane with other components (e.g., a β-lactamase inhibitor, such as tazobactam, or a lyophilized β-lactamase inhibitor, such as a lyophilized tazobactam) to obtain the pharmaceutical composition. The resulting pharmaceutical composition can be a powder for reconstitution to obtain an injectable pharmaceutical composition that can be intravenously administered to a patient. In yet a further embodiment, the method comprises adding 189 mg sodium from sodium chloride per 1000 mg of ceftolozane active in an aqueous solution, then lyophilizing the solution to obtain a lyophilized material comprising sodium chloride and ceftolozane sulfate in a ratio effective to provide a product with less than 0.03% of the RT63 Impurity as detected by the HPLC method of Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









A. Blending
In other embodiments, pharmaceutical compositions comprising ceftolozane can be obtained by methods that include the steps of: (1) adding a stabilizing amount of sodium chloride to ceftolozane optionally followed by co-lyophilizing or spray drying the ceftolozane and sodium chloride; and (2) combining the product of step (1) with other components. For example, the product of step (1) can be combined with a β-lactamase inhibitor, such as tazobactam (CAS#: 89786-04-9), avibactam (CAS#1192500-31-4), Sulbactam (CAS#68373-14-8) and/or clavulanate (CAS#58001-44-8). The beta lactamase inhibitor can be included in a crystalline or amorphous form, such as a lyophilized tazobactam or crystalline tazobactam (e.g., U.S. Pat. Nos. 8,476,425 and 5,763,603) to obtain the pharmaceutical composition.
Pharmaceutical compositions comprising ceftolozane and tazobactam with reduced or even undetectable levels of the compound of RRT 1.22 (e.g., including levels of RRT 1.22 that are not detectable by HPLC according to Example 1 and/or comprise less than 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC according to Example 1) can be obtained by blending a first composition comprising a therapeutically effective amount of ceftolozane in the absence of tazobactam with a second composition comprising a therapeutically effective amount of tazobactam in the absence of ceftolozane to form a blended pharmaceutical composition.
Without being bound by theory, the compound RRT 1.22 can be formed by a reaction between ceftolozane and formylacetic acid, a by-product of tazobactam as illustrated in Marunaka et al. (Chem. Pharm. Bull. 1988, Vol. 36 (11), pp. 4478-4487.
FIG. 1 is a flowchart showing the steps for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a blending process, wherein the ceftolozane and tazobactam are lyophilized separately prior to blending as described herein.
The (first) ceftolozane composition can be prepared in the absence of tazobactam by forming a first aqueous solution comprising ceftolozane sulfate and other components including excipients, stabilizers, pH adjusting additives (e.g., buffers) and the like. Non-limiting examples of these additives include sodium chloride, citric acid and L-arginine. For example, the use of sodium chloride results in greater stability; L-arginine is used to adjust pH of the aqueous solution (e.g., to pH 6-7) and to increase the solubility of ceftolozane; and citric acid is used to prevent discoloration of the product, due to its ability to chelate metal ions. Preferably, the first aqueous solution comprises about 125 mg-500 mg sodium chloride per 1,000 mg of ceftolozane active. The ceftolozane can be included as an amount of ceftolozane sulfate of formula (I) containing at least about 1,000 mg ceftolozane active. The (first) aqueous solution is then lyophilized to form a first lyophilized ceftolozane composition, which is combined with tazobactam, e.g., the lyophilized tazobactam (e.g., lyophilized tazobactam sodium) or crystalline tazobactam.
The (second) tazobactam composition can be prepared in the absence of ceftolozane by forming a second solution comprising tazobactam. The tazobactam can be included in an amount providing about 500 mg of tazobactam active per 1,000 mg ceftolozane active (i.e., a 1:2 weight ratio of tazobactam active to ceftolozane active). Unless otherwise indicated, tazobactam can be a free acid, a sodium salt, an arginine salt, or a hydrate or solvate thereof. In one embodiment, the tazobactam in the (second) tazobactam composition is tazobactam acid and the second composition further comprises sodium bicarbonate or sodium hydroxide. Lyophilizing tazobactam in the presence of sodium bicarbonate or sodium hydroxide forms a lyophilized tazobactam sodium, which can then be further blended with the (first) lyophilized ceftolozane composition.
Pharmaceutical compositions with reduced or undetectable amounts of the compound of RRT 1.22 can be obtained by lyophilizing ceftolozane without formylacetic acid and/or tazobactam under conditions that prevent formation of RRT 1.22 (e.g., Example 9). The presence of RRT 1.22 can be detected by HPLC (e.g., Examples 1 and 7). Specific methods of lyophilization are described in Remington's Pharmaceutical Sciences, Chapter 84, page 1565, Eighteenth Edition, A. R. Gennaro, (Mack Publishing Co., Easton, Pa., 1990). The formation of the compound of formula (III) can be avoided by preventing the reaction of ceftolozane and formylacetic acid. In one embodiment, the compound of formula (III) can be suppressed by separately lyophlizing ceftolozane sulfate and tazobactam in separate solutions, and then blending the lyophilized compositions to form a pharmaceutical composition.
In one aspect, antibiotic pharmaceutical compositions comprising ceftolozane and tazobactam with less than about 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC of the compound of formula (III) are obtained by a process comprising the steps of: (a) lyophilizing ceftolozane in the absence of tazobactam to obtain a lyophilized ceftolozane composition, and (b) blending the lyophilized ceftolozane composition with a composition comprising tazobactam under conditions suitable for attaining the aforementioned purity levels, e.g., by blending with crystalline tazobactam or lyophilized tazobactam.
In another aspect, antibiotic pharmaceutical compositions comprising ceftolozane and tazobactam and less than about 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC of the compound of formula (III) are obtained by a process comprising the steps of: (a) lyophilizing tazobactam in the absence of ceftolozane to obtain a lyophilized tazobactam composition, and (b) blending the lyophilized tazobactam composition with a composition comprising ceftolozane (e.g., lyophilized ceftolozane sulfate).
In a third aspect, antibiotic pharmaceutical compositions comprising ceftolozane and tazobactam and less than about 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC of the compound of formula (III) are obtained by a process comprising the steps of: (a) lyophilizing tazobactam in the absence of ceftolozane to obtain a lyophilized tazobactam composition, (b) lyophilizing ceftolozane in the absence of tazobactam to obtain a lyophilized ceftolozane composition, and (c) blending the lyophilized tazobactam composition with the lyophilized ceftolozane composition.
Pharmaceutical compositions comprising the compound of formula (III), ceftolozane and tazobactam can be formulated to treat infections by parenteral administration (including subcutaneous, intramuscular, and intravenous) administration. Pharmaceutical compositions may additionally comprise excipients, stabilizers, pH adjusting additives (e.g., buffers) and the like. Non-limiting examples of these additives include sodium chloride, citric acid and L-arginine. For example, the use of sodium chloride results in greater stability; L-arginine is used to adjust pH and to increase the solubility of ceftolozane; and citric acid is used to prevent discoloration of the product, due to its ability to chelate metal ions. In one particular embodiment, the pharmaceutical compositions described herein are formulated for administration by intravenous injection or infusion.
Other pharmaceutical antibiotic compositions can include ceftolozane sulfate and the compound of formula (III). For example, pharmaceutical compositions comprising 0.13%, 0.15%, 0.30%, 0.38%, 0.74% or 0.97% of the compound of formula (III) are herein. The pharmaceutical antibiotic compositions can be provided in a unit dosage form (e.g., in a container). The unit dosage form can be dissolved with a pharmaceutically acceptable carrier, and then intravenously administered. The unit dosage form comprises 1000 mg of ceftolozane active and 500 mg tazobactam, typically 1000 mg ceftolozane active as ceftolozane sulfate and 500 mg of tazobactam active as tazobactam sodium, argininate or free acid. The unit dosage forms are commonly stored in containers.
In another aspect, provided herein is a unit dosage form of a pharmaceutical composition comprising 1,000 mg ceftolozane and 500 mg tazobactam, the pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising ceftolozane sulfate and tazobactam, obtained by a process comprising the steps of: lyophlizing an aqueous solution to obtain a lyophilized ceftolozane composition, wherein the aqueous solution comprises water, ceftolozane sulfate, 125-500 mg sodium chloride per 1,000 mg ceftolozane active in the aqueous solution, an amount of L-arginine to provide a pH of 6-7 in the solution prior to lyophilization; and blending the lyophilized ceftolozane composition with a lyophilized tazobactam composition in an amount providing the ratio of about 500 mg tazobactam free acid per 1,000 mg of ceftolozane active to obtain the unit dosage form.
Another embodiment of the invention is a container containing a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections. The container can be obtained by a process comprising the steps of: a) lyophilizing an aqueous solution comprising 189 mg sodium from sodium chloride per 1000 mg ceftolozane active in the form of ceftolozane sulfate and further comprising citric acid, and L-arginine to obtain a lyophilized ceftolozane composition; and b) filling a sufficient quantity of the lyophilized composition into a container to obtain a unit dosage form comprising 189 mg sodium from sodium chloride and 1,000 mg of ceftolozane active in the form of ceftolozane sulfate. In one aspect, the pH of the aqueous solution is 6.0 to 7.0. In another aspect the pharmaceutical composition is formulated for parenteral administration by reconstituting the pharmaceutical composition in the container (e.g., with 10 mL of diluent such as water for injection or isotonic saline) followed by addition of the reconstituted pharmaceutical composition to a carrier for injection (e.g., about 100 mL of isotonic saline or other pharmaceutically acceptable carrier for intravenous administration). Optionally, the container is also filled with tazobactam (e.g., a lyophilized tazobactam such as tazobactam sodium). In yet another aspect, the pharmaceutical composition is a liquid composition comprising 189 mg sodium from sodium chloride, 1,000 mg of ceftolozane active and tazobactam in an amount providing about 500 mg tazobactam acid equivalent per 1,000 mg of ceftolozane active, formulated for parenteral administration and the pH of the aqueous solution is 6.0 to 7.0.
The pharmaceutical composition in the container can also be a Ceftolozane/Tazobactam for Injection Drug Product, 1000 mg/500 mg. It is presented as a combination of two sterile active powders in a single container intended for reconstitution and intravenous infusion. In an embodiment, the drug product is prepared by converting ceftolozane sulfate to a sterile drug product intermediate (composition) powder with excipients citric acid, sodium chloride and L-arginine. This can be done by lyophilization, as described herein. Tazobactam sodium drug substance can be presented as a sterile powder without any excipients. The tazobactam sodium drug substance can be lyophilized, spray dried or provided as a crystalline material. The drug product is then prepared by aseptically filling the two powders (e.g., the two separately lyophilized drug powders) sequentially into a single container.
In an embodiment, the container of ceftolozane/tazobactam for injection contains approximately 2255 mg ceftolozane sterile composition powder that contains 1147 mg ceftolozane sulfate, which is equivalent to 1000 mg ceftolozane free base, as well as approximately 537 mg tazobactam sodium sterile drug substance, equivalent to 500 mg tazobactam free acid. At the time of administration, the container is reconstituted with 10 mL vehicle, sterile 5% Dextrose Injection USP, Water for Injection or 0.9% Sodium Chloride Injection USP, then the container contents further diluted in an infusion bag of 0.9% Sodium Chloride Injection USP or 5% Dextrose Injection USP, for administration. The constituents are shown in Table 29.
A pharmaceutical composition can include ceftolozane sulfate and tazobactam in an amount providing 1,000 mg of ceftolozane active per 500 mg of tazobactam active, and 0.03% to 0.15% by HPLC of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using the method described in Example 1. Optionally, the pharmaceutical composition can further include 125 mg to 500 mg of sodium chloride per 1,000 mg of ceftolozane active, and L-arginine. The tazobactam in the composition can be tazobactam sodium.
In one embodiment of these methods of preparing, 125-500 mg sodium chloride per 1000 mg of ceftolozane is combined. In another embodiment of these methods of preparing, the amount of the sodium chloride combined is 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane. In another embodiment of these methods of preparing, the amount of the sodium chloride combined is about 487 mg sodium chloride per 1000 mg of ceftolozane.
In another embodiment of these methods of preparing, the method further comprises lyophilizing the ceftolozane in the absence of the tazobactam. In yet another embodiment, the method can further comprise lyophilizing the tazobactam in the absence of the ceftolozane.
Accordingly, in one aspect, provided herein is a pharmaceutical composition comprising ceftolozane and tazobactam, wherein the composition comprises less than 0.5%, 0.4%, 0.3%, 0.25%, 0.2%, 0.15%, 0.1%, or 0.05% by weight of the compound RRT 1.22. In another aspect, provided herein is a pharmaceutical composition comprising ceftolozane and tazobactam, wherein the composition comprises less than 0.1% by weight of the compound RRT 1.22. In one embodiment, the pharmaceutical composition comprises less than 0.05% by weight of the compound RRT 1.22. In another embodiment, the pharmaceutical composition comprises less than 0.15% by weight of the compound RRT 1.22. In yet another embodiment, the pharmaceutical composition comprises no detectable amount of the compound RRT 1.22 as measured by HPLC.
In contrast, a greater amount of compound RRT 1.22 was found in compositions of ceftolozane and tazobactam, wherein the compositions were formed through co-lyophilization, i.e., the ceftolozane and tazobactam were combined and co-lyophilized together (see, e.g., Examples 6, 7a, and 7b), as opposed to being individually lyophilized and blended together (see, e.g., Examples 9 and 10).
In one aspect, provided herein is an antibacterial pharmaceutical composition comprising ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of: lyophilizing a first aqueous solution in the absence of tazobactam, the first aqueous solution comprising ceftolozane sulfate prior to lyophilization to obtain a first lyophilized ceftolozane composition; and blending the first lyophilized ceftolozane composition with tazobactam to obtain an antibacterial composition comprising less than 0.13% by HPLC of a compound of formula (III) (compound RRT 1.22) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using the method described in Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









In one embodiment, the antibacterial composition comprises less than 0.03% of the compound of formula (III) detected by HPLC. In another embodiment, the first aqueous solution further comprises L-arginine in an amount effective to adjust the pH of the first aqueous solution to 6-7 prior to lyophilization to obtain a first lyophilized ceftolozane composition.
In another embodiment, the antibacterial pharmaceutical composition is obtained by a process further comprising the steps of: lyophilizing a second solution comprising tazobactam in the absence of ceftolozane to form a second lyophilized tazobactam composition; and blending the first lyophilized ceftolozane composition and the second lyophilized tazobactam composition to obtain the antibacterial composition.
In another embodiment, the tazobactam in the second solution is tazobactam acid, and wherein the tazobactam acid in the second solution is lyophilized in the presence of sodium bicarbonate to form the second lyophilized tazobactam solution.
In another embodiment, the first aqueous solution comprises L-arginine in an amount effective to provide a pH of about 5-7, e.g., 6-7. In another embodiment, the first aqueous solution comprises 125 mg to 500 mg of sodium chloride per 1,000 mg of ceftolozane active.
In another embodiment, the first aqueous solution further comprises citric acid. In another embodiment, the first aqueous solution consists of ceftolozane sulfate, citric acid, sodium chloride, L-arginine, and water.
In another aspect, provided herein is a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of: lyophilizing a first aqueous solution in the absence of tazobactam, the first aqueous solution comprising ceftolozane sulfate, 125 mg to 500 mg of sodium chloride per 1,000 mg of ceftolozane active, at a pH of 6-7 prior to lyophilization to obtain a first lyophilized ceftolozane composition; lyophilizing a second solution comprising tazobactam in the absence of ceftolozane to form a second lyophilized tazobactam composition; and blending the first lyophilized ceftolozane composition and the second lyophilized tazobactam composition to obtain the antibacterial composition.
In another embodiment, the unit dosage form comprises a total of not more than 0.03% by HPLC of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using the method described in Example 1. i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









to detect:





In another embodiment, the unit dosage form comprises a total of 1,000 mg of ceftolozane active and a total of 500 mg of tazobactam active.
In another embodiment, the unit dosage form comprises a total of not more than 0.03% by HPLC of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using the method described in Example 1, i.e.:













Column
Develosil ODS-UG-5, 5 μm, 250 × 4.6 mm


Column temperature
45° C. ± 2° C.


Wavelength:
254 nm for ceftolozane and ceftolozane-related



substances in Table 1A


Mobile phase A
50 mM Sodium Perchlorate Monohydrate in



water, adjusted to pH 2.5 with Diluted Perchloric



Acid


Mobile phase B
63 mM Sodium Perchlorate Monohydrate in water,



adjusted to pH 2.5 with Diluted Perchloric



Acid:Acetonitrile, 4:1 (v/v)











Time (min.)
% A
% B


Gradient Profile
0
75
25



30
70
30



60
0
100



85
0
100



85.1
75
25



110
75
25









to detect:





In another aspect, provided herein is a compound of formula (III):




or a pharmaceutically acceptable salt thereof.
In still another aspect, provided herein is a pharmaceutical composition comprising a compound of formula (III):




or a pharmaceutically acceptable salt thereof.

In one embodiment, the pharmaceutical composition further comprises ceftolozane sulfate. In another embodiment, the pharmaceutical composition further comprises tazobactam.
In another embodiment, the compound of formula (III) is obtained by a process comprising the step of reacting ceftolozane and formylacetic acid to obtain the compound of formula (III). In another embodiment, the compound of formula (III) is obtained by a process comprising the step of reacting ceftolozane and tazobactam acid to obtain the compound of formula (III).
In another embodiment, the compound of formula (III) is obtained by a process comprising the steps of: forming an aqueous solution comprising ceftolozane and tazobactam acid; and lyophilizing the aqueous solution to obtain a lyophilized composition comprising the compound of formula (III). In another embodiment, the aqueous solution comprises ceftolozane sulfate and tazobactam acid in a 2:1 weight ratio between the amount of ceftolozane active and the amount of tazobactam acid. In another embodiment, the aqueous solution comprises sodium chloride, ceftolozane sulfate, tazobactam acid and L-arginine.
In another embodiment, the aqueous solution has a pH of about 5.0 to 7.0, e.g., 6.0 to 7.0. In another embodiment, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the compound of formula (III) is obtained by a process further comprising the step of performing high performance liquid chromatography (HPLC) on the lyophilized composition to isolate the compound of formula (III).
In another embodiment, the pharmaceutical composition comprises about 0.13-0.97% of the compound of formula (III). In another embodiment, the pharmaceutical composition comprises up to about 0.15% of the compound of formula (III). In another aspect, provided herein is a pharmaceutical composition comprising a compound of formula (III),




the compound of formula (III) obtained by a process comprising the steps of: forming an aqueous solution comprising tazobactam acid and ceftolozane sulfate in an amount providing 1,000 mg of ceftolozane active per 500 mg of tazobactam acid in the aqueous solution; lyophilizing the aqueous solution of step (a) to obtain a lyophilized composition comprising a compound of formula (III); and formulating the lyophilized composition as a pharmaceutical composition for parenteral delivery.

In one embodiment, the pH of the aqueous solution is 5.0 to 7.0, e.g., 6.0 to 7.0. In another embodiment, the pharmaceutical composition is formulated for parenteral administration.
In another aspect, provided herein is a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising a compound of formula (III) in a lyophilized composition obtained by lyophilizing an aqueous solution comprising tazobactam and an amount of ceftolozane sulfate containing 1,000 mg of ceftolozane active per 500 mg of tazobactam acid.
B. Co-Lyophilization
In one aspect, provided herein is a method of preparing a composition comprising ceftolozane and sodium chloride, comprising combining sodium chloride with ceftolozane, wherein 125-1000 mg sodium chloride per 1000 mg of ceftolozane is combined, followed by lyophilization of the sodium chloride ceftolozane mixture. The process is referred to herein as “co-lyophilization”. In another aspect, provided herein is a method of preparing a composition comprising sodium chloride, tazobactam, and ceftolozane, comprising combining sodium chloride, tazobactam, and ceftolozane, wherein 125-1000 mg sodium chloride per 1000 mg of ceftolozane is combined, followed by lyophilization of the mixture of sodium chloride, tazobactam, and ceftolozane.
Also provided herein is a method of preparing a pharmaceutical composition comprising sodium chloride, ceftolozane, and tazobactam, comprising combining sodium chloride, tazobactam, and ceftolozane, followed by spray-drying the mixture of sodium chloride, ceftolozane, and tazobactam.
FIG. 2 is a flowchart showing the steps for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a co-lyophilization process, as described herein.
In another aspect, provided herein is a pharmaceutical composition comprising stabilized ceftolozane sulfate obtained by a process comprising lyophilizing an aqueous solution comprising 125 mg to 500 mg sodium chloride with an amount of ceftolozane sulfate providing 1,000 mg of ceftolozane active, to obtain the lyophilized stabilized ceftolozane sulfate composition.
In one embodiment, the stabilized ceftolozane is obtained by lyophilizing the sodium chloride and ceftolozane sulfate with L-arginine. In another embodiment, the stabilized ceftolozane is obtained by lyophilizing an aqueous solution having a pH of about 5.0 to 7.0, e.g., 6.0 to 7.0.
In another embodiment, the stabilized ceftolozane is obtained by lyophilizing the sodium chloride and ceftolozane sulfate with L-arginine and citric acid. In another embodiment, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the composition is a unit dosage form in a container comprising 125 mg to 500 mg sodium chloride, 1,000 mg of ceftolozane in the form of ceftolozane sulfate, and L-arginine. In another embodiment, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the pH of the aqueous solution is 6.0 to 7.0.
In another aspect, provided herein is a container comprising a pharmaceutical composition of stabilized ceftolozane sulfate, obtained by a process comprising the step of: lyophilizing an aqueous solution comprising 125 mg to 500 mg sodium chloride with an amount of ceftolozane sulfate providing 1,000 mg of ceftolozane active, to obtain the lyophilized stabilized ceftolozane sulfate composition; filling the lyophilized stabilized ceftolozane composition into a container.
IV. Manufacturing for the Prevention of Cross-Contamination
Recent FDA manufacturing guidance (published in April 2013) states that manufacturing facilities dedicated to manufacturing a sensitizing non-penicillin beta-lactam compound should be “completely and comprehensively separated” from areas in the facility in which any class of sensitizing beta-lactam is manufactured. See U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research, Non-Penicillin Beta-Lactam Drugs: A CGMP Framework for Preventing Cross-Contamination (April 2013) (“FDA Guidance”). The FDA also considers separation of production facilities for penicillins to be good manufacturing practice. The FDA Guidance can be understood to require the use of a dedicated facility to manufacture antibiotic compounds comprising a non-penicillin beta-lactam compound (e.g., a cephalosporin) and a BLI compound with a beta-lactam ring (e.g., tazobactam). Accordingly, a facility that manufactures a product containing both cephalosporin and a beta-lactam containing BLI such as tazobactam for sale in the United States cannot be subsequently used to manufacture any other products containing beta-lactam ring, other than additional combinations of other cephalosporins with the same BLI compound (e.g., other non-penicillin beta-lactam compounds including other cephalosporin antibiotics cannot be subsequently manufactured in the facility).
Beta-lactam antibiotics, including penicillin and the non-penicillin classes, share a basic chemical structure that includes a three-carbon, one-nitrogen cyclic amine structure known as the beta-lactam ring. The side chain associated with the beta-lactam ring is a variable group attached to the core structure by a peptide bond; the side chain variability contributes to antibacterial activity. As of the date of this publication, FDA has approved over 34 beta-lactam compounds as active ingredients in drugs for human use. (see, e.g., FDA's Approved Drug Products with Therapeutic Equivalence Evaluations, generally known as the Orange Book) Beta-lactam antibiotics include the following five classes: penicillins (e.g., ampicillin, oxacillin); cephalosporins (e.g., cephalexin, cefaclor); penems (e.g., imipenem, meropenem); carbacephems (e.g., loracarbef); and monobactams (e.g., aztreonam). (Yao, J D C, and R C Moellering, Jr., Antibacterial agents, in Manual of Clinical Microbiology, 9th edition, edited by PR Murray et al., Washington D.C., ASM Press, 2007.)
Under the FDA Guidance, a manufacturing facility handling a product for sale in the United States containing both a cephalosporin (e.g, ceftolozane) and a penicillin nucleus (e.g., tazobactam) cannot be subsequently used in the manufacture of any other class of beta-lactam products, including all other penicillins, cephalosporins, penems, carbacephems and monobactams or in the manufacture of other finished pharmaceuticals or active pharmaceutical ingredients. The FDA Guidance states that (non-penicillin) cephalosporin beta-lactam compounds (e.g., such as ceftolozane) for sale in the United States must be “completely and comprehensively separated from” manufacturing areas that handle any other class of beta-lactam compound (e.g., compounds in the penicillin class).
A product containing ceftolozane and tazobactam includes both a non-penicillin beta-lactam cephalosporin (ceftolozane) and a beta-lactamase inhibitor with a beta-lactam moiety (tazobactam). Under the FDA Guidance, these two compounds must be “completely and comprehensively separated.” Accordingly, there is a need for methods of manufacturing antibiotic compositions comprising ceftolozane and tazobactam for sale in the United States in compliance with the FDA Guidance, as well as antibiotic compositions manufactured in accordance with the FDA Guidance without affecting the purity, stability, and safety of the resulting composition.
Provided herein are methods of manufacturing or preparing pharmaceutical compositions containing two or more beta-lactam compounds in accordance with FDA Guidance, as well as pharmaceutical compositions manufactured in compliance with FDA Guidance. Specifically, certain manufacturing methods are provided herein that conform to standards recommended by FDA Guidance for the avoidance of cross-contamination of non-penicillin beta-lactam drugs.
In one aspect, provided herein is an antibacterial pharmaceutical composition formulated for parenteral administration for the treatment of infections, the pharmaceutical composition comprising a therapeutically effective amount of ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of:

    
    
        a. lyophilizing a first aqueous solution in the absence of tazobactam, the first aqueous solution comprising ceftolozane sulfate, to obtain a first lyophilized ceftolozane composition;
        b. blending the lyophilized ceftolozane composition with a tazobactam composition comprising tazobactam prepared and provided in the absence of ceftolozane;
    
    


wherein the process is completed in the absence of other non-cephalosporin beta-lactam compounds.
In another aspect, provided herein is a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of

    
    
        a. lyophilizing a first aqueous solution in the absence of tazobactam, the first aqueous solution comprising ceftolozane sulfate, 125 mg to 500 mg of sodium chloride per 1,000 mg of ceftolozane active, to obtain a first lyophilized ceftolozane composition,
        b. lyophilizing a second solution comprising tazobactam in the absence of ceftolozane to form a second lyophilized tazobactam composition; and
        c. blending the first lyophilized ceftolozane composition and the second lyophilized tazobactam composition to obtain the antibacterial composition;
    
    


wherein the process is completed in the absence of other non-cephalosporin beta-lactam compounds.
V. Methods of Treatment
Pharmaceutical compositions comprising ceftolozane/tazobactam are being developed as an intravenous (IV) formulation for the treatment of complicated urinary tract infections (cUTIs) and complicated intra-abdominal infections (cIAIs).
Ceftolozane/tazobactam is an antibacterial composition including ceftolozane, a cephalosporin with potent antipseudomonal cephalosporin activity, in combination with tazobactam, a beta (β)-lactamase inhibitor (BLI). Like other members of the cephalosporin class, ceftolozane is believed to exert its bactericidal activity by inhibiting essential penicillin-binding proteins (PBPs), resulting in inhibition of cell wall synthesis and subsequent cell death. Ceftolozane has activity against Pseudomonas aeruginosa including strains that are resistant to carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides, and other common Gram-negative pathogens, including most extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. Tazobactam inhibits chromosomal- and plasmid-mediated bacterial class A and C β lactamases. Tazobactam is believed to protect ceftolozane from hydrolysis by covalently binding these enzymes, and broadens coverage to include most ESBL-producing Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae, including some Enterobacteriaceae overexpressing AmpC. Tazobactam inhibits or decreases the activity of beta-lactamases (e.g., bacterial beta-lactamases), and can be combined with beta-lactam compounds (e.g., antibiotics), thereby broadening the spectrum of the beta-lactam compound and increasing the beta-lactam compound's efficacy against organisms that produce beta-lactamase. A compound or a composition possesses efficacy against an organism if it kills or weakens the organism, or inhibits or prevents reproduction the organism.
The CXA-201 product (ceftolozane/tazobactam for injection) is undergoing regulatory review for the treatment of complicated intra-abdominal infections (cIAI) caused by susceptible isolates of the Gram-negative and Gram-positive microorganisms such as: Citerobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumonia, Klebsiella oxytoca, Proteus mirabilis, and Pseudomonas aeruginosa. In patients who are at risk of mixed aerobic-anaerobic infection, concurrent therapy with an anti-anaerobic agent can also be used.
The CXA-201 product (ceftolozane/tazobactam for injection) is undergoing regulatory review for the treatment of complicated urinary tract infections (cUTI), including pyelonephritis caused by susceptible isolates of the following Gram-negative microorganisms: Enterobacter spp, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis and Pseudomonas aeruginosa. In one embodiment, a pharmaceutical composition comprising a CXA-201 product (e.g, the unit dosage container of Table 29 below) is reconstituted in a pharmaceutically acceptable carrier (e.g., a total volume of about 90-150 mL, preferably about 110 mL, of 0.9% aqueous sodium chloride for injection or in initial volume of 10-20 mL of water for injection or 0.9% aqueous sodium chloride for injection, followed by dilution of this solution into a 100 mL volume of 0.9% aqueous sodium chloride for injection). The resulting pharmaceutical composition can be infused into a patient in need thereof for treatment of a complicated intra-abdominal infection (e.g, using 1 hour infusion times) three times per day (e.g., once every 8 hours) for a recommended duration of treatment (e.g. 4-10 days).
The preferred dosage of (ceftolozane/tazobactam for injection) for cUTI and cIAI is 1.5 g administered every 8 hours by intravenous (IV) infusion over 1 hour in patients≧18 years of age. The duration of therapy should be guided by the severity and site of infection and the patient's clinical and bacteriological progress. In one embodiment, a pharmaceutical composition comprising a CXA-201 product (e.g, the unit dosage container of Table 29 below) is reconstituted in a pharmaceutically acceptable carrier (e.g., a total volume of about 90-150 mL, preferably about 110 mL, of 0.9% aqueous sodium chloride for injection or in initial volume of 10-20 mL of water for injection or 0.9% aqueous sodium chloride for injection, followed by dilution of this solution into a 100 mL volume of 0.9% aqueous sodium chloride for injection). The resulting pharmaceutical composition can be infused into a patient in need thereof for treatment of a Complicated Urinary Tract Infections (cUTI), Including Pyelonephritis (e.g, using 1 hour infusion times) three times per day (e.g., once every 8 hours) for a recommended duration of treatment (e.g. 7 days).
Ceftolozane/tazobactam displays potent antibacterial activity against common Gram-negative organisms, including Enterobacteriaceae and Pseudomonas aeruginosa; select Gram-positive organisms, including streptococci; the majority of pathogenic enteric bacilli and select Gram-positive anaerobic species, thus making ceftolozane/tazobactam a potentially practical choice for pathogens involved in gastro-intestinal, urinary and community acquired as well as nosocomial respiratory infections. In general, the Gram-positive and Gram-negative spectrum of ceftolozane is similar to ceftazidime, but its antipseudomonal activity is the most potent among all currently available β-lactams, including the cephalosporins and carbapenems. Most importantly, ceftolozane has been shown to be active against strains of P. aeruginosa that are resistant to carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides, including the majority of multi-drug resistant isolates. Indeed, the minimum inhibitory concentration (MIC) required to inhibit the growth of 90% of organisms (MIC90) for P. aeruginosa (MIC90≦2 μg/mL) is the lowest among all systemically administered antipseudomonal antibiotics.
In vitro studies have demonstrated that ceftolozane/tazobactam has a broad spectrum of activity against Gram-negative bacteria. The in vitro activity of ceftolozane and ceftolozane/tazobactam was evaluated against a broad range of Gram-positive and Gram-negative bacteria. It was observed that tazobactam potentiated the activity of ceftolozane against Acinetobacter spp. and common species of Enterobacteriaceae, including Citrobacter spp., Enterobacter cloacae, E. coli, K. pneumoniae, Proteus mirabilis, and Serratia marcescens. These surveillance data demonstrate that 88% to 100% of these Enterobacteriaceae species are inhibited at <8 μg/mL.
In one aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition prepared according to the methods described herein. In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of tazobactam and ceftolozane. In certain embodiments of the above methods, the bacterial infection is caused by an extended-spectrum beta-lactamase-producing organism. In certain embodiments, the bacterial infection is caused by an antibiotic-resistant organism. In yet another aspect, the invention is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising both tazobactam and ceftolozane. In certain embodiments of the above methods, the bacterial infection is caused by an extended-spectrum beta-lactamase-producing organism. In certain embodiments, the bacterial infection is caused by an antibiotic-resistant organism.
In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising tazobactam, ceftolozane, and less than 0.1% by weight of the compound RRT 1.22. In another embodiment of the treatment method, the pharmaceutical composition comprises tazobactam, ceftolozane, and less than 0.05% by weight of the compound RRT 1.22.
In certain embodiments of the treatment methods, the pharmaceutical composition further comprises 125 to 1000 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane. In one specific embodiment of the treatment methods, the pharmaceutical composition further comprises about 487 mg sodium chloride per 1000 mg of ceftolozane.
In other embodiments of the treatment methods, the pharmaceutical composition comprises 250-750 mg tazobactam per 1000 mg of ceftolozane, e.g., 250-700 mg tazobactam per 1000 mg of ceftolozane, 300-700 mg tazobactam per 1000 mg of ceftolozane, 300-650 mg tazobactam per 1000 mg of ceftolozane, 350-650 mg tazobactam per 1000 mg of ceftolozane, 350-600 mg tazobactam per 1000 mg of ceftolozane, 400-600 mg tazobactam per 1000 mg of ceftolozane, 400-550 mg tazobactam per 1000 mg of ceftolozane, 450-550 mg tazobactam per 1000 mg of ceftolozane, or about 500 mg tazobactam per 1000 mg of ceftolozane.
Non-limiting examples of the bacterial infections that can be treated by the methods of the invention include infections caused by: aerobic and facultative gram-positive microorganisms (e.g., Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Viridans group streptococci), aerobic and facultative gram-negative microorganisms (e.g., Acinetobacter baumanii, Escherichia coli, Haemophilus influenza, Klebsiella pneumonia, Pseudomonas aeruginosa, Citrobacter koseri, Moraxella catarrhalis, Morganella morganii, Neisseria gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Serratia marcescens, Providencia stuartii, Providencia rettgeri, Salmonella enterica), gram-positive anaerobes (Clostridium perfringens), and gram-negative anaerobes (e.g., Bacteroides fragilis group (e.g., B. fragilis, B. ovatus, B. thetaiotaomicron, and B. vulgates), Bacteroides distasonis, Prevotella melaninogenica).
In certain embodiments of the methods described herein, the bacterial infections resulting from beta-lactamase-producing organisms are treated or controlled. Non-limiting examples of beta-lactamase-producing organisms include:
(1) ESBL (extended-spectrum beta-lactamase)-producing organisms selected from the group consisting of Enterobacteriaceae spp.: Escherichia coli, Klebsiella spp. (including K. pneumoniae and K. oxytoca), Proteus mirabilis, Proteus vulgaris, Enterobacter spp., Serratia spp., Citrobacter spp., Pseudomonas spp., Acinetobacter spp.) and Bacteroides spp.;
(2) CSBL (conventional-spectrum beta-lactamase)-producing organisms, known to those of skill in the art; and
(3) Inducible-AmpC-type beta-lactamases, such as Citrobacter spp., Serratia spp., Morganella morganii, Proteus vulgaris, and Enterobacter cloacae. 
In certain embodiments of the methods described herein, the bacterial infection is associated with one or more of the following conditions:
Appendicitis (complicated by rupture or abscess) and peritonitis caused by piperacillin-resistant beta-lactamase producing strains of Escherichia coli or the following members of the Bacteroides fragilis group: B. fragilis, B. ovatus, B. thetaiotaomicron, or B. vulgates; 
Uncomplicated and complicated skin and skin structure infections, including cellulitis, cutaneous abscesses, and ischemic/diabetic foot infections caused by piperacillin-resistant, beta-lactamase producing strains of Staphylococcus aureus; 
Postpartum endometritis or pelvic inflammatory disease caused by piperacillin-resistant, beta-lactamase producing strains of Escherichia coli; 
Community-acquired pneumonia (moderate severity only) caused by piperacillin-resistant, beta-lactamase producing strains of Haemophilus influenza; 
Nosocomial pneumonia (moderate to severe) caused by piperacillin-resistant, beta-lactamase producing strains of Staphylococcus aureus and by Acinetobacter baumanii, Haemophilus influenzae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Nosocomial pneumonia is also known as hospital acquired/ventilator-associated bacterial pneumonia (HABP/VABP);
Complicated intra-abdominal infections (cIAI);
Complicated urinary tract infections (cUTIs);
Acute Pyelonephritis; and
Systemic Inflammatory Response Syndrome (SIRS).
Also provided herein is the use of tazobactam, and hydrates and solvates thereof, in combination with ceftolozane, for the preparation of a medicament for the treatment of bacterial infections. The bacterial infections can result from either gram-negative or gram-positive organisms.
The compositions provided herein can be used in the treatment of infections caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumonia. 
In one embodiment of the treatment methods, the bacterial infections are Gram-negative bacterial infections. In one embodiment, the gram-negative infections are complicated Urinary Tract Infections (cUTI) and complicated intra-abdominal infections (cIAI). In another embodiment, the gram-negative bacterial infections are caused by Pseudomonas aeruginosa, E. coli, and/or Klebsiella pneumonia. 
In a further embodiment, provided herein is a method for the treatment of gram-negative bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane, about 500 mg tazobactam free acid per 1000 mg of ceftolozane, about 476 mg sodium chloride per 1000 mg of ceftolozane, about 587 mg L-arginine per 1000 mg of ceftolozane, and about 21 mg anhydrous citric acid per 1000 mg of ceftolozane. In one embodiment, the gram-negative bacterial infections are selected from the group consisting of complicated Urinary Tract Infections (cUTI) and complicated intra-abdominal infections (cIAI). In another embodiment, the gram negative bacterial infection is nosocomial pneumonia.
In another specific embodiment, provided herein is a method for the treatment of gram-negative bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane, about 500 mg tazobactam free acid equivalents per 1000 mg of ceftolozane, about 487 mg sodium chloride per 1000 mg of ceftolozane, about 600 mg L-arginine per 1000 mg of ceftolozane, and about 21 mg anhydrous citric acid per 1000 mg of ceftolozane. In one embodiment, the gram-negative bacterial infections are selected from the group consisting of complicated Urinary Tract Infections (cUTI) and complicated intra-abdominal infections (cIAI). In another embodiment, the gram negative bacterial infection is nosocomial pneumonia.
In one embodiment, provided herein is a method for the treatment of an infection in a mammal, wherein the infection is caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumoniae comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane, corresponding to approximately 1000 mg of the free base form of ceftolozane; tazobactam, corresponding to approximately 500 mg of the tazobactam acid form; and 400-500 mg sodium chloride. In one embodiment, the pharmaceutical composition further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.
In another embodiment, provided herein is a method for the treatment of urinary tract infection, intra-abdominal infection, or nosocomial pneumonia in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane, corresponding to approximately 1000 mg of the free base form of ceftolozane; tazobactam, corresponding to approximately 500 mg of the tazobactam acid form; and 400-500 mg sodium chloride. In an embodiment, the pharmaceutical composition comprises 487 mg sodium chloride. In one embodiment, the pharmaceutical composition further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.
In one embodiment, provided herein is a method for the treatment of an infection in a mammal, wherein the infection is caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumoniae comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; and 400-500 mg sodium chloride. In one embodiment, the pharmaceutical composition further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.
In another embodiment, provided herein is a method for the treatment of urinary tract infection, intra-abdominal infection, or nosocomial pneumonia in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; and 400-500 mg sodium chloride. In an embodiment, the pharmaceutical composition comprises 487 mg sodium chloride. In one embodiment, the pharmaceutical composition further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.
In one embodiment, provided herein is a method for the treatment of an infection in a mammal, wherein the infection is caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumoniae comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; approximately 487 mg sodium chloride; and approximately 600 mg L-arginine. In one embodiment, the pharmaceutical composition further comprises 15-30 mg anhydrous citric acid.
As used herein, “treating”, “treat” or “treatment” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a pharmaceutical composition of the present invention to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.
By a “therapeutically effective amount” of a compound of the invention is meant a sufficient amount of the compound to treat the disorder (e.g., bacterial infection). The specific therapeutically effective amount that is required for the treatment of any particular patient or organism (e.g., a mammal) will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound or composition employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see, for example, Goodman and Gilman's, “The Pharmacological Basis of Therapeutics”, Tenth Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001, which is incorporated herein by reference in its entirety). The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.
As used herein, “189 mg sodium from sodium chloride per 1000 mg of ceftolozane” refers to a ratio of sodium from the sodium chloride to ceftolozane active. For example, “189 mg sodium from sodium chloride per 1000 mg of ceftolozane” includes, for example, 94.5 mg sodium from sodium chloride per 500 mg of ceftolozane, as well as, for example, 47.25 mg sodium from sodium chloride per 250 mg ceftolozane. In addition, “1,000 mg of ceftolozane as ceftolozane sulfate” refers to an amount of ceftolozane sulfate effective to provide 1,000 mg of ceftolozane. “189 mg sodium from sodium chloride” refers to the amount of sodium chloride (e.g., 480 mg) effective to provide 189 mg of sodium. The amount of sodium from sodium chloride per gram of ceftolozane activity in a pharmaceutical composition containing ceftolozane sulfate, chloride and sodium chloride can be calculated using the relevant molecular weights of ceftolozane, ceftolozane sulfate, sodium chloride and sodium. For example, a composition comprising about 1,147 mg ceftolozane sulfate and 189 mg sodium from sodium chloride contains 480 mg sodium chloride per 1,000 mg ceftolozane active.
Unless otherwise indicated, as used herein, the term “Related Substances” with respect to HPLC detection refers to all the ceftolozane related process impurities and degradation products other than ceftolozane separated and detected by HPLC according to Example 1. Unless otherwise indicated, as used herein, the term “% Related Substances” refers to the % of the total HPLC peak area obtained by Example 1 attributed to all the ceftolozane related process impurities and degradation products other than ceftolozane.
EXAMPLES
Example 1: HPLC Analysis of Compositions Comprising Ceftolozane
The purity of ceftolozane in the pharmaceutical compositions was measured using the analytical HPLC method described below.
The HPLC methodologies described herein were used to acquire the data provided in Examples 5 and 8.
Analytical HPLC Method
A. Operative Conditions


    Column: Develosil ODS-UG-5; 5 μm, 250×4 6 mm (Nomura Chemical, Japan)
    Wavelength: 254 nm (determination of CXA-101 impurities)
    
        210 nm (determination of Tazobactam Related Compound A)
    
    
    Flow rate: 1.0 mL/min
    Column Temperature: 45° C.
    Injection volume: 10 μL
    Run Time: 85 minutes

Gradient Profile:















Time (min)
A %
B %

















0
75
25


30
70
30


60
0
100


85
0
100


85.1
75
25


110
75
25









    Mobile phase A: Sodium Perchlorate Buffer Solution (pH 2.5) (also referred to herein as “50 mM Sodium Perchlorate Monohydrate in water, adjusted to pH 2.5 with Diluted Perchloric Acid”
    
        Preparation: Dissolve 7.02 g of Sodium Perchlorate Monohydrate in 1000.0 mL of water and adjust to pH 2.5 with Diluted Perchloric Acid (1 in 20).
    
    
    Mobile phase B: Sodium Perchlorate Buffer Solution (pH 2.5):Acetonitrile in a ratio of 4:1 (v/v) (also referred to herein as “63 mM Sodium Perchlorate Monohydrate in water, adjusted to pH 2.5 with Diluted Perchloric Acid:Acetonitrile, 4:1 (v/v)”)
    
        Preparation: Dissolve 8.78 g of Sodium Perchlorate Monohydrate in 1000.0 mL of water and adjust to pH 2.5 with Diluted Perchloric Acid (1 in 20). Mix the resulting solution with Acetonitrile in the ratio 4:1 (v/v).

B. System Suitability Solution (1%)

    
    


Transfer 33 mg, accurately weighed, of Sample (use first sample if more are present) into a 10.0 mL volumetric flask. Dissolve and dilute to volume with water. Into a 100.0 mL volumetric flask, transfer 1.0 mL of this Solution and dilute with water to volume and mix.
C. Sample Preparation.
Transfer 33 mg, accurately weighed, of Sample into a 10.0 mL volumetric flask. Dissolve and dilute to volume with water. Prepare just before injection into the HPLC system.
D. HPLC Analysis Procedure
1. Inject Blank (water)
2. Inject System Suitability Solution and check for tailing factor and theoretical plates number for CXA-101 and Tazobactam peaks:


    
    
        The tailing factor must not be greater than 1.5
        Theoretical plates number must not be less than 10000

3. Inject Sample Solution

4. Identify the peaks of CXA-101 Related Substances in the Sample chromatogram, recorded at 254 nm, on the basis of the RRT values reported in Table 1.

5. Identify the peak of Tazobactam Related Compound A in the Sample chromatogram, recorded at 210 nm, on the basis of the RRT values reported in Table 1A.

    
    









TABLE 1A







Relative retention times of ceftolozane related substances










Impurity
RRT














Peak 1 (P1)
~0.15



Peak 2 (P2)
~0.16



Peak 3 (P3)
~0.4



Peak 4 (P4)
~0.6



Peak 5 (P5)
~0.89



CXA-101
1.0



Peak 6 (P6)
~1.2



Peak 7 (P7)
~1.3



Peak 8 (P8)
~1.4



Peak 9 (P9)
~1.7



Peaks 10, 11 (P10, P11)
~2.3

















TABLE 1B







Related Substances of CXA-101 Drug Substance and Drug Product











Proposed


Compound
Proposed Structure
Source












Peak 1 3-side chain




Degradation product and process impurity


 


Peak 2
Unidentified
Process impurity


Peak 3
Unidentified
Process impurity


Peak 4
Unidentified
Process impurity


 


Peak 5 7- Epimer type




Degradation product and process impurity


 


Peak 6
NA
Process impurity


 


Peak 7 Δ3 Isomer type




Degradation product and process impurity


 


Peak 8
Unidentified
Process impurity


 


Peak 9 Anti-Isomer type




Process impurity and Degradation impurity


 


Peaks 10, 11
Unidentified
Process impurity
















TABLE 1C







Relative retention time of tazobactam related compound A










Impurity
RRT














Related Compound A
0.34



Tazobactam
1.0










E. Calculations

Only peaks with a value≧LOQ are integrated.

I. CXA-101 Impurities Determination at 254 nm.

(Tazobactam and Tazobactam Related compound A peaks are integrated separately, see below)

Each related substance at 254 nm is reported for its amount as expressed by area percent.





  
    C
    i
  
  =
  
    
      
        A
        i
      
      ×
      100
    
    
      
        A
        t
      
      +
      
        Σ
        ⁢
        
          
        
        ⁢
        
          A
          i
        
      
    
  




where:
Ci=Amount of related substance i in the Sample calculated at 254 nm, area %
Ai=Peak area of related substance i in the Sample chromatogram
At=Area of CXA-101 peak in the Sample chromatogram
At+ΣAi=Total peaks area in the Sample chromatogram
Consider as any Unspecified Impurity, each peak in the chromatogram except CXA-101, peaks from 1 to 11 and every peak present in the blank chromatogram and report the largest.
II. Total CXA-101 Related Impurities at 254 nm: Report the Total Impurities Content as Expressed by the Following Formula:




  
    C
    
      T
      ⁢
      
        
      
      ⁢
      254
    
  
  =
  
    
      Σ
      ⁢
      
        
      
      ⁢
      
        A
        i
      
      ×
      100
    
    
      
        A
        t
      
      +
      
        Σ
        ⁢
        
          
        
        ⁢
        
          A
          i
        
      
    
  




wherein:
CT254=total impurities content in the Sample calculated at 254 nm, area %
At=area of CXA-101 peak in the sample chromatogram
ΣAi=total peak areas of impurities in the sample chromatogram
At+ΣAt=total peaks area in the sample chromatogram
III. Tazobactam Related Compound a Determination at 210 nm:
Integrate only the Tazobactam and Tazobactam Related Compound A Peaks




  
    C
    a
  
  =
  
    
      
        A
        a
      
      ×
      100
    
    
      
        A
        a
      
      +
      
        A
        T
      
    
  




where:
Ca=Amount of Tazobactam Related Compound A in the Sample, are %
Aa=Peak area of Tazobactam Related Compound A in the Sample chromatogram
At=Area of Tazobactam in the Sample chromatogram
IV. Total Impurities
Make the sum of Tazobactam Related Compound A (III) and the Total CXA-101 impurities determined (II)
V. Quantitation Limit














Component
LOQ %









Tazobactam
0.03



CXA-101
0.03










Example 2: Screening of Stabilizing Agents
Nine stabilizing agents were screened, including sodium chloride, fructose, xylitol, sorbitol, dextran 40, lactose, glucose, maltose, and D-mannitol. The purity of the ceftolozane in a composition comprising 100 mg ceftolozane and 100 mg of one of the stabilizing agents after 3 days at 70° C. was compared to a composition comprising 100 mg ceftolozane but no stabilizing agent.
As shown in Table 2A, the ceftolozane compositions comprising sodium chloride, dextran 40, lactose, or maltose were demonstrated to be more stable than the other ceftolozane compositions comprising the other stabilizing agents, or no stabilizing agent. Sodium chloride and maltose were selected for further investigation.







TABLE 2A





Screening of Stabilizing Agents

















Stabilizing agent













Sodium chloride
Fructose
Xylitol
Sorbitol
Dextran 40









Storage



















70° C.

70° C.

70° C.

70° C.

70° C.



Initial
3 days
Initial
3 days
Initial
3 days
Initial
3 days
Initial
3 days





Appearance
White
Pale
White
Orange
White
Orange
White
Pale
White
Pale



mass
yellow
mass
paste
mass
paste
mass
yellow
mass
yellow




mass





paste

mass


Color and
Pale
Pale
Pale
Orange
Pale
Orange
Pale
Yellow
Pale
Yellow


clarity
yellow
yellow
yellow
and
yellow
and
yellow
and
yellow
and



and
and
and
clear
and
clear
and
clear
and
clear



clear
clear
clear

clear

clear

clear


pH
5.58
4.23
6.04
3.81
5.96
4.18
6.01
4.00
5.60
4.36


Residual rate (%)
100.0
75.7
100.0
4.29
100.0
0.41
100.0
0.00
100.0
72.2


Reconstitution
15
30
20
40
15
180<   
15
160
170
160


time(s)












Stabilizing agent









No stabilizing













Lactose
Glucose
Maltose
D-Mannitol
agent (Control)









Storage



















70° C.

70° C.

70° C.

70° C.

70° C.



Initial
3 days
Initial
3 days
Initial
3 days
Initial
3 days
Initial
3 days





Appearance
Pale
Pale
White
Pale
White
Pale
White
Pale
Pale
Pale



yellow
yellow
mass
yellow
mass
yellow
mass
yellow
yellow
yellow



mass
mass

mass

mass

mass
mass
mass


Color and
Pale
Pale
Pale
Pale
Pale
Pale
Pale
Yellow
Pale
Yellow


clarity
yellow
yellow
yellow
yellow
yellow
yellow
yellow
and
yellow
and



and
and
and
and
and
and
and
clear
and
clear



clear
clear
clear
clear
clear
clear
clear

clear


pH
5.86
4.70
6.23
4.32
6.08
5.06
6.13
3.97
5.10
4.02


Residual rate (%)
100.0
80.5
100.0
37.3
100.0
80.9
100.0
1.38
100.0
51.2


Reconstitution
15
15
15
15
15
15
30
50
15
15


time(s)









The stabilizing effect of other non-reducing sugars such as sucrose and trehalose, as well as polyvinylpyrrolidone (PVP), was also evaluated in a ceftolozane formulation.
Five samples were prepared, the components of which are shown in Table 2B below. Each sample contained 1000 mg of ceftolozane active, 40 mg citric acid monohydrate (equivalent of 36 mg citric acid anhydrous), and the same amount of L-arginine. Stabilizing reagents in four samples are 480 mg sodium chloride, 300 mg of trehalose, 300 mg of sucrose, and 300 mg of PVP, respectively. One sample was a control that contained no stabilizing reagent. The samples were in lyophilized form and stored at 60° C. for 7 days. The purities of the samples were monitored by HPLC on day 0, day 1, day 3 and day 7.







TABLE 2B







Comparison between stabilizing reagents












Excipient
NaCl
Trehalose
Sucrose
PVP
None















Ceftolozane
1000
1000
1000
1000
1000


amount


Excipient
480
300
300
300
N/A


amount


Purity: t0
98.42
98.09
98.14
97.89
97.94


60° C./1 d
97.85
96.73
96.97
96.05
96.15


60° C./3 d
97.21
95.36
95.81
94.57
94.53


60° C./7 d
95.65
94.21
94.19
92.78
92.06


Purity Δ
−2.77%
−3.88%
−3.95%
−5.11%
−5.88%


(0-7 d)









As shown in Table 2B, the sample containing sodium chloride exhibited the best stability. The purity of ceftolozane in the sample containing sodium chloride had the slightest purity drop over 7 days. This experiment further supports the discovery that sodium chloride provides surprisingly better stabilizing effect than the other reagents.
Example 3: Stability Study of Ceftolozane Compositions Comprising Sodium Chloride, or Maltose, or No Stabilizing Agent
Three ceftolozane compositions were prepared, the components of which are shown in Table 3. These compositions were put in a stressed stability study at 70° C. for 3 days and 6 days. The purity of the ceftolozane in the compositions was analyzed using the HPLC method described in Example 1.







TABLE 3





Ceftolozane Compositions




















CEF/no stabilizer
9.5
g active
Ceftolozane




5.7
g
L-Arginine




200
mg
Citric acid



CEF/maltose
9.5
g active
Ceftolozane




5.7
g
L-Arginine




200
mg
Citric acid




5
g
Maltose H2O



CEF/sodium chloride
9.5
g active
Ceftolozane




5.7
g
L-Arginine




200
mg
Citric acid




4.6
g
Sodium Chloride










The results are shown in Table 4 where only the most significant composition peaks (P1, P7, and P12) are shown. It was found that the composition comprising maltose (CEF/maltose) contained a significantly large amount of the composition P12 peak, which was identified as having the following formula:




In addition, the presence of maltose produced a particularly aggregated powder after lyophilization, which has a potentially negative impact to manufacturing ceftolozane compositions.
In contrast, the ceftolozane composition comprising sodium chloride (CEF/sodium chloride) was much more stable than the ceftolozane composition comprising maltose or the ceftolozane composition comprising no stabilizing agent. Therefore, sodium chloride was, unexpectedly, a better stabilizing agent for ceftolozane compositions.







TABLE 4







Stability Study of Ceftolozane Compositions Comprising


Sodium Chloride, or Maltose, or No Stabilizing Agent











Time (days)
P1
P7
P12
Total










CEF/no stabilizer











0
0.49
0.69
0.00
1.98


3
3.06
1.29
0.00
8.48


6
4.11
1.49
0.00
10.83







CEF/maltose











0
0.41
0.65
0.15
1.91


3
2.85
1.02
3.44
10.08


6
3.45
1.12
4.01
11.65







CEF/sodium chloride











0
0.20
0.62
0.00
1.64


3
1.70
0.85
0.00
4.29


6
2.86
1.05
0.00
6.70









Example 4a: Manufacturing Procedure of Mono Product for Injection
4a.1. Preparation of the Compound Solution of CXA-101 Lyophilized Product
1) Weigh 30 kg of water for injection into the compounding vessel;
2) Add 100 g of citric acid, anhydrous and 150 g of sodium bicarbonate into the compounding vessel and dissolve them with mixing;
3) Weigh 5,000 g potency of CXA-101 drug substance and suspend it with mixing. (Note any generation of carbon dioxide.)
4) Slowly add 1,100 g of sodium bicarbonate and dissolve CXA-101 with mixing. (Again, note any generation of carbon dioxide.)
5) Add 1,146 g of sodium chloride and 10,000 g of maltose, dissolve with mixing.
6) Purge dissolved carbon dioxide in the solution with nitrogen until the pH of the solution does not change.
7) Adjust the pH of the solution to 6.0±0.1 with 5%-sodium bicarbonate solution.
8) Adjust the total weight to 56,850 g (D20=1.137) with water for injection.
9) Confirm the pH of the compounded solution within the range 6.0±0.1.
4a.2. Prefiltration and Sterile-Filtration
10) Filtrate the compounded solution with a sterile tilter-set which consists of a 0.2 um polyvinylidene fluoride membrane filter (DURAPORE®, Millipore) and a 0.1 urn polyvinylidene fluoride membrane filter (DURAPORE®, Millipore) connected in tandem. Confirm the integrity of each filter before and after the filtration. Take approximately 100 mL of the filtrate in order to check bioburden.
11) Filter the prefiltered compounded solution through a sterile filter-set which consists of a 0.2 um polyvinylidene fluoride membrane filter and a 0.1 urn polyvinylidene fluoride membrane filter connected in tandem, and introduce the final filtrate into an aseptic room. Confirm the integrity of each filter before and after the filtration.
4a.3. Processing of Container, Stopper and Flip-Off Cap
12) Wash a sufficient quantity of 28 mL containers with water for injection and sterilize the washed containers by a dry-heat sterilizer. Then transfer the sterilized containers into a Grade A area located in an aseptic room.
13) Wash a sufficient quantity of stoppers with, water for injection. Sterilize and dry the washed stoppers by steam sterilizer. Then transfer the sterilized stoppers into a Grade A area located in an aseptic room.
14) Sterilize a sufficient quantity of flip-off caps by steam sterilizer. Then transfer the sterilized flip-off caps into a Grade A or B area located in an aseptic room.
4a.4. Filling and Partially Stoppering
15) Adjust the fill weight of the filtered compounded solution to 11.37 g (corresponds to 10 mL of the compounded solution), then start filling operation. Check the filled weight in sufficient frequency and confirm it is in target range (11.37 g±1%, 11.26 to 11.43 g). When deviation from the control range (11.37 g±2%, 11.14 to 11.59 g) is occurred, re-adjust the filling weight.
16) Immediately after a container is filled, partially stopper the container with a sterilized stopper. Load the filled and partially stoppered containers onto the shelves of a lyophilizer aseptically.
4a.5. Lyophilization to Crimping, Visual Inspection, Labeling and Packaging
17) After all filled and partially stoppered containers are loaded into a lyophilizer, start the lyophilization program. Freeze the loaded containers at −40° C. and keep at temperature until all containers freeze. Forward the program to primary drying step (shelf temperature; −20° C., chamber pressure; 100 to 150 mTorr). Primary drying time should be determined by monitoring the product temperature. Forward the program to secondary drying step (shelf temperature; 30° C., chamber pressure; not more than 10 mTorr) after completion of the primary drying step. After all containers are dried completely, return the chamber pressure to atmospheric pressure with sterilized nitrogen. Then stopper containers completely.
Example 4b: Manufacturing Procedure of Bulk (Tray) Lyophilized Ceftolozane
There are four main steps in the manufacture of a CXA-101 pharmaceutical composition: dissolution, sterile filtration, bulk lyophilization, and packaging into Sterbags®. These four main steps are composed of a total of 19 minor steps. The CXA-101 bulk drug product manufacturing process is presented below.
I. Dissolution
1. The prescribed amount of water for injection (“WFI”) is charged into the dissolution reactor.
2. A prescribed amount of citric acid is added.
3. The solution is cooled at 5° C. to 10° C.
4. A prescribed amount of CXA-101 drug substance is added to the solution.
5. A prescribed amount of L-arginine is slowly added to the solution.
6. A check for complete dissolution is performed. Solution pH is verified to be in the target range of 6.5 to 7.0.
7. A prescribed amount of sodium chloride is added to the solution, wherein the preferred amount of sodium chloride is 125-500 mg of sodium chloride per 1000 mg of ceftolozane active.
8. A check for complete dissolution is performed. Solution pH is verified to be in the target range of 6.0 to 7.0. If the pH is out of this range adjust with either L-Arginine or citric acid.
9. WFI is added to bring the net weight to adjust compounded solution concentration.
10. Samples are withdrawn for testing of final pH.
II. Sterile Filtration
11. The solution is passed through the filter (pore size 0.45 μm) followed by double filters (pore size 0.22 μm) onto a shelf on the Criofarma lyophilizer.
12. The line is washed with WFI.
13. The washing solution is passed from Step 12 through sterile filtration.
III. Bulk Lyophilization
14. The washing solution is loaded onto a separate shelf in the lyophilizer (and later discarded).
15. The solution is lyophilized until dry.
16. The product shelf is cooled to 20° C.±5° C.
IV. Packaging into STERBAGS®
17. The lyophilized bulk drug product powder is delumped.
18. The milled powder is sieved.
19. The powder is then discharged into STERBAGS®
Example 5: Stabilizing Effect of Sodium Chloride in CXA-101 Compositions
A. Improvement in the Purity of the Ceftolozane in CXA-101 Pharmaceutical Compositions with Varying Amounts of Sodium Chloride
A stability study was carried out at 30° C. and 60° C. and analyzed by HPLC. The sodium chloride content in the CXA-101 compositions is described in Table 5. The HPLC data are summarized in Tables 6-8. The data are also plotted in FIGS. 3-6 to show the trends of the purity, and the amounts of the composition peak 1, the composition with a RRT of 0.43 and the composition peak 3, and the composition peak 7 in the CXA-101 compositions with respect to NaCl.







TABLE 5







Sodium Chloride Content in the CXA-101 Compositions










Samples
NaCl content







A1
481.0 mg NaCl per 1000 mg of ceftolozane



A2
190.0 mg NaCl per 1000 mg of ceftolozane



A3
125.0 mg NaCl per 1000 mg of ceftolozane



A4
 75.0 mg NaCl per 1000 mg of ceftolozane



A5
 50.0 mg NaCl per 1000 mg of ceftolozane

















TABLE 6 







Purity of Ceftolozane in CXA-101 Compositions


with Varying Amounts of Sodium Chloride














Day
A1
A2
A3
A4
A5

















t0/60° C.
0
96.6
98.0
97.9
97.8
97.7


t0/30° C.
0
98.1

97.8
97.8
97.7


1 day/60° C.
1
95.9
96.9
96.5
95.7
95.5


1 day/30° C.
1
98.2

97.7
97.7
97.6


3 days/60° C.
3
94.9
95.7
94.8
93.9
93.6


(Δt0-t3)

(1.7)
(2.3)
(3.1)
(3.9)
(4.1)


3 day/30° C.
3
98.0

97.5
97.5
97.3


7 days/60° C.
7
93.6
94.0
94.2
92.3
91.9


7 day/30° C.
7
97.8

97.2
97.1
97.0


Total Δ/60° C.

3.07
4.06
3.7
5.48
5.83


Total Δ/30° C.

0.3

0.6
0.7
0.7
















TABLE 7







HPLC Peak Area of Composition Peak 1 in CXA-101 Compositions


with Varying Amounts of Sodium Chloride














Day
A1
A2
A3
A4
A5

















t0/60° C.
0
0.95
0.31
0.3
0.36
0.39


t0/30° C.
0
0.47

0.36
0.36
0.39


1 day/60° C.
1
1.36
0.86
0.94
1.36
1.39


1 day/30° C.
1
0.48

0.40
0.42
0.48


3 days/60° C.
3
1.71
1.31
1.73
2.06
2.1


3 day/30° C.
3
0.53

0.50
0.52
0.58


7 days/60° C.
7
2.26
2.14
2.07
2.86
2.93


7 day/30° C.
7
0.62

0.63
0.66
0.72


INCREASE %/60° C.

1.31
1.83
1.77
2.5
2.54


INCREASE %/30° C.

0.15

0.27
0.30
0.33
















TABLE 8A







HPLC Peak Area of the Composition with a RRT of


0.43 and Composition Peak 3 in CXA-101 Compositions


with Varying Amounts of Sodium Chloride














Day
A1
A2
A3
A4
A5

















t0/60° C.
0
0.28
0.10
0.09
0.10
0.11


t0/30° C.
0
0.15

0.10
0.10
0.11


1 day/60° C.
1
0.37
0.13
0.16
0.35
0.36


1 day/30° C.
1
0.13

0.09
0.09
0.10


3 days/60° C.
3
0.68
0.21
0.31
0.71
0.71


3 day/30° C.
3
0.17

0.13
0.13
0.14


7 days/60° C.
7
1.04
0.36
0.30
0.81
0.81


7 day/30° C.
7
0.19

0.16
0.16
0.17


INCREASE %/60° C.

0.76
0.26
0.21
0.71
0.7


INCREASE %/30° C.

0.04

0.06
0.06
0.06
















TABLE 8B







The HPLC Peak Area of Composition Peak 7 in CXA-101


Compositions with Varying Amounts of Sodium Chloride














Day
A1
A2
A3
A4
A5

















t0/60° C.
0
1.31
0.95
0.96
1.01
1.02


t0/30° C.
0
0.69

1.00
1.01
1.02


1 day/60° C.
1
1.37
1.10
1.10
1.23
1.29


1 day/30° C.
1
0.68

0.99
1.01
1.02


3 days/60° C.
3
1.43
1.19
1.27
1.41
1.46


3 day/30° C.
3
0.68

1.03
1.01
1.05


7 days/60° C.
7
1.49
1.31
1.35
1.55
1.57


7 day/30° C.
7
0.68

1.01
1.03
1.07


INCREASE %/60° C.

0.18
0.36
0.39
0.54
0.55


INCREASE %/30° C.

NC

0.01
0.02
0.05









Conclusion:
The stability test demonstrates that high sodium chloride content enhances stability of CXA-101 Compositions.
The HPLC measurements on day 3 were used to analyze the stability of the CXA-101 compositions.
CXA-101 compositions comprising high amounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane) were found to be more chemically stable than CXA-101 compositions comprising low amounts of sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mg of ceftolozane). Table 6 shows that, by day 3 of heating at 60° C., sample A1, which has the highest salt concentration, is most stable, i.e., has the lowest Δt0-t3 of all samples. By day 3, the sample with the lowest salt concentration, A5, has the highest Δt0-t3 indicating the most degradation. Overall, A5 has degraded 141% more than A1. Further, Table 6 shows that, by day 3 of heating at 60° C., sample A3, which contains a lower salt concentration within the limits of the invention at 125 mg, is still significantly more stable than A4, a composition containing 75.0 mg of the salt. A3 has a Δt0-t3 of 3.1, while A4 has a Δt0-t3 of 3.9, meaning that A4 has degraded 26% more than A3.
B. Long-Term Stability Study of CXA-101 Pharmaceutical Compositions with Varying Amounts of Sodium Chloride
Another stability study was carried out at 5° C. and 25° C. The sodium chloride content in the CXA-101 compositions is described in Table 9. The amounts of citric acid and L-arginine in each composition were the same. These samples were in lyophilized form and were placed on long-term (24-36 months), real time stability programs.
The composition peak 1 is considered “diagnostic” for formulation failure because it is the first peak to go out of trend or specification (1.5%). Thus, the stability of these CXA-101 compositions was also measured by the length of storage until formulation failure as indicated by the composition peak 1. The data in Table 9 were extrapolated from data collected after 4 months. Clearly, based on the amount of the composition peak 1 in the compositions, the composition with about 480 mg sodium chloride per 1 gram ceftolozane active was significantly more stable than the compositions containing 125 mg or 62.5 mg sodium chloride per 1 gram of active ceftolozane (i.e., stability of ceftolozane compositions: 480>>125 mg>62.5 mg).







TABLE 9







The Peak 1 Failure Points of CXA-101 Compositions


with Varying Amounts of Sodium Chloride










Peak 1 failure
Peak 1 failure


Ceftolozane active, 1 g+
point at 5° C.
point at 25° C.





480 mg NaCl
245 months 
15 months 


125 mg NaCl
70 months
5 months


62.5 mg NaCl 
25 months
 3 months*





*Results at 3 months = 1.34%,


4 months = 1.15%






Example 6: Manufacturing Process of a CXA-201 Composition Comprising Tazobactam and CXA-101/Ceftolozane by Co-Lyophilization
The manufacturing process of a CXA-201 composition comprising tazobactam and ceftolozane by co-lyophilization is shown in FIG. 2. Non-sterile bulk tazobactam and bulk ceftolozane were mixed, followed by dissolution and sterile filtration. The filtrate was then tray-lyophilized to obtain the CXA-201 composition. The CXA-201 composition can be container-filled as a final drug product. The components of a CXA-201 composition prepared by co-lyophilization are shown in Table 10.







TABLE 10







Components of a CXA-201 Composition


Prepared by Co-lyophilization









Component
Function
Amount (mg/container)





Ceftolozane
Active pharmaceutical
1000 (potency)



ingredient


L-arginine
Alkalization reagent
587


Citric acid (anhydrous)
Buffer
 21


Sodium chloride
Stabilizer
476


Tazobactam (free acid)
Active pharmaceutical
500



ingredient


Sodium bicarbonate
Alkalization reagent
Quantity sufficient1 for




pH 4.8 to 7.0


Water
Dissolution solvent
Not more than 4% by




weight2


Nitrogen
Inert gas
Sufficient quantity





1Sodium content is approximately 78 mg/g of tazobactam in drug product after lyophilization.


2Water is removed during the lyophilization process and is controlled at no more than 4% by weight.






Example 7a: Assessment of Co-Lyophilized Combo Drug Product (i.e., a CXA-201 Composition)
A. Preparation of the Co-Lyophilized Combo Drug Product (i.e. the CXA-201 Composition)
The components of the co-lyophilized CXA-201 composition are shown in Table 11. This composition was prepared, as described above in Example 6.







TABLE 11





Components of the CXA-201 Compo-


sition Prepared by Co-Lyophilization


CXA-201 Comp.

















16.3
g active
ceftolozane


8.1
g active
Tazobactam free ac.


15.5
g
L-Arginine


350
mg
Citric acid


7.9
g
NaCl










6.1
pH compounded solution










B. Stressed Stability Test

Stability studies of this CXA-201 composition prepared by co-lyophilization were carried out at 25° C. and 40° C. The composition was analyzed using HPLC. The following Tables 12 and 13 are summaries of the HPLC measurements at time zero, after one month (T1), and after three months (T2).







TABLE 12







Stability Data of Co-Lyophilized CXA-201 Composition


at 25° C./RH = 60%











Test items
Spec. D.P.
T0
T1 25° C.
T2 25° C.














Related Substances






Peak1
≦1.50%
0.31%
0.54%
0.71%


Peak2
≦0.40%
0.07%
0.07%
0.09%


Peak3
≦0.30%
<0.03%
<0.03%
<0.03%


Peak4
≦0.80%
0.08%
0.08%
0.09%


Peak5
≦1.00%
0.27%
0.26%
0.29%


Peak6
≦0.15%
<0.03%
<0.03%
<0.03%


Peak7
≦2.00%
0.64%
0.65%
0.66%


Peak8
≦0.15%
<0.03%
<0.03%
<0.03%


Peak9
≦0.60%
0.05%
0.11%
0.10%


Peak10, 11
≦0.15% each
0.04%
0.04%
0.04%


Peak12
≦2.00%
<0.03%
<0.03%
<0.03%


Others (RRT 0.43)
≦0.15%
<0.03%
<0.03%
0.04%


Others (RRT 1.22)
≦0.15%
0.13%
0.30%
0.38%


Others (RRT 2.18)
≦0.15%
0.03%
<0.03%
0.05%


Others (RRT 2.77)
≦0.15%
<0.03%
0.03%
0.03%


Sing. Unk.
≦0.15%
0.05%
0.07%
0.05%


Total
≦5.00%
1.67%
2.19%
2.77%


pH
report value
5.5

4.83
















TABLE 13







Stability Data of Co-Lyophilized CXA-201 Composition


at 40° C./RH = 75%











Test items
Spec. D.P.
T0
T1 40° C.
T2 40° C.














Related Substances






Peak1
≦1.50%
0.31%
1.77%
2.22%


Peak2
≦0.40%
0.07%
0.10%
0.16%


Peak3
≦0.30%
<0.03%
<0.03%
0.06%


Peak4
≦0.80%
0.08%
0.09%
0.09%


Peak5
≦1.00%
0.27%
0.27%
0.30%


Peak6
≦0.15%
<0.03%
<0.03%
<0.03%


Peak7
≦2.00%
0.64%
0.69%
0.78%


Peak8
≦0.15%
<0.03%
<0.03%
0.10%


Peak9
≦0.60%
0.05%
0.09%
0.09%


Peak10, 11
≦0.15% each
0.04%
0.04%
0.05%


Peak12
≦2.00%
<0.03%
<0.03%
<0.03%


Others (RRT 0.43)
≦0.15%
<0.03%
0.09%
0.15%


Others (RRT 1.22)
≦0.15%
0.13%
0.74%
0.97%


Others (RRT 2.18)
≦0.15%
0.03%
<0.03%
0.08%


Others (RRT 2.77)
≦0.15%
<0.03%
<0.03%
0.04%


Sing. Unk.
≦0.15%
0.05%
0.11%
0.25%


Total
≦5.00%
1.67%
4.49%
6.32%


pH
report value
5.5

4.09









C. Conclusion:

A new compound having RRT=1.22 was observed in the co-lyophilized CXA-201 compositions. While not wishing to be bound by theory, the compound RRT 1.22 was identified as a compound formed by a reaction between ceftolozane and formylacetic acid, which was a by-product of tazobactam as illustrated in Marunaka et al. (Chem. Pharm. Bull. 1988, Vol. 36 (11), pp. 4478-4487). The stability data at 25° C. and at 40° C. have confirmed the continued formation of the compound RRT 1.22 over the course of time.
Example 7b: Identifying the Compound of Formula (III)
The Co-Lyophilized Combo Drug Product was prepared as described above in Example 6. The formulation composition of the Co-Lyophilized Combo drug product is shown in Table 11 (Example 7a). This sample maintained at 25° C./RH=60% and 40° C./RH=75% after one month (T1) and three months (T2). Samples were analyzed using a HPLC method as described in Example 1. The data for analysis of the samples by HPLC is shown in Example 10 in Table 23 (Stability data of Co-Lyo Combo Drug Product at 25° C.) and Table 24 (Stability data Co-Lyo Combo Drug Product at 40° C.). The presence of the compound of Formula (III) was identified has having a retention time of about 1.22 as measured by HPLC (see Example 2). RRT=1.22 was observed in co-lyophilized drug product. The compound of formula (III) is believed to be formed by a reaction between ceftolozane and formylacetic acid, which was a degradation product of tazobactam. The amount of the compound of formula (III) in a composition comprising ceftolozane and tazobactam can be increased over time at 25° C. and at 40° C.
The material obtained from the RRT 1.22 peak was analyzed by LC/MS, providing the spectra shown in FIG. 14. FIG. 15 (below) is the corresponding structures for the peaks shown in FIG. 14.
A test sample prepared from ceftolozane and tazobactam acid co-compounding solution containing RRT 1.22 impurity was used on the LC/MS experiment. Liquid chromatography separation was performed on a Zorbax SB C8, 3.5 μm, 3.0 mm×150 mm column, using gradient elution with 20 mM ammonium formate containing 0.1% Heptofluorobutyric acid pH 3.2 as mobile phase A and 0.1% Heptofluorobutyric acid in acetonitrile as mobile phase B. The gradient starts from 3% (initial) to 15% mobile phase B in 20 minutes (with RRT 1.22 eluting at about 10.7 minutes). Mass detection was performed using electrospray ionization technique under positive mode. The column effluent was also monitored at 254 nm using a photodiode-array detector. MS/MS fragmentation was performed on m/z 737.3 positive ion using nitrogen as collision gas, with collision energy set at 35V.
Example 8: Stabilizing Effect of Sodium Chloride in CXA-201 Compositions
A. Reduction of the Composition at RT=63 Minutes in CXA-201 Compositions
A stability study was carried out at 25° C. and analyzed by HPLC. CXA-201 compositions comprise ceftolozane and tazobactam, further comprising high, mid, or low amounts of sodium chloride (480, 125, or 62.5 mg NaCl per 1000 mg of ceftolozane, respectively). Comparison of the compositions are listed in Table 14. The amounts of the composition RT 63′, as measured by the HPLC method, are summarized in Table 15.







TABLE 14







Comparison of the CXA-201 Compositions












Lot
CXA-101
NaCl
Tazobactam







C1
10%
High
Na



C2
20%
Mid
Na



C3
20%
Low
Na



C4
20%
Mid
Arginate



C5
20%
Low
Arginate

















TABLE 15







RT 63′ Peak Area at t = 3 months, 25° C./60% RH storage











1st data
2nd data
3rd data



collection
collection
collection














Sam-


Area

Area

Area


ple
Summary
RT
%
RT
%
RT
%





C1
High salt +
63.90
0.03
63.30
0.08
62.49
0.14



Tazo Na


C2
Mid salt +
63.78
0.06
63.12
0.12
62.45
0.28



Tazo Na


C3
Low salt +
63.75
0.12
63.11
0.14
62.46
0.29



Tazo Na


C4
Mid salt +
63.76
0.10
63.16
0.13
62.44
0.28



Tazo Arg


C5
Low salt +
63.72
0.08
63.14
0.16
62.46
0.33



Tazo Arg









Conclusion:
At the three month time point, the reduced salt formulations were observed to be not as stable as the full salt formulation; and trends indicate that reduction in salt causes at least 1.5-fold greater composition at RT=63 minutes, as measured by HPLC. The compositions comprising 480 mg NaCl per 1000 mg of ceftolozane had the least amount of the composition RT 63′ after 3 months at 25° C. The amount of the composition RT 63′ in the compositions comprising 125 mg NaCl per 1000 mg of ceftolozane was 1.5-fold or greater than the amount of the composition 63′ in the compositions comprising 480 mg NaCl per 1000 mg of ceftolozane. The amount of the composition RT 63′ in the compositions comprising 62.5 mg NaCl per 1000 mg of ceftolozane was 2-fold or greater than the amount of the composition RT 63′ in the compositions comprising 480 mg NaCl per 1000 mg of ceftolozane. Thus, the CXA-201 compositions comprising high amounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane) were more chemically stable than the compositions comprising low amounts of sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mg of ceftolozane).
B. Improvement in the Purity of Ceftolozane in CXA-201 Pharmaceutical Compositions with Varying Amounts of Sodium Chloride
A stability study was carried out at 30° C. and 60° C. analyzed by HPLC. The sodium chloride content in the CXA-201 compositions is described in Table 16. The HPLC data at 60° C. are summarized in Tables 17-20. The data are also plotted in FIGS. 7-10 to show the trends of the purity, and the amounts of the composition peak 1, the composition with a RRT of 0.43 and the composition peak 3, and the composition peak 7 in the CXA-201 compositions with respect to NaCl.







TABLE 16







The Sodium Chloride Content in the CXA-201 Compositions










Samples
NaCl content







B1
481.0 mg sodium chloride per 1000 mg of ceftolozane



B2
125.0 mg sodium chloride per 1000 mg of ceftolozane



B3
 75.0 mg sodium chloride per 1000 mg of ceftolozane



B4
 50.0 mg sodium chloride per 1000 mg of ceftolozane

















TABLE 17







The Purity of Ceftolozane in CXA-201 Compositions


with Varying Amounts of Sodium Chloride













Day
B1
B2
B3
B4
















t0
0
98.1
97.8
97.8
97.7


1 day/60° C.
1
97.2
96.3
96.2
96.0


1 day/30° C.
1
98.2
97.7
97.6
97.6


3 days/60° C.
3
95.4
94.9
94.7
94.6


(Δt0−t3)

 (2.7)
 (2.9)
 (3.1)
 (3.1)


3 day/30° C.
3
98.0
97.5
97.4
97.3


7 days/60° C.
7
92.7
93.8
93.6
93.4


7 day/30° C.
7
97.8
97.2
97.0
96.9


Total Δ/60° C.

 5.3
 4.0
 4.2
 4.3


Total Δ/30° C.

 0.3
 0.6
 0.8
 0.8
















TABLE 18







The HPLC Peak Area of Composition Peak 1 in CXA-201


Compositions with Varying Amounts of Sodium Chloride













Day
B1
B2
B3
B4
















t0
0
0.47
0.38
0.38
0.41


1 day/60° C.
1
1
1.08
1.09
1.14


1 day/30° C.
1
0.48
0.44
0.45
0.49


3 days/60° C.
3
1.85
1.64
1.66
1.71


3 day/30° C.
3
0.53
0.53
0.56
0.61


7 days/60° C.
7
3.3
2.28
2.25
2.29


7 day/30° C.
7
0.62
0.67
0.71
0.77


INCREASE %/60° C.

2.83
1.9
1.87
1.88


INCREASE %/30° C.

0.15
0.29
0.33
0.36
















TABLE 19







The Total HPLC Peak Area of the Composition with a RRT of 0.43 and


Composition Peak 3 in CXA-201 Compositions with Varying Amounts


of Sodium Chloride













Day
B1
B2
B3
B4





t0
0
0.15
0.12
0.12
0.12


1 day/60° C.
1
0.36
0.35
0.31
0.32


1 day/30° C.
1
0.13
0.12
0.13
0.12


3 days/60° C.
3
0.92
0.67
0.65
0.62


3 days/30° C.
3
0.17
0.16
0.17
0.16


7 days/60° C.
7
1.29
0.78
0.75
0.71


7 days/30° C.
7
0.19
0.19
0.20
0.20


INCREASE %/60° C.

1.14
0.66
0.63
0.59


INCREASE %/30° C.

0.04
0.07
0.08
0.08
















TABLE 20







The HPLC Peak Area of Composition Peak 7 in CXA-201


Compositions with Varying Amounts of Sodium Chloride













Day
B1
B2
B3
B4















t0
0
0.69
1.01
1.01
1.01


1 day/60° C.
1
0.73
1.12
1.15
1.18


1 day/30° C.
1
0.68
1.00
0.99
0.95


3 days/60° C.
3
0.8
1.24
1.27
1.27


3 days/30° C.
3
0.68
1.00
1.01
1.03


7 days/60° C.
7
0.94
1.32
1.35
1.4


7 days/30° C.
7
0.68
1.02
1.05
1.06


INCREASE %/60° C.

0.25
0.31
0.34
0.39


INCREASE %/30° C.

NC
0.01
0.04
0.05









Conclusion:
The stability data shows that high sodium chloride content enhances stability of CXA-201 compositions.
Similarly to CXA-101 compositions, CXA-201 compositions comprising high amounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane) were found to be more chemically stable than CXA-201 compositions comprising low amounts of sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mg of ceftolozane). Table 17 shows that, by day 3 of heating at 60° C., sample B1 containing the highest salt concentration is most stable, i.e., has the lowest Δt0-t3 of all samples. By day 3, the sample with the lowest salt concentration, B4, has the highest Δt0-t3 indicating the most degradation. Overall, B4 has degraded 15% more than B1.
Example 9: Manufacturing Process of a CXA-201 Composition (Comprising Tazobactam and Ceftolozane) by Blending
A. Sterile Dry Blending of Bulk Lyophilized Ceftolozane and Bulk Lyophilized Tazobactam
A low energy drum blender that agitates the material by tumbling and also moving the bed up and down is used. A representative process of blending is described as follows, also shown in FIG. 1. The blender was charged with 23.4 kg of CXA-101 bulk product, and 5.4 kg of tazobactam bulk product. Both the CXA-101 and tazobactam were individually lyophilized beforehand. The material was blended for 180 minutes. In-process tests of content assay for both CXA-101 and tazobactam were performed to assess the homogeneity using the samples of blend materials taken from three places. The relative standard deviation (RSD) for each of CXA-101 and tazobactam content assay was no greater than 2% and the RSD for the ratio of CXA-101/tazobactam was no greater than 2% (See Table 21).







TABLE 21







In-Process Testing of Blending Samples of a CXA-201 Composition at Three Places









Results













Acceptance Limits

60
120
180


Test
(expected value)
Sampling
minute
minute
minute















Content: Ceftolozane1
30.4%-37.2%
1
34.24
34.07
34.42




2
34.62
34.21
34.66




3
34.71
34.60
34.85




Mean3
34.52
34.30
34.64




RSD %
0.72
0.80
0.63


Content: Tazobactam2
15.2%-18.6%
1
17.96
18.20
17.12




2
16.90
18.26
16.51




3
17.27
16.93
17.02




Mean3
17.38
17.80
16.89




RSD %
3.10
4.22
1.96


Ratio of Content (w/w)
2.004
1
1.91
1.87
2.01


ceftolozane/tazobactam

2
2.05
1.87
2.10




3
2.01
2.04
2.05




Mean3
1.99
1.93
2.05




RSD %
3.69
5.12
2.2





RSD = relative standard deviation


1Theoretical value: 33.96% Acceptance limits are 90%-110% of the theoretical value.


2Theoretical value: 16.99% Acceptance limits are 90%-110% of the theoretical value.


3Three samples are taken at each time point at three places to measure the percentage by weight of ceftolozane and tazobactam. The “Mean” is the average of the percentages or the weight ratios of Ceftolozane/tazobactam.


4Acceptance limits were established based on batch history.






B. Packaging into STERBAGS®

The blended powder is then discharged into STERBAGS®.

C. Finished CXA-201 Drug Product

A fill and finish process is utilized for the final drug product, which is a pharmaceutical composition comprising CXA-101 and tazobactam at a ratio of 1000 mg/500 mg. Glass containers are washed with WFI and depyrogenated in a Class 100 depyrogenation tunnel at a temperature of 320° C. Pre-washed and pre-siliconized stoppers are autoclaved for 40 minutes at 121° C. The bulk drug product is packaged in a STERBAG® system comprised of three bags. The outer bag is cleaned with disinfectant in a Class 10,000 clean room. The bag system is placed in a pass-through UV box where it is subjected to UV radiation (>20 μW/cm2) for 20 minutes to sterilize the surface of the outer bag. The outer bag is removed and left in the UV box. The middle bag is placed in a Class A laminar airflow (LAF) hood. The sterile middle bag is removed under LAF. The sterile, bottle-shaped inner bag is then placed in a sterile stainless steel carrier and attached to the filling machine.
Sterile bulk CXA-101/tazobactam drug product is filled under a nitrogen blanket into 30-mL, Type I clear glass containers. The sterile drug product is gravity-fed into the filling machine under LAF. container fill weights are periodically checked throughout the filling operation to ensure proper operation of the filling line. Filling and stoppering operations are performed under Class 100 LAF conditions. Capping and container washing are done in the Class 10,000 clean room.
Example 10: Assessment of Blend Combination Drug Product
A. Preparation of the Blend Combination Drug Product (CXA-201 Composition)
The blend drug product was prepared, as described above in Example 9, on lab scale using a small blender. The components of the blend composition are shown in Table 22.







TABLE 22







Components of the Blend Composition











Quantity as active


Component
Composition
components





CXA-101 for Injection
Ceftolozane
10.8 g


Bulk (25 g)




CXA-201
L-Arginine
 6.7 g


Comp.
Citric acid
 233 mg



Sodium chloride
 5.2 g


Tazobactam sodium

 5.4 g (as Tazo free acid)


sterile Bulk (6 g)









B. Stressed Stability Test

Stability studies of this CXA-201 composition prepared by the blending process were carried out at 25° C. and 40° C. The composition was analyzed using HPLC method described in Example 1. The following Tables 23 and 24 are summaries of the HPLC measurements at time zero, after one month (T1), and after three months (T2).







TABLE 23







Stability Data of Blend CXA-201 Composition at 25° C./RH = 60%















T2


Test items
Specifications
T0
T1 25° C.
25° C.














Related Substances






Peak1
≦1.50%
0.61%
0.93%
1.08%


Peak2
≦0.40%
<0.03% 
<0.03%
<0.03%


Peak3
≦0.30%
<0.03% 
<0.03%
<0.03%


Peak4
≦0.80%
0.03%
0.03%
0.04%


Peak5
≦1.00%
0.09%
0.12%
0.13%


Peak6
≦0.15%
<0.03% 
<0.03%
<0.03%


Peak7
≦2.00%
1.28%
1.34%
1.35%


Peak8
≦0.15%
<0.03% 
<0.03%
<0.03%


Peak9
≦0.60%
0.03%
<0.03%
0.03%


Peak10, 11
≦0.30%
<0.03% 
0.04%
0.05%


Sing. Unk.
≦0.15%
0.13%
0.13%
0.14%


Total
≦5.00%
2.49%
3.03%
3.28%


Assay CXA-101
Teor. % = 32.6%
32.5%
n.a.
n.a.


Assay Tazobactam
Teor. % = 17.4%
18.2%
n.a.
n.a.


Tazobactam Related
 ≦4.0%
0.07%
0.12%
0.14%


Compound A






K.F.
 ≦4.0%
 2.6%
n.a.
n.a.


pH
5.0-7.0
6.0
5.6
5.1
















TABLE 24







Stability Data of Blend CXA-201 Composition at 40° C./RH = 75%















T2


Test items
Specifications
T0
T1 40° C.
40° C.














Related Substances






Peak1
≦1.50%
0.61%
1.66%
2.28%


Peak2
≦0.40%
<0.03% 
<0.03%
<0.03%


Peak3
≦0.30%
<0.03% 
<0.03%
0.04%


Peak4
≦0.80%
0.03%
0.04%
0.05%


Peak5
≦1.00%
0.09%
0.13%
0.14%


Peak6
≦0.15%
<0.03% 
<0.03%
<0.03%


Peak7
≦2.00%
1.28%
1.41%
1.46%


Peak8
≦0.15%
<0.03% 
<0.03%
<0.03%


Peak9
≦0.60%
0.03%
<0.03%
0.03%


Peak10, 11
≦0.30%
<0.03% 
0.08%
0.09%


Sing. Unk.
≦0.15%
0.13%
0.14%
0.13%


Total
≦5.00%
2.49%
4.21%
5.27%


Assay CXA-101
Teor. % = 32.6%
32.5%
n.a.
n.a.


Assay Tazobactam
Teor. % = 17.4%
18.2%
n.a.
n.a


Tazobactam Related
 ≦4.0%
0.07%
0.35%
0.54%


Compound A






K.F.
 ≦4.0%
 2.6%
n.a.
n.a.


pH
5.0-7.0
6.0
5.0
4.4









C. Conclusion

The data at both 25° C. and at 40° C. have shown that the blending process completely inhibits formation of the compound RRT=1.22.
Example 11: Alkalizing Agent Selection
Compositions for intravenous administration should be formulated to resemble the and pH of human blood to reduce vascular complications. The recommended pH is between 5 and 9 (ideal pH is as close to 7.4 as possible). Departing from this recommended pH ranges of an intravenously administered composition can result in the development of complications such as phlebitis, or inflammation of the veins. Marc Stranz, A Review of pH and Osmolarity, 6 Int'l J. of Pharm. Compounding 216, 218 (May/June 2002). Unfortunately, few drug infusions are stable at a suitable pH for intravenous administration. Depending on the molecular structure, a drug is most stable or has the best solubility at a particular pH range (e.g., pH<6) and divergence from this pH range may lead to increased drug decomposition. It is thus challenging to find a balance between the safe limits of pH and optimum drug stability in compositions for intravenous administration. Marc Stranz, The Implications of Osmolality, Osmolarity and pH in Infusion Therapy, INS Annual Conference (May 2005).
A formulation close to physiologic pH was targeted. This necessitates an alkalizing agent due to intrinsic pH 1.92 of ceftolozane in solution (2%). The initial study of alkalizing agents included sodium hydroxide, L-arginine, tris, sodium bicarbonate, meglumine, diethanolamine, and triethanolamine. Samples containing 100 mg ceftolozane sulfate, 22.9 mg sodium chloride, 200 mg maltose, and 2 mg citric acid anhydrous were prepared and adjusted to  ̃pH 4. The samples were lyophilized and powders stored at 70° C. for 3 days, 60° C. for 3, 6 and 9 days and at 40° C. for one month. The stored samples were then analyzed for ceftolozane content. Results are reported below in Table 25:







TABLE 25







Effect of Alkalizing Agent on Ceftolozane Recovery















Sodium


Sodium

Diethanol-
Triethanol-


Storage
hydroxide
L-arginine
Tris
bicarbonate
Meglumine
amine
amine





70° C., 3 d
93.3
93.0
83.1
93.8
71.2
52.7
28.0


60° C., 3 d
97.0
96.3
93.5
93.9
94.4
91.6
67.2


60° C., 6 d
95.7
95.5
89.8
96.0
89.8
83.6
59.0


60° C., 9 d
93.9
93.1
87.5
93.8
88.7
82.0
75.9


40° C./75%
97.3
97.0
95.1
97.6
97.6
94.4
94.4


RH, 1 mo









Ceftolozane recovery was consistently above 90% in the presence of sodium hydroxide, L-arginine, or sodium bicarbonate. Although sodium hydroxide performed well, as a strong base, it could promote base hydrolysis of the active more readily during scale up and be more difficult to dry during lyophilization than other alkalizing agents. Accordingly sodium hydroxide was not considered for further formulation development. L-arginine was thus chosen as the alkalizing agent for the formulation.
To ensure suitability of L-arginine as an alkalizing agent, a study was conducted to compare L-arginine against sodium bicarbonate. In this study, solutions were prepared to contain ceftolozane in the presence of sodium chloride and citric acid adjusted to approximately pH 6 with either L-arginine or sodium bicarbonate. The solutions were then lyophilized and samples distributed for accelerated and regular storage. A summary of the total additional compounds and pH for the various conditions after 1 month is presented in Table 26.







TABLE 26







Effect of L-Arginine and Sodium Bicarbonate on Ceftolozane


Related Substances during Storage, pH 6









Bulk solution composition



per 1000 mg ceftolozane free base










632 mg L-arginine
288 mg sodium



485 mg
bicarbonate



sodium chloride
481 mg sodium chloride



21 mg citric acid
21 mg citric acid












Total Related
Sample
Total Related
Sample


Storage condition
Substances
pH
Substances
pH





Initial
1.42%
5.8
2.12%
5.8


 5° C., 1 month
1.38%
5.8
2.66%
5.6


25° C., 1 month
1.74%
5.5
4.99%
4.8


40° C., 1 month
2.32%
5.0
5.93%
4.5









As seen in the table the bicarbonate-adjusted sample showed a larger increase in related substances and a less stable pH profile. Accordingly, it was decided to maintain L-arginine as the alkalizing agent in the formulation.
Example 12: Components of a CXA-201 Composition
An example of a batch formulae for ceftolozane composition (compounding of ceftolozane substance with excipients such as citric acid, sodium chloride, and L-arginine followed by sterile lyophilization) is found below in Table 27.







TABLE 27







Batch Formula for Ceftolozane composition











Amount per



Target Composition
Batch (kg)










Component
mg/g
1
2













Ceftolozane Sulfatei)
172.1
114.0
202.6


Citric Acid, Anhydrous, USP
3.2
2.1
3.7


Sodium Chloride, USP
73.1
48.3
86.0


L-Arginine, USP
~90
59.7
106.0



QS to achieve target pHii)




Water for Injection, USP
QS to 1000
QS
QS









Total Batch Size
660
1175





1)Ceftolozane sulfate is charged based on its measured potency to obtain 150 mg free base/g solution.


2)L-arginine is added as needed to obtain pH 6.5 ± 0.5 in the bulk solution; 90 mg per gram solution is considered a representative amount.






An example of a batch formula for the ceftolozane/tazobactam drug product is presented in Table 28 below.







TABLE 28







Batch Formula Ceftolozane/Tazobactam Drug Product Composition












Amount per
Amount per Batch,



Component
container, mg
kg














Ceftolozane
2255
112.8



compositioni)





Tazobactamii)
537
26.9



Nitrogen, NFiii)
—
—



Total
2792
139.7








Total Batch Size, kg
139.7


Total container Quantity
50,000





i)The target fill for ceftolozane is 1000 mg free base, added to the container as the composition. The amount 2255 mg is based on 100% theoretical potency of the composition. Actual weight will vary based on composition measured potency.


ii)The target fill for tazobactam is 500 mg free acid, added to the container as its sodium salt form. The amount 537 mg is based on 100% theoretical potency.


iii)Nitrogen is used as a processing aid to blanket containers after powder filling and prior to insertion of stopper.






The unit composition of a dosage for reconstitution is described in Table 29.








TABLE 29







Unit Compositions of Ceftolozane/Tazobactam for Injection,


1000 mg/500 mg











Nominal Composition


Component
Function
mg per container













Ceftolozane
Ceftolozane
Active
1147


composition1)
Sulfate





Citric Acid,
Chelating Agent
21



Anhydrous





Sodium
Stabilizing Agent
487



Chloride





L-Arginine
Alkalizing Agent
6002) Q.S. for pH





adjustment









Tazobactam Sodium3)
Active
537


Nitrogen
Processing Aid(a)
Q.S.








Total Weight
2792





1)Actual amount of ceftolozane composition will vary based on the measured potency. Ceftolozane sulfate, 1147 mg, corresponds to 1000 mg ceftolozane free base.


2)L-arginine is added as needed to achieve pH 6.5 ± 0.5; 600 mg per container is considered a representative total amount.


3)Actual weight of tazobactam sodium will vary based on the measured potency. Tazobactam sodium 537 mg, corresponds to 500 mg tazobactam free acid


4)Nitrogen blanket is applied after powders are dispensed to the container and prior to insertion of stopper.






Example 12a: Development and Implementation of a System to Prevent Cross-Contamination in Accordance with FDA Guidance
A recently published (April 2013) Food and Drug Administration Guidance for Industry Non-Penicillin Beta-Lactam Drugs: A CGMP Framework for Preventing Cross-Contamination provides direction on prevention of cross-contamination for facilities that manufacture non-penicillin beta-lactam drugs. Provided herein are steps for the development and implementation of a system to prevent cross-contamination due to the introduction of both sterile ceftolozane drug product intermediate and tazobactam sodium into a facility that is in conformance with FDA Guidance.
Segregation steps to conform with FDA Guidance can include, but are not limited to:

    
    
        Relocation all other drug products to other sites
        Separating the ceftolozane/tazobactam product filling line and the veterinary cephapirin product filling line
        Creating separate HVAC systems
        Establishing separate warehouse areas
        Formalizing separate material, waste and personnel flows
        Constructing temporary facilities for gowning and entrance to the line used for the ceftolozane/tazobactam drug product.
        Constructing new walls, modifying and reinforcing existing walls
        Equipping the existing emergency egress with alarms and gaskets to completely separate both lines throughout all the floors of the building
        Creating the permanent separation of locker, rest and break rooms for both lines of the facility:
        Dedicated maintenance and operations personnel for each part of the facility including different uniform colors for each part of the facility
        Dedicated equipment and tools for each part of the facility
        An Emergency Recovery plan
    
    


Example 13: Physicochemical and Biological Properties Ceftolozane/Tazobactam for Injection, 1000 mg/500 mg
As a product intended for intravenous use, several properties are important for physiological compatibility. These include particulate matter, sterility, endotoxin limit, pH, and osmolality. Particulate matter and sterility are controlled at the point of manufacture. The drug product is processed aseptically throughout the entire manufacturing process, inclusive of ceftolozane, tazobactam sodium, and ceftolozane/tazobactam in-container drug product.
The ceftolozane/tazobactam drug product is controlled to approximately pH 6, to provide physiological comfort, while still assuring adequate stability for the drug substances. The ceftolozane drug product intermediate is controlled during compounding to pH 6.5±0.5 and is controlled at release to pH 5 to 7. The tazobactam sodium is controlled at release to pH 5 to 7.
Ceftolozane/tazobactam following reconstitution with normal saline and dilution for infusion also in normal saline (10 mg/mL ceftolozane; 5 mg/mL tazobactam) is slightly hypertonic, with osmolality approximately 500 mOsm/kg. However, slightly hypertonic intravenous infusion solutions are not uncommon as drug products are commonly prepared and diluted with already-isotonic solutions, such as normal saline. The generally accepted maximum upper limit for peripheral intravenous administration is approximately 900 mOsm/kg, though admixtures 600 to 900 mOsm/kg are typically administered through a central line. Therefore, to be within the limits of this range, the infusion product is less than 600 mOsm/kg.
Example 14: Determining Osmolality of CXA-201 Compositions
CXA-101 and Tazobactam Sodium samples (#1-#3) were reconstituted as follows:


    Sample#1: Weighed 0.103 g of Tazobactam Sodium and 0.462 g of CXA-101 dissolved in 4 mL of WFI Water and 6 mL of USP Normal Saline.
    Sample#2: Weighed 0.103 g of Tazobactam Sodium and 0.462 g of CXA-101 dissolved in 4 mL of WFI Water added 10 mL of USP Normal Saline.
    Sample#3: Weighed 0.103 g of Tazobactam Sodium dissolved in 1 mL of WFI Water and 0.462 g of CXA-101 dissolved in 1 mL of WFI Water then mixed together added 10 mL of USP Normal Saline.

Tazobactam Sodium (Potency: 97.5%)

CXA-101 (Potency: 43.3%)

WFI Water

USP Normal Saline



The osmolality of CXA-101 and Tazobactam Sodium samples (#1-#3) was then determined using a freezing point depression Osmometer (available from Advanced Instruments, Inc.).







TABLE 30A







Osmolality of Reconstituted Solutions














Tazobactam






CXA-101
Sodium






Conc.
Conc.


Osmolality


Sample#
(mg/mL)
(mg/mL)
WFI Water
Saline
(mOsm/kg)















1
20.0
10.0
40% (40 mL)
60% (60 mL) 
589


2
14.3
7.1
29% (40 mL)
71% (100 mL)
512


3
16.7
8.3
17% (20 mL)
83% (100 mL)
604









A unit dosage form composition of Table 29 was reconstituted with 10 mL of Sterile WFI or USP Normal Saline then added into 100 mL 5% Dextrose Injection (D5W) or 0.9% Sodium Chloride (NS) bags and the osmolality of the resulting bag solution was determined as shown in Table 30B below.







TABLE 30B







Osmolarity of Ceftolozane Bag Solution (mOsm/kg)











Time Points
WFI-D5W
NS-D5W
sWFI-NS
NS-NS





RT T0
446
470
449
478









In Table 30B, data for osmolality of the following product reconstitution scenarios was determined using the composition from Table 29

    
    
        5% Dextrose Injection USP, 100 mL Bag (Baxter)
        0.9% Sodium Chloride Injection USP, 100 mL Bag (Baxter)
        sWFI-D5W: reconstituted with Sterile WFI then added into 5% Dextrose Injection bag
        NS-D5W: reconstituted with USP Normal Saline then added into 5% Dextrose Injection bag
        sWFI-NS: reconstituted with Sterile WFI then added into 0.9% Sodium Chloride Injection Bag
        NS-NS: reconstituted with USP Normal Saline then added into 0.9% Sodium Chloride Injection bag
    
    


Example 15: Excipients in Ceftolozane Drug Product Intermediate
The excipients in exemplary ceftolozane compositions were chosen to ensure stability and processability of the ceftolozane drug substance into the drug product. The specific excipients, their quantities and functions are provided in Table 31. All excipients are compendial and typical for sterile pharmaceutical dosage forms, requiring no additional treatment prior to use in the formulation. The excipients are used in levels within the range established in other FDA approved products as described in the Inactive Ingredients Database (IID).







TABLE 31







Excipients Used in Ceftolozane Composition

















Inactive




Amount,
Concentration

Ingredients




mg/
in Infusion
Rationale for
Database


Component
Function
container
Solution, %
Inclusion
(IID) Range





Citric acid
Chelating
 21
0.02
Used to prevent
0.0025 to 50%



agent


discoloration and







degradation



Sodium
Stabilizing
487
0.49
Used as a stabilizing
 0.187 to 45%


Chloride
agent


agent for







ceftolozane sulfate



L-arginine
Alkalizing
600(a)
0.60
Used to adjust
 0.29 to 88%



agent
Q.S. for pH

ceftolozane solution





adjustment

pH





(a)L-arginine is added as needed to achieve pH 6.5 ± 0.5; 600 mg per container is considered a representative total amount.






Example 16: Manufacturing Process of a CXA-201 Composition (Comprising Tazobactam and Ceftolozane) by Co-Filling
The ceftolozane/tazobactam finished drug product is a sterile powder fill of lyophilized active ingredients ceftolozane drug product intermediate (composition) and tazobactam sodium together into a sterile single container. The lyophilized form of the sterile tazobactam sodium contains no excipients. Ceftolozane sulfate drug substance is converted first into a sterile drug product intermediate, composition, by formulation with citric acid, sodium chloride and L-arginine, followed by lyophilization.
The full manufacturing process includes unit operations typical of an aseptic lyophilization process and aseptic powder filling process. The overall process can be outlined in two stages, as presented in the manufacturing flow chart of FIG. 12. The first stage is the manufacturing of the sterile ceftolozane composition. The second stage is the filling of the sterile drug powders into containers for the final drug product. The major process steps are:
Preparation of the sterile ceftolozane composition comprises

    
    
        compounding the bulk solution for lyophilization;
        sterile filtering the bulk solution;
        lyophilizing the bulk solution into bulk powder;
        grinding and sieving of the sterile bulk powder; and
        aseptic packaging of the sterile bulk powder in Sterbags®.
    
    


Filling of the sterile bulk powders comprises

    
    
        receipt of ceftolozane and tazobactam sterile powders at site;
        aseptic filling both sterile powders into the container sequentially;
        blanketing the container with a nitrogen headspace;
        stoppering and crimping the container; and
        inspecting the container prior to secondary packaging.

## Claims

1. A solid pharmaceutical composition comprising ceftolozane sulfate and tazobactam sodium, said composition comprising less than 0.15% of a compound of formula (III)




relative to ceftolozane sulfate, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%, wherein the ceftolozane sulfate and tazobactam sodium provide ceftolozane active and tazobactam active in a ratio of 2:1 by weight.

2. The composition of claim 1, which contains less than 0.1% of the compound of formula (III) relative to ceftolozane sulfate, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.

3. The composition of claim 1, which contains less than 0.03% of the compound of formula (III) relative to ceftolozane sulfate, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.

4. The composition of claim 1, wherein the amount of ceftolozane active is 1,000 mg and the amount of tazobactam active is 500 mg.

5. The composition of claim 1, wherein the amount of ceftolozane active is 2,000 mg and the amount of tazobactam active is 1,000 mg.

6. The pharmaceutical composition of claim 1, comprising 1,147 mg ceftolozane sulfate and 537 mg tazobactam sodium.

7. The composition of claim 1, which is provided in a unit dosage form.

8. An injectable composition comprising the pharmaceutical composition of claim 1 dissolved in a pharmaceutically acceptable vehicle.

9. A pharmaceutical composition comprising ceftolozane sulfate and tazobactam sodium, said composition comprising less than 0.15% of the compound of formula (III)



relative to ceftolozane sulfate, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, and less than 1.5% of a compound of formula (IV)




relative to ceftolozane sulfate, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, when stored for 3 months at a temperature of 25° C. and at a relative humidity of 60%, wherein the ceftolozane sulfate and tazobactam sodium provide ceftolozane active and tazobactam active in a ratio of 2:1 by weight.

10. The composition of claim 9, which contains less than 0.05% of the compound of formula (III) relative to ceftolozane sulfate, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, when stored for 3 months at a temperature of 25° C. and at a relative humidity of 60%.

11. An injectable composition comprising the pharmaceutical composition of claim 9 dissolved in a pharmaceutically acceptable vehicle.

12. A pharmaceutical composition comprising ceftolozane sulfate and tazobactam sodium, said composition comprising less than 0.15% relative to ceftolozane sulfate of the compound having a mass spectra depicted in FIG. 14, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%, wherein the ceftolozane sulfate and tazobactam sodium provide ceftolozane active and tazobactam active in a ratio of 2:1 by weight.

13. The composition of claim 12, which contains less than 0.1% relative to ceftolozane sulfate of the compound with the mass spectra depicted in FIG. 14, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.

14. The composition of claim 12, which contains less than 0.03% relative to ceftolozane sulfate of the compound with the mass spectra depicted in FIG. 14, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.

15. The composition of claim 12, wherein the amount of ceftolozane active is 1,000 mg and the amount of tazobactam active is 500 mg.

16. The composition of claim 12, wherein the amount of ceftolozane active is 2,000 mg and the amount of tazobactam active is 1,000 mg.

17. The pharmaceutical composition of claim 12, comprising 1,147 mg ceftolozane sulfate and 537 mg tazobactam sodium.

18. An injectable composition comprising the pharmaceutical composition of claim 12 dissolved in a pharmaceutically acceptable vehicle.

19. A pharmaceutical composition comprising ceftolozane sulfate and tazobactam sodium, said composition comprising less than 0.15% relative to ceftolozane sulfate of a compound having a mass spectra depicted in FIG. 14, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, and less than 1.5% of a compound of formula (IV):



relative to ceftolozane sulfate, as determined by high performance liquid chromatography (HPLC) at a wavelength of 254 nm, when stored for 3 months at a temperature of 25° C. and at a relative humidity of 60%, wherein the ceftolozane sulfate and tazobactam sodium provide ceftolozane active and tazobactam active in a ratio of 2:1 by weight.

20. An injectable composition comprising the pharmaceutical composition claim 19 dissolved in a pharmaceutically acceptable vehicle.