# Patent

## Title
Aztreonam L-lysine and methods for the preparation and analysis thereof

## Abstract

The present invention relates to the L-lysine salt of aztreonam and methods for making the L-lysine salt of aztreonam.

## Background
CROSS-REFERENCED TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application Ser. Nos. 60/484,861 filed Jul. 2, 2003 and 60/550,098 filed Mar. 4, 2004 and U.S. application Ser. No. 10/882,591 filed Jul. 1, 2004, the disclosures of which are incorporated by reference in their entirety herein.
FIELD OF THE INVENTION
The present invention relates to the L-lysine salt of aztreonam and methods for making the L-lysine salt of aztreonam.
BACKGROUND OF THE INVENTION
Aztreonam is a monobactam antibiotic disclosed in U.S. Pat. No. 4,775,670, which is incorporated by reference herein in its entirety. Aztreonam has the chemical name (Z)-2-[[[(2-amino-4-thiazolyl)[[(2S,3S)-2-methyl-4-oxo-1-sulfo-3-azetidinyl]carbamoyl]methylene]amino]oxy]-2-methylpropionic acid. Aztreonam is also known as [3S-[3α(Z),4β]]-3-[-[(2-amino-4-thiazolyl)[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid and (2S,3S)-3-[[2-[2-amino-4-thiazolyl]-(Z)-2-[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-1-azetidine-1-sulfonic acid. Aztreonam has the structure:




U.S. Pat. No. 4,775,670 discloses a process for making Aztreonam and pharmaceutically acceptable salts thereof. However, U.S. Pat. No. 4,775,670 does not teach how to prepare salts of Aztreonam with amines or amino acids.
Applicants encountered unexpected difficulties when trying to prepare salts of Aztreonam with amines and amino acids by dissolution of the acid and base in a solvent and precipitation of the salt. In the majority of experiments an oil, which was impossible to crystallize and which decomposed very rapidly, was obtained.
Applicants have discovered methods that enable the preparation of a solid, stable Aztreonam L-lysine salt.
SUMMARY OF THE INVENTION
The invention relates to an amorphous, solid Aztreonam L-lysine salt. The invention also relates to methods for making the amorphous L-lysine salt. The first method comprises freeze-drying an aqueous solution of Aztreonam L-lysine. The second method comprises spray-drying an aqueous solution of Aztreonam L-lysine. The third method comprises precipitating Aztreonam L-lysine from an aqueous solution.


DETAILED DESCRIPTION OF THE INVENTION
Aztreonam is converted into its L-lysine salt in aqueous solution. The pH plays an important role in the stability of the Aztreonam L-lysine aqueous solution and it should not be more than 5.5. The salt may be isolated from the aqueous solution as an amorphous solid by three different techniques. The three techniques include freeze-drying, spray-drying and precipitation in an organic solvent. All three techniques provide an amorphous product.
Aztreonam L-lysine salt may be obtained by freeze drying an aqueous solution of aztreonam L-lysine. The ratio of Aztreonam and L-lysine used to form the aqueous solution is preferably between 1:1 and 1:2.1. The product obtained by this method appears as white to yellowish flakes and contains about 3 to about 6% water.
Aztreonam L-lysine may also be obtained by spray drying an aqueous solution of Aztreonam L-lysine. The Aztreonam L-lysine salt obtained by spraying is a white to off-white powder. The water content of the product obtained by this method is between about 4 to about 7%. The preferred spray drying parameters are listed in the following table. The parameters apply to a Buchi laboratory spray-drier B-191 (Aspirator rate: 31.5 m3/h).















Inlet

Concentration
Outlet


Pump speed
temperature
Spray flow
of solution
temperature*


(ml/h)
(° C.)
(l/h)
(m/m %)
(° C.)







240 . . . 750
115 . . . 195
400 . . . 800
7 . . . 29
45 . . . 127





*The outlet temperature depends mainly on the pump speed, inlet temperature, spray flow and concentration of solution.






Using optimal drying parameters, the product exhibited good handling properties, i.e., it was free-flowing. The particle size can also be influenced by regulating the specific drying parameters.
The L-lysine salt of aztreonam can also be isolated by precipitating Aztreonam L-lysine from an aqueous solution. The aqueous solution of aztreonam L-lysine is preferably dropped into an aqueous or anhydrous organic solvent, e.g., ethanol, acetone, etc. The water content of the alcohol used for the precipitation is preferably between about 0 and 9% (m/m).
The Aztreonam L-lysine obtained using these methods was stable in the sense that during 3 months at 2-8° C.:
1. the assay of Aztreonam was maintained constant in the limit 60-66%; and
2. no impurity exceed 0.3 area %.
EXAMPLES
The impurity content of Aztreonam lysine salt using the HPLC method is determined as follows:
a. Aztreonam Lysine salt sample is dissolved in 0.02 M KH2PO4 buffer solution (pH adjusted 2.0 with 25 w/w % phosphoric acid) diluent,
b. The sample solution (ca. 10 μl) is injected into a 100.0 mm×4.0 mm, 3 μm RP-18 HPLC column,
c. Gradient eluting with a mixture of 0.02 M KH2PO4 buffer solution (pH adjusted 3.0 with 25 w/w % phosphoric acid) (A) and acetonitrile (B) according to the following profile:
















Flow rate
Time
Eluent A
Eluent B



[ml/min]
[min]
[v/v %]
[v/v %]





















1.2
0.0
100.0
0.0



1.2
16.0
84.0
16.0



1.2
25.0
70.0
30.0



1.2
25.1
100.0
0.0



1.2
30.0
100.0
0










d. The amounts of each impurity was measured at 230 nm wavelength with a UV detector and appropriate recording device.
e. The amount of each impurity was calculated referring to an Aztreonam working standard at a concentration of 2.5 g/ml.
In the above method, Aztreonam has a retention time of about 10.2 minutes.
The assay of Aztreonam Lysine salt using the HPLC method was determined as follows:
a. Dissolving Aztreonam Lysine salt sample in a mixture of 0.02 M KH2PO4 buffer solution (pH adjusted 3.0 with 25 w/w % phosphoric acid) and methanol (80:20) diluent,
b. Injecting the sample solution (ca. 10 μl) into a 50.0 mm×4.6 mm, 3 μm RP-18 HPLC column,
c. Isocratic eluting at 1.5 ml/min with a mixture of 0.02 M KH2PO4 buffer solution (pH adjusted 3.0 with 25 w/w % phosphoric acid) and methanol in a 83:17 v/v % ratio.
d. Measuring of the amounts of each impurity at 270 nm wavelength with a UV detector and appropriate recording device.
e. Calculating of the assay referring to the Aztreonam working standard at a concentration of 100 μg/ml.
In the above method, Aztreonam has a retention time of about 2.3 minutes
Example 1 
Aztreonam (5.00 g, water content: 12.2%) was suspended in 25 ml distilled water at 0-5° C. A solution of 2.70 g L-lysine in 13.5 ml distilled water was added dropwise to the above suspension with cooling (ice-water bath). The solution of Aztreonam L-lysine salt obtained by this method was filtered on a glass filter and freeze dryed.
Product: white flakes.
Yield: 6.8 g (quant.)
Example 2 
Aztreonam (35.0 g, water content: 12.6%) was suspended in 230 ml distilled water at 0-5° C. A solution of 17.5 g L-lysine in 45 ml distilled water was added dropwise to the above suspension with cooling (ice-water bath). The solution of Aztreonam L-lysine salt obtained by this method was decolorized with charcoal, filtered on a glass filter and spray dryed using laboratory spray dryer Buchi B-191.
Product: white powder.
Yield: 31 g (62%)
The Aztreonam L-lysine salt produced according to this example, does not contain any impurity exceeding 0.3 area %, and/or maintains at least about 63 weight % of the Aztreonam, after storage for three months at about 2-8° C.
Example 3 
Aztreonam (3.50 g, water content: 11.3%) was suspended in 8 ml distilled water at 0-5° C. A solution of 1.80 g L-lysine in 3.5 ml distilled water was added dropwise to the above suspension with cooling (ice-water bath). The solution of Aztreonam L-lysine salt obtained by this method was diluted with 23 ml ethanol and added dropwise to the stirred mixture of 60 ml ethanol and 4.75 ml water at 0-5° C. in 15 min. 120 ml pure ethanol was added dropwise together with the Aztreonam L-lysine salt solution but from another dropping funnel in the same time period. The precipitation was filtered off and dried in air-circulated oven at 38° C.
Product: white powder.
Yield: 3.86 g (77%)
Having thus described the invention with reference to particular preferred embodiments and illustrated it with examples, those of skill in the art may appreciate modifications to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification.

## Claims

1. A method for determining a level of impurities in an Aztreonam Lysine salt, the method comprising:
forming a sample solution by dissolving the Aztreonam Lysine salt in a KH2PO4 buffer solution, having a concentration of about 0.02 M and a pH adjusted to about 2 with about 25 weight percent phosphoric acid;
injecting the sample solution of the Aztreonam Lysine salt into a HPLC column;
eluting the injected sample with a mixture of a KH2PO4 buffer solution (A) and acetonitrile (B), wherein the buffer solution (A) has a concentration of about 0.02 M and a pH adjusted to about 3 with about 25 weight percent phosphoric acid, and the injected sample is gradient eluted; and
determining the amount of each impurity by measuring at a wavelength of about 230 nm with a UV detector and referring to an Aztreonam working standard at a concentration of about 2.5 g/ml.

2. A method for determining a level of impurities in an Aztreonam Lysine salt, the method comprising:
forming a sample solution by dissolving the Aztreonam Lysine salt in a mixture of a KH2PO4 buffer solution, having a concentration of about 0.02 M and a pH adjusted to about 3 with about 25 weight percent phosphoric acid, and methanol;
eluting the injected sample at about 1.5 ml/min with a mixture of a KH2PO4 buffer solution, having a concentration of about 0.02 M and a pH adjusted to about 3 with about 25 weight percent phosphoric acid, and methanol in a volume ratio of about 83:17, and the injected sample is isocratic eluted; and
the amount of the at least one impurity is measured at about 270 nm wavelength with a UV detector; and
determining the amount of each impurity by measuring at about 270 nm wavelength with a UV detector and referring to an Aztreonam working standard at a concentration of about 100 μg/ml.