Description:TECHNICAL FIELD
[001] The present subject matter generally relates to pharmaceutical formulations of Polymyxin B Sulfate injection. More specifically, the development of a sterile, ready-to-use, isotonic, Polymyxin B Sulfate injection formulation having a preferred pH between 3.0 and 6.0 suitable for intramuscular, intravenous and intrathecal administration, and capable of long-term storage at room temperature.

BACKGROUND OF THE INVENTION
[002] Polymyxin B Sulfate, sulfate salt of polymyxin B1 and B2 which is produced by the growth of Bacillus polymyxa (Prazmowski) Migula (Fam. Bacillaceae), is an antibiotic used to treat bacterial infections such as urinary tract infections, meningitis, blood infections, and eye infections. It is effective against susceptible strains such as Pseudomonas aeruginosa, Haemophilus influenzae, Escherichia coli, Aerobacter aerogenes or Klebsiella pneumonia.

[003] Polymyxin B is a complex polypeptide antibiotic comprising of a cyclic heptapeptide group attached to a tripeptide side chain with fatty acyl residue on the N-terminus. Polymyxin B is subdivided into four components differing only in fatty acyl chain, namely Polymyxin B1, B2, B3, and B4.

[004] Polymyxin B sulfate is susceptible to pH and temperature. Polymyxin B sulfate is unstable in highly acidic and alkaline conditions. Under more acidic or alkaline conditions, the polymyxin B sulfate loses its activity after 36 hours at room temperature. The USP monograph of Polymyxin B drug substance and Polymyxin B for Injection specifies a pH limit of 5.0 to 7.5 for a 0.5% (5 mg/mL) solution.

[005] The Reference product, Polymyxin B for Injection, USP is a lyophilized powder, containing 500,000 Polymyxin B units per vial, which upon reconstitution and dilution is suitable for preparation of sterile solutions for intramuscular, intravenous drip, intrathecal, or ophthalmic use. For intramuscular administration, the vials are reconstituted with 2 mL sterile water for injection or sodium chloride injection or procaine hydrochloride injection 1%. For intravenous infusions, 500,000 polymyxin B units is diluted with 300 to 500 mL solutions of 5% dextrose injection for continuous drip. For intrathecal administration, 500,000 polymyxin B units is diluted in 10 mL sodium chloride injection USP.

[006] Though lyophilization partly addresses the issues related to stability of polymyxin B sulfate, lyophilization is a complex process which increases the cost of production. The lyophilized powder must be reconstituted with suitable fluids to achieve desired concentration for administration, which is an additional step that leads to inconvenience and safety related issues. As per the label instructions of the marketed product, aqueous solutions of polymyxin B sulfate may be stored up to 12 months without significant loss of potency if kept under refrigeration. In the interest of safety, solutions for parenteral use should be stored under refrigeration and any unused portion should be discarded after 72 hours. Polymyxin B sulfate should not be stored in alkaline solutions since they are less stable.

[007] Since the currently marketed products are available only in lyophilized form, there exists a need for a ready-to-use, isotonic, polymyxin B sulfate injection suitable for intravenous, intramuscular and intrathecal administration. Further, a room-temperature stable formulation is preferred.

[008] In order to address the above challenges, the inventors of the present disclosure worked out several approaches and came up with a ready-to-use, isotonic, polymyxin B sulfate injection by optimizing the composition, allowing for long-term storage at room temperature without the need for lyophilization and identifying pH range to stabilize the formulation in aqueous solution.

SUMMARY OF THE INVENTION
[009] In the light of the disadvantages mentioned in the previous section, the following summary is provided to facilitate an understanding of some of the innovative features unique to the present invention and is not intended to be a full description. A full appreciation of the various aspects of the invention can be gained by taking the entire specification and drawings as a whole.
[0010] In one embodiment, a stable, isotonic ready-to-use injectable formulation is disclosed. The formulation comprises of concentration from 1000 units/mL to 250,000 units/mL of polymyxin B sulfate thereof. The formulation also consists of a tonicity adjusting agent, a pH adjusting agent, and optionally a buffering agent and a quantity sufficient of water to make up the volume.
[0011] According to an embodiment of the present invention, the composition may comprise a tonicity agent selected from sodium chloride, dextrose, sucrose, xylitol, fructose, glycerol, propylene glycol, sorbitol, mannitol to obtain a tonicity between 250 mOsm/ kg to 350 mOsm/kg.
[0012] According to an embodiment of the present disclosure, acids not limited to sulfuric acid, phosphoric acid, acetic acid or hydrochloric acid may be used for pH adjustment between 3.0 to 6.0, preferably between 4.0 to 5.5. The pH adjusting agent may also comprise sodium hydroxide or a suitable alkali, if required to adjust the pH to a preferred range.
[0013] According to an embodiment of the present invention, the compositions may further comprise a buffering agent selected from citrate, acetate, glutamate, tartrate, benzoate, lactate, histidine, amino acids, gluconate, phosphate, succinate, or a combination thereof. In some embodiments, the buffering agent can function as a tonicity agent. According to one embodiment of the present disclosure, pH of the formulation is stabilized by the use of a buffer system, in the concentration of 0.25 mMol to 100 mMol, preferably using citrate between 0.5mMol to 10 mMol.
[0014] According to an embodiment of the present invention, the composition may further comprise a co-solvent selected from propylene glycol, polyethylene glycol, ethanol or a combination thereof. Some of the co-solvents may also function as tonicity adjusting agents. According to one embodiment of present disclosure, the solvent system comprises about 100%v/v of water for injection.
[0015] According to an embodiment of the present disclosure, the formulation potency claimed to be not less than (NLT) 80% as per the USP monograph, over the stability period atleast 12 months at room temperature (25°C/60%RH).
[0016] According to an embodiment of the present disclosure, the formulation comprises preferably not more than 15% of polymyxin B1-I and not more than 6% of polymyxin B3 and any individual impurities at a level of Not more than 3% and not more than 17% total impurities over the stability period at room temperature conditions.

[0017] In an embodiment, a method for preparing a ready-to-use, isotonic, room-temperature stable polymyxin B sulfate injection formulation for intramuscular, intravenous and intrathecal administration is disclosed. The method comprises collecting water for injection and maintaining it at 20°C to 30°C, adding the tonicity adjusting agent and buffering agents (as applicable) and mixing to obtain a clear solution. Then, addition of polymyxin B sulfate and mixing for about 10 to 20 minutes and further adjustment of pH, as necessary, using pH adjusting agents, preferably between pH 4.0 to 5.5 and then finally making the volume up to 100% of the batch size using water for injection and then final mixing for about 10 to 20 minutes. Then aseptically filtering the solution through 0.2 µm sterilizing grade filters, filling the filtered solution in suitable container closure system.
[0018] In the above-described process, the fill volume and container size of the product may be varied according to the required dose. For example, a dose of 500,000 units in 2 mL (250,000 units/mL) is filled in a 2 mL or 10 mL USP type-1 glass containers such as ampule, vial or prefilled syringes or in appropriate plastic containers. The concentration of 250,000 units/mL is suitable for intramuscular administration. The 500,000 units in 2mL solution may be further diluted to 10 mL using sodium chloride solution to obtain a concentration of 50,000 units/mL suitable for intrathecal and ophthalmic use.
[0019] In another embodiment, a ready to use solution of 500,000 units in 50 mL (10,000 units/mL) is filled in 50 mL vial or plastic bags. Similarly, a ready to use solution of 500,000 units in 100 mL (5,000 units/mL) may be filled in a 100 mL vial or plastic bag and a ready to use solution of 500,000 units in 250 mL (2,000 units/mL) may be filled in a 250 mL vial or plastic bag and a ready to use solution of 500,000 units in 500 mL (1,000 units/mL) may be filled in a 500 mL vial or plastic bags.
[0020] According to an embodiment of the present disclosure, the sterilizing grade filters used for aseptic filtration of solution comprises of a pore size of 0.2 µm.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0021] The detailed description is provided with reference to the accompanying figures. These and other features and advantages of the present invention will be readily appreciated, as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:
[0022] Fig. 1 is a flowchart illustrating a method for preparing a ready-to-use, isotonic, room temperature storage, pH stabilized formulation of polymyxin B sulfate injection for intramuscular, intravenous and intrathecal administration.
DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0023] For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Such alterations and further modifications in the illustrated system, and such further applications of the principles of the disclosure as would normally occur to those skilled in the art are to be construed as being within the scope of the present disclosure.
[0024] The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more devices or sub-systems or elements or structures or components preceded by “comprises” does not, without more constraints, preclude the existence of other devices, sub-systems, elements, structures, components, additional devices, additional sub-systems, additional elements, additional structures, or additional components. Appearances of the phrase "in an embodiment”, "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.
[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.
[0026] In the following specification and the claims, reference will be made to a number of terms, which shall be defined to have the following meanings. The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.
[0027] As used herein, the term “Polymyxin B Sulfate” refers to a medication used to treat bacterial infections such as urinary tract infections, meningitis, blood infections. It belongs to a class of drugs called antibiotics, which work by disrupting the outer cell membrane of Gram-negative bacteria, binding and neutralizing lipopolysaccharide, and inhibits respiration of Gram-negative bacterial cells.
[0028] As used herein, the term “ready-to-use” refers to substance or material that can be directly administered devoid of reconstitution or dilution.
[0029] As used herein, the term “Room-temperature stable” refers to a substance or material that can be stored and maintained at controlled room temperature (usually around 20-25°C or 68-77°F) without significant degradation or loss of quality over an extended period of time.
[0030] As used herein, the term “pH stabilized” refers to a substance or solution that is adjusted to a particular pH range using suitable pH adjusting agent and/or buffering agents and the desired pH is maintained throughout the shelf life. The pH stabilization in a narrow range is preferred as the degradation is found to be pH dependent and polymyxin B sulfate is unstable at highly acidic or alkaline conditions. For this purpose, the pH of the formulation to be maintained between 3.0 to 6.0, preferably between 4.0 and 5.5.
[0031] As used herein, the term “isotonic” refers to a solution that has same osmotic pressure as body fluid, preferably between 250 mOsm/ kg to 350 mOsm/kg.
[0032] As used herein, the term “Intramuscular administration”, abbreviated as IM administration, refers to the delivery of medication into the deep muscles. This allows the bloodstream to absorb the medication quickly.
[0033] As used herein, the term “intravenous administration”, often abbreviated as IV administration, refers to the delivery of medication or fluids directly into a vein through a needle or catheter. This method of administration allows for the rapid and efficient delivery of medication or fluids into the bloodstream, bypassing the digestive system and allowing for more immediate effects.
[0034] As used herein, the term “Intrathecal administration”, abbreviated as IT administration, refers to introduction of a medication or substance into the cerebrospinal fluid by injection into the subarachnoid space of the spinal cord to bypass the blood-brain barrier. Intrathecal administration of antibiotics is indicated in central nervous system infections by multi-resistant pathogens to achieve adequate cerebrospinal fluid (CSF) concentrations.
[0035] Embodiments described herein disclose a ready-to-use, isotonic, room-temperature storage, pH stabilized polymyxin B sulfate injection formulation for intravenous, intramuscular and intrathecal administration and a method thereof.
[0036] According to an embodiment of the present disclosure, the liquid, isotonic, ready-to-use, room-temperature storage, pH stabilized Polymyxin B sulfate injection formulation for intravenous administration comprises of varying concentrations of polymyxin B from 1000 units/mL to 250,000 units/mL. The concentration may vary depending on the route of administration, dose and course of treatment.
[0037] According to an embodiment of the present invention, the composition may comprise a tonicity agent selected from sodium chloride, dextrose, sucrose, xylitol, fructose, glycerol, propylene glycol, sorbitol, mannitol to obtain a tonicity between 250 mOsm/ kg to 350 mOsm/kg. A preferred tonicity adjusting agent is sodium chloride at a concentration of 0.6% to 0.9% w/v or dextrose or mannitol or sorbitol at a concentration of about 3% to 5% w/v.
[0038] According to an embodiment of the present invention, the compositions may optionally comprise a buffering agent selected from citrate, acetate, glutamate, tartrate, benzoate, lactate, histidine, amino acids, gluconate, phosphate, succinate, or a combination thereof. In some embodiments, the buffering agent can function as a tonicity agent. According to one embodiment of the present disclosure, pH of the formulation is stabilized by the use of a buffer system, in the concentration of 0.25 mMol to 100 mMol, preferably using citrate buffer between 0.5 mMol to 10 mMol.
[0039] According to an embodiment of the present disclosure, acids not limited to sulfuric acid, phosphoric acid, acetic acid or hydrochloric acid may be used for pH adjustment between 3.0 to 6.0, preferably between 4.0 and 5.5. The pH adjusting agent may comprise sodium hydroxide or suitable alkali, if required to adjust the pH to a preferred range.
[0040] According to an embodiment of the present invention, the composition may further comprise a co-solvent selected from propylene glycol, polyethylene glycol, ethanol or a combination thereof. Some of the co-solvents may also function as tonicity adjusting agents. According to one embodiment of present disclosure, the solvent system comprises about 100%v/v of water for injection.
[0041] According to an embodiment of the present disclosure, the formulation comprises preferably not more than 15% of polymyxin B1-I and not more than 6% of polymyxin B3 and any individual impurities at a level of Not more than 3% and not more than 17% total impurities over the stability period at room temperature conditions.

[0042] Fig. 1 is a flowchart illustrating methods for preparing a ready-to-use, isotonic, room temperature storage, pH stabilized formulation of polymyxin B sulfate injection for intramuscular, intravenous and intrathecal administration in accordance with an embodiment of the present invention.
[0043] In an embodiment, a method for preparing a ready-to-use, isotonic, room-temperature storage, pH stabilized Polymyxin B sulfate injection formulation for intramuscular, intravenous and intrathecal administration is disclosed. The method comprises collecting approximately 80% of the batch size of water for injection into a manufacturing vessel and maintaining the temperature at about 20°C to about 30°C at step 102. Then at step 104, adding a buffering agent optionally, selected from citric acid, sodium citrate, or a combination thereof, in the batch quantity, and mixing for about 10 minutes to dissolve and obtain a clear solution. Next at 106, adding a tonicity-adjusting agent, preferably sodium chloride, in the batch quantity, and mixing for about 10 minutes to dissolve and obtain a clear solution. Then at 108, adding Polymyxin B sulfate in the batch quantity, and mixing for about 20 minutes to dissolve and obtain a clear solution. Next step at 110, adjusting the pH using a pH-adjusting agent, preferably sulfuric acid, to achieve a pH in the range of about 3.0 to about 6.0, preferably between 4.0 and 5.5. Then at 112, making up the volume to 100% of the batch size using water for injection, stirring for about 15 minutes, and aseptically filtering the solution through a series of sterilizing-grade filters with a pore size of about 0.2 µm. Finally at 114, aseptically filling the sterile solution into suitable containers and sealing the containers.
[0044] According to an embodiment of the present disclosure, the water for injection is sparged with nitrogen throughout the process except during the addition of the materials.
[0045] According to an embodiment of the present disclosure, the sterilizing grade filters used for aseptically filtering solution comprises of a pore size of 0.2 µm.

[0046] In the above-described process, the fill volume and container size of the product may be varied according to the required dose. For example, a dose of 500,000 units in 2 mL (250,000 units/mL) is filled in a 2 mL or 10 mL USP type-1 glass containers such as ampule, vial or prefilled syringes or in appropriate plastic containers. The concentration of 250,000 units/mL is suitable for intramuscular administration. The 500,000 units in 2mL solution may be further diluted to 10 mL using sodium chloride solution to obtain a concentration of 50,000 units/mL suitable for intrathecal and ophthalmic use.
[0047] In another embodiment, a ready to use solution of 500,000 units in 50 mL (10,000 units/mL) is filled in 50 mL vial or plastic bags. Similarly, a ready to use solution of 500,000 units in 100 mL (5,000 units/mL) may be filled in a 100 mL vial or plastic bag and a ready to use solution of 500,000 units in 250 mL (2,000 units/mL) may be filled in a 250 mL vial or plastic bag and a ready to use solution of 500,000 units in 500 mL (1,000 units/mL) may be filled in a 500 mL vial or plastic bags.
[0048] WORKING EXAMPLES

Method of preparation for Claimed Formulation of the Present Disclosure
Step 1: Collect Water for injection (about 80%) of batch size in the manufacturing vessel and maintain at a temperature of 20-30°C. The water for injection and the solution is sparged with nitrogen throughout the process except during the addition of the materials.
Step 2: Add batch quantity of buffering agent (optionally used), preferably citric acid and/ or sodium citrate, as applicable and mix for about 10 minutes to dissolve and obtain a clear solution.
Step 3: Add batch quantity of tonicity adjusting agent, preferably sodium chloride and mix for about 10 minutes to dissolve and obtain a clear solution
Step 4: Add batch quantity of Polymyxin B Sulfate and mix for about 20 minutes to dissolve and obtain a clear solution.
Step 5: Adjust the pH using pH adjusting agents, preferably sulfuric acid and/or sodium hydroxide to obtain a pH between 3.0 and 6.0, preferably between 4.0 and 5.5.
Step 6: Make up the volume to 100% of batch size using the Water for injection and stir for 20 minutes.
Step 7: Aseptically filter the solution through 0.2 µm sterilizing grade filters and fill the filtered solution in a vial or suitable containers and seal the containers.

[0049] Examples formulations suitable for intramuscular administration and intravenous administration are provided in the following tables.

Examples of Ready to use compositions suitable for IM administration
[0050] Examples of formulations without any buffering agents (unbuffered compositions) suitable for intramuscular administration having a concentration of 250,000 Units/ mL are provided in Table 1.
Table 1. Examples of Ready to use, unbuffered compositions suitable for IM administration

Sr. No. Ingredient Function Example Formulation 1 Example Formulation 2
Each mL contains
1 Polymyxin B Sulfate USP Active substance 250,000 units 250,000 units
2 Sodium Chloride USP Tonicity adjusting agent 9 mg Not Used
3 Mannitol USP Not used 50 mg
4 Sulfuric acid USP/BP pH Adjusting agent q.s. to adjust pH between 4.0 to 5.5
(as necessary)
6 Sodium hydroxide NF
7 Water for Injection USP Vehicle q.s. to 1 mL q.s. to 1 mL
q.s : Quantity Sufficient
[0051] The example formulations containing a dose of 500,000 units in 2 mL (250,000 units/mL) is filled in a suitable 2 mL or 10 mL USP Type-1 glass or plastic containers, such as ampule, vial or prefilled syringes.
[0052] The comparative stability data of unbuffered formulation of Example Formulation 1 manufactured at pH 3, pH 4, pH 5 and pH 7 are provided in Table 4.
[0053] The comparative stability data of unbuffered formulation of Example Formulation 2 having pH 3, pH 4, pH 5 and pH 7 are provided in Table 5.
 
[0054] Examples of buffered compositions having different buffering agents suitable for IM administration are provided in Table 2.
Table 2. Examples of Ready to use, buffered compositions suitable for IM administration
Sr.
No. Ingredient Function Example 3
(17 mM Acetate buffer) Example 4
(2 mM Citrate buffer) Example 5
(10 mM Citrate buffer) Example 6
(20 mM Citrate buffer)
Each mL contains
1 Polymyxin B Sulfate USP Active substance 250,000
units 250,000
units 250,000
units 250,000
units
2 Acetic acid USP Buffering agent 1.0 mg Not used Not used Not used
3 Citric acid monohydrate USP Not used 0.4 mg 0.945 mg 1.89 mg
4 Sodium citrate dihydrate USP Not used Not used 1.617 mg 3.235 mg
5 Sodium Chloride USP Tonicity adjusting agent 9.0 mg 9.0 mg 7.5 mg 6.5 mg
6 Water for Injection USP Vehicle q.s. to 1 mL q.s. to 1 mL q.s. to 1 mL q.s. to 1 mL
q.s : Quantity Sufficient
[0055] The example formulations containing a dose of 500,000 units in 2 mL (250,000 units/mL) is filled in a suitable 2 mL or 10 mL USP Type-1 glass or plastic containers, such as ampule, vial or prefilled syringes.
[0056] The comparative stability data of Example formulations 4 to 5 having 2 mMol and 10 mMol concentrations of citrate buffer is provided in Table 6.
Examples of Ready to use compositions suitable for IV administration
[0057] The Example Formulations 7 to 10 of the present disclosure having different concentration of polymyxin B suitable for intravenous administration are provided in Table 3.
 
Table 3. Examples of Ready to use compositions suitable for IV administration
Sr. No. Ingredient Function Example 7 Example 8 Example 9 Example 10
Each mL contains
1 Polymyxin B Sulfate USP Active substance 1000 units 5000 units 10000 units 10000 units
2 Sodium Chloride USP Tonicity adjusting agent 9 mg 9 mg 9 mg Not used
3 Dextrose USP Not used Not used Not used 50 mg
4 Sulfuric acid USP/BP pH Adjusting agent q.s. to adjust pH to 4.5 to 5.5 (as necessary)
5 Sodium hydroxide NF
6 Water for Injection USP Vehicle q.s. to
1 mL q.s. to
1 mL q.s. to
1 mL q.s. to
1 mL
q.s : Quantity Sufficient
[0058] The above-mentioned unbuffered ready to use solutions can be either filled aseptically in vials or flexible plastic bags. For example, the formulation containing 1000 Units/mL Polymyxin B may be filled in 250 mL or 500 mL flexible bag to obtain 250,000 Units/ 250 mL or 500,000 units/500 mL bag. Similarly, the formulation containing 10,000 Units/mL Polymyxin may be either filled a 50 mL vial or flexible plastic bags to 500,000 units/50 mL container.
[0059] The stability data of unbuffered Example compositions 7 to 9 suitable for intravenous administration are provided in Table 7.
Table 4. Stability data of Example formulation 1: Effect of pH on the product stability of unbuffered formulation (having Sodium Chloride as tonicity adjusting agent and Sulfuric acid as pH adjusting agent) suitable for IM administration

Example formulation 1 having pH about 3.0 Example formulation 1 having pH about 4.0 Example formulation 1 having pH about 5.0 Example formulation 1 having pH about 7.0
Test Specification Initial 6 Month at 40°C 6 Month at 25°C 6 Month
at 5°C Initial 6 Month at 40°C 6 Month at 25°C 6 Month
at 5°C Initial 6 Month at 40°C 6 Month at 25°C 6 Month
at 5°C Initial 6 Month at 40°C 6 Month at 25°C 6 Month
at 5°C
Description A pale yellow colour solution Complies Complies Complies Complies
pH 3.0 to 6.0 3.00 3.69 3.62 3.38 4.00 4.92 4.81 4.55 5.00 5.65 5.43 5.39 6.99 7.12 7.05 7.00
Osmolality 250 to 350 mOsm/kg 328 322 323 324 322 326 324 324 331 324 324 324 358 357 362 361
Composition of Polymyxins/ Potency
Polymyxin B3 NMT 6.0% 1.54 1.26 1.35 1.42 1.58 1.34 1.40 1.44 1.60 1.37 1.41 1.45 1.71 1.28 1.36 1.55
Polymyxin B1-I NMT 15.0% 5.77 4.93 5.36 5.64 5.85 5.24 5.59 5.75 5.93 5.04 5.49 5.72 6.35 1.36 2.35 6.10
Sum of Polymyxins B2, B3, B1-I and B1 NLT 80% 97.4 85.5 97.5 102.5 99 88.6 101.8 104.7 100.2 92.5 101.4 105.1 107.6 39.6 68.7 111.2
Degradation products
Total Impurities NMT 17.0% 5.54 13.83 10.61 7.68 5.54 11.10 7.97 6.93 5.56 10.93 8.09 6.87 5.94 47.29 29.19 9.65

Observation: The stability data of Example Formulation 1 at different pH shows that formulation is stable at a pH range of 3.0 to 5.5. However, there was a significant drop in potency and increase in impurities observed at pH about 7.0. Further, there was a slight increase in pH observed over the stability in unbuffered composition.

Table 5. Stability data of Example formulation 2: Effect of pH on the product stability of unbuffered solution (having mannitol as tonicity adjusting agent and Sulfuric acid as pH adjusting agent) suitable for IM administration

Example formulation 2 having pH about 3.0 Example formulation 2 having pH about 4.0 Example formulation 2 having pH about 5.0 Example formulation 2 having pH about 7.0
Test Specification Initial 6 Month at 40°C 6 Month at 25°C 6 Month
at 5°C Initial 6 Month at 40°C 6 Month at 25°C 6 Month
at 5°C Initial 6 Month at 40°C 6 Month at 25°C 6 Month
at 5°C Initial 6 Month at 40°C 6 Month at 25°C 6 Month
at 5°C
Description A pale yellow colour solution Complies Complies Complies Complies
pH 3.0 to 6.0 3.00 3.79 3.71 3.58 4.00 5.15 4.92 4.83 5.00 5.77 5.67 5.64 6.99 6.99 6.97 7.01
Osmolality 250 to 350 mOsm/kg 328 298 299 299 322 301 300 302 331 306 304 304 358 327 325 326
Composition of Polymyxins/ Potency by HPLC
Polymyxin B3 NMT 6.0% 1.65 1.31 1.39 1.47 1.63 1.37 1.39 1.48 1.62 1.33 1.42 1.48 1.70 1.32 1.34 1.50
Polymyxin B1-I NMT 15.0% 6.13 5.19 5.56 5.84 6.09 5.18 5.56 5.84 6.05 4.87 5.54 5.93 6.34 1.72 2.48 5.83
Sum of Polymyxins B2, B3, B1-I and B1 NLT 80% 103.8 95.4 102.7 108.3 102.8 96.4 102.7 109.0 102.3 91.2 102.9 109.5 107.5 49.4 71.0 109.5
Degradation products by HPLC
Total Impurities NMT 17.0% 5.48 13.55 11.58 13.47 5.52 10.97 8.58 7.09 5.56 13.58 9.66 7.06 5.87 40.41 27.63 9.92

Observation: The stability data of Example Formulation 2 at different pH shows that formulation is stable at a pH range of 3.0 to 5.5. However, there was a significant drop in potency and increase in impurities observed at pH about 7.0. Further, there was a slight increase in pH observed over the stability in unbuffered composition.

Table 6. Stability data of Ready to use, Buffered compositions Example Formulation 4 and Formulation 5 suitable for IM administration
Details Example formulation 4
(with 2 mM citrate buffer) Example formulation 5
(with 10 mMol of citrate buffer)
Specification Initial 3 Months at 40°C 3 Months at 25°C Initial 3 Months at 40°C 3 Months at 25°C
Description A pale yellow colour solution Complies Complies
pH 3.0 to 6.0 4.5 4.78 4.71 4.69 5.02 4.84
Osmolality 250 to 350 mOsm/kg 328 328 331 306 301 303
Composition of Polymyxins
Polymyxin B3 NMT 6.0% 1.35 1.32 1.29 1.50 1.34 1.46
Polymyxin B1-I NMT 15.0% 5.13 4.86 5.07 10.13 9.44 10.25
Sum of Polymyxins B2, B3, B1-I and B1 NLT 80% 94.0 85.6 93.1 106.5 93.0 101.5
Degradation products by HPLC
Total Impurities NMT 17.0% 5.79 10.22 7.78 3.46 9.17 5.97

Observation: The stability data of formulations having different concentrations of a buffering agent shows minimal shift in pH. The stability of the buffered formulations maintained at pH of about 4.5 is found satisfactory.

Table 7. Stability profile of Example formulation 7 to 9 suitable for Intravenous administration

Details Example formulation 7
(1000 units/mL with sodium chloride as tonicity agent) Example formulation 8
(5000 units/mL with sodium chloride as tonicity agent) Example formulation 9
(10000 units/mL with sodium chloride as tonicity agent)
Test Specification Initial 1 Month at 40°C 1 Month at 25°C Initial 1 Month at 40°C 1 Month at 25°C Initial 1 Month at 40°C 1 Month at 25°C
Description A pale yellow colour solution Complies Complies Complies
pH 4.0-5.5 5.09 5.09 5.03 5.06 5.16 4.9 5.02 5.24 5.17
Osmolality For information 286 285 285 278 277 277 287 287 289
Composition of Polymyxins
Polymyxin B3 NMT 6.0% 1.45 1.31 1.35 1.52 1.41 1.44 1.49 1.43 1.45
Polymyxin B1-I NMT 15.0% 5.45 5.02 5.08 5.61 5.28 5.44 5.60 5.35 5.54
Sum of Polymyxins B2, B3, B1-I and B1 NLT 80% 90.1 89.8 97.7 94.6 92.6 95.0 94.7 99.0 96.4
Degradation products by HPLC
Total Impurities NMT 17.0% 9.05 10.66 9.31 8.25 11.50 10.34 8.15 10.33 10.15

Observation:
• The stability data of formulations having 1000 Units/mL to 5,000 Units/mL concentrations is found satisfactory and there was minimal pH shift over the stability in these unbuffered compositions.

[0060] The stability trend data demonstrates that the product is stable in the pH range of 3.0 to 6.0 and more preferably in the range of 4.0 to 5.5. The formulation characteristics such as pH, osmolality, composition of polymyxins and impurities were found suitable over the stability.
[0061] The stability studies demonstrate that the formulation of the present disclosure is suitable for refrigeration and room temperature storage. The stability data demonstrates that pH stabilization can be achieved using a suitable buffering system in the concentration of 0.25 mMol to 100 mMol, preferably between 0.5 mMol to 10 mMol.
[0062] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the disclosure and are not intended to be restrictive thereof. While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended.
[0063] The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, the order of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all the acts need to be necessarily performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.
, Claims:1. A ready-to-use, sterile aqueous solution of Polymyxin B sulfate Injection suitable for Intramuscular administration comprising of:
a) about 250,000 Units/mL Polymyxin B;
b) an osmolality between 250 mOsm/ kg to 350 mOsm/kg; and
c) a pH between 3.0 and 6.0;
wherein the ready-to-use solution is stable at room temperature storage and does not require dilution prior to administration to a patient.

2. A ready-to-use, sterile aqueous solution of Polymyxin B sulfate Injection suitable for Intravenous administration comprising of
a) about 1,000 Units/mL and 50,000 Units/mL Polymyxin B;
b) an osmolality between 250 mOsm/ kg to 350 mOsm/kg; and
c) a pH between 3.0 and 6.0;
wherein the ready-to-use solution is stable at room temperature storage and does not require dilution prior to administration to a patient.

3. The formulations as claimed in claim 1 and claim 2, wherein a tonicity adjusting agent is selected from at least one of sodium chloride, dextrose, sucrose, xylitol, fructose, glycerol, propylene glycol, sorbitol, mannitol to obtain a tonicity between 250 mOsm/ kg to 350 mOsm/kg.

4. The formulation as claimed in claim 1 and claim 2, wherein acids comprise at least one of sulfuric acid, phosphoric acid, acetic acid, citric acid, tartaric acid or hydrochloric acid for pH adjustment between 3.0 and 6.0, preferably between 4.0 and 5.5.

5. The formulation as claimed in claim 1 and claim 2, wherein the buffering agents are optionally selected from at least one of citrate, acetate, glutamate, tartrate, benzoate, lactate, histidine, amino acids, gluconate, phosphate, succinate, in the concentration of 0.25 mMol to 100 mMol, preferably citrate buffer between 0.5 mMol to 10 mMol to achieve pH stabilization over the stability period.

6. A method for preparing a stable, ready-to-use, sterile Polymyxin B sulfate solution suitable for intramuscular administration, the method comprising:
collecting approximately 80% of the batch size of water for injection into a manufacturing vessel and maintaining the temperature at about 20°C to about 30°C;
adding a buffering agent (optionally), selected from citric acid, sodium citrate, or a combination thereof, in the batch quantity, and mixing for about 10 minutes to dissolve and obtain a clear solution;
adding a tonicity-adjusting agent, preferably sodium chloride, in the batch quantity, and mixing for about 10 minutes to dissolve and obtain a clear solution;
adding Polymyxin B sulfate in the batch quantity, and mixing for about 20 minutes to dissolve and obtain a clear solution;
adjusting the pH using a pH-adjusting agent, preferably sulfuric acid, to achieve a pH in the range of about 3.0 to about 6.0, more preferably about 4.0 to about 5.5;
making up the volume to 100% of the batch size using water for injection, stirring for about 15 minutes, and aseptically filtering the solution through a series of sterilizing-grade filters with a pore size of about 0.2 µm; and
aseptically filling the sterile solution into suitable containers and sealing the containers.
Dated this 30th day of January 2025
Signature

Gokul Nataraj E
Patent Agent (IN/PA-5309)
Agent for the Applicant