FORM 2
THE PATENT ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
PRO VISIONAL/COMPLETE SPECIFICATION
(See section 10; rule 13)
1 TITLE OF THE INVENTION : Composition and Manufacturing Process For
Tigecycline Reconstitute Powder For Injection
2 APPLICANT(S)
(a) Name: VHB Pharmaceuticals Pvt. Ltd.
(b) Nationality: Indian
(c) Address: 40-B/I. Shankar Smruti, Sir Bhalchandra Road,Dadar (E),
Mumbai-400 014
3 PREAMBLE TO THE DESCRIPTION
COMPLETE
The following specification
particularly describes the invention
and the manner in which it is to be
performed. :
4 DESCRIPTION (Description shall start from next page)
Attached
5 CLAIMS (not applicable for provisional specification. Claims should start with
the preamble - "I/We claim" on separate page)
Attached
~6 DATE AND SIGNATURE (to be given at the end of last page of specification)
Attached
~7 ABSTRACT OF THE INVENTION (to be given along with complete specification
on separate page) Attached
Note-
* Repeat boxes in case of more than one entry
* To be signed by the applicant(s) or by authorized registered patent agent.
* Name of the applicant should be given in full, family name in the beginning.
* Complete address of the applicant should be given stating the postal index no. / code State ad country.
* Strike out the column which is/are not applicable.

FIELD OF INVENTION:
The present invention relates to a composition and manufacturing process for the tigecycline reconstitute powder for injection. The present invention is a formulation of tigecycline i.e. stable when dissolved, lyophilized, and reconstituted.
BACKGROUND OF INVENTION:
Tigecycline is a glycylcycline antibiotic for intravenous infusion. It is a semisythetic analog of tetracycline The chemical name of tigecycline is (4S,4aS,5aR,12aS)-9-[2-(tert-butylamino)acetamido]-4,7bis(dimethylamino)-l,4,4a,5,5a,6,11,12a-octahydro3,10,12,12a-tetrahydroxy-1,11-dioxo-2-naphthacenecarboxamide. The empirical formula is C29H39N5O8 and the molecular weight is 585.65. The chemical structure of tigecycline is as follows:

Tigecycline is an antibiotic with a broad spectrum of bacteriostatic activity. It is structurally similar to the tetracyclines in that it contains a central four-ring carbocyclic skeleton and is actually a derivative of minocycline. Tigecycline has a substitution at the D-9 position, which is believed to confer broad-spectrum activity.
Tigecycline inhibits protein translation in bacteria by binding to the 30S ribosomal subunit and blocking entry of amino-acyl tRNA molecules into the A

site of the ribosome. This prevents incorporation of amino acid residues into elongating peptide chains. Tigecycline carries a glycylamido moiety attached to the 9-position of minocycline. The substitution pattern is not present in any naturally occurring or semisynthetic tetracycline and imparts certain microbiologic properties to tigecycline. In general, tigecycline is considered bacteriostatic.
Tigecycline is not affected by the two major tetracycline-resistance mechanisms, ribosomal protection and efflux. Additionally, tigecycline is not affected by resistance mechanisms such as beta-lactamases (including extended spectrum beta-lactamases), target-site modifications, macrolide efflux pumps or enzyme target changes (e.g. gyrase/topoisomerases).
Tigecycline has historically been administered intravenously because it exhibits generally poor bioavailability when given orally. Intravenous solutions have largely been prepared immediately prior to use, e.g. administration to a patient, from lyophilized powders because tigecycline degrades in solution principally via oxidation.
However, tigecycline is difficult to manufacture because it is susceptible to degradation including oxidative degradation from atmospheric oxygen and epimer formation. The epimer form of tigecycline is non toxic but it lacks anti bacterial efficacy therefore it is considered as an undesirable degradation product. Therefore there is a need for a manufacturing process, which controls epimerization and oxidative degradation of tigecycline.



NH

OH O OK O O

NH:

Structure of active tigecycline has N- dimethyl group at C 4 position is cis to the adjacent hydrogen atom (as shown above). Whereas, in the epimer form, the N-dimethyl group at C 4 position is trans to the adjacent hydrogen atom (as shown below).


DESCRIPTION OF INVENTION:
This invention provides a manufacturing process of tigecycline in water for injection (WFI) in a concentration of 50 mg/3ml wherein the oxygen level in the water. This invention provides the manufacture of tigecycline reconstitute powder for injection with very less degradants by controlling the temperature and level of dissolved oxygen in bulk solution. Dissolved oxygen level is controlled by nitrogen flushing in water for injection (WFI).
This invention is to provide tigecycline reconstitute powder for injection having the shelf life of 24 months from the date of manufacturing. Prior to being administered to patients, the tigecycline reconstitute powder is reconstituted with 0.9% saline. After reconsritution with saline, tigecycline is ready for intravenous administration to a patient. However, tigecycline should be used as early as possible after dilution because tigecycline undergoes rapid degradation via oxidation. In tigecycline structure, it possesses a phenol group, which is become deprotonated while dissolved in water and saline solution. And these deprotonated phenol groups are highly prone to react with oxygen hence process of tigecycline preparation occurs under nitrogen blanket. The temperature of water for injection used to prepare a solution of tigecycline is about 2° C to 10° C.
The composition of present invention containing tigecycline, carbohydrate, suitable anti-oxidant agent in water for injection and adjust pH with buffering agent are expected to possess greater stability on reconstitution.

DETAIL DESCRIPTION OF INVENTION:
The present invention provides a manufacturing process and composition for tigecycline as a reconstitutable powder. The preparations of tigecycline reconstitute powder for injection by using solutions of tigecycline, lactose monohydrate and sodium metabisulphite in water for injection (WFI) on a 70L to 90L batch size or greater than that
This invention is providing a manufacturing process of tigecycline in water for injection (WFI) in a concentration of 50 mg/3ml wherein the oxygen level in the water is very less by controlling nitrogen flushing. This invention provides the manufacture of tigecycline reconstitute powder for injection with very less degradants by controlling the temperature and level of dissolved oxygen in bulk solution. Dissolved oxygen level is controlled by nitrogen flushing in water for injection (WFI).
This invention is to provide tigecycline reconstitute powder for injection having the shelf life of 24 months from the date of manufacturing. Prior to being administered to patients, the tigecycline reconstitute powder is reconstituted with 0.9% saline. After reconstitution with saline, tigecycline is ready for intravenous administration to a patient. However, tigecycline should be used as early as possible after dilution because tigecycline undergoes rapid degradation via oxidation. In tigecycline structure, it possesses a phenol group, which is become deprotonated while dissolved in water and saline solution. And these deprotonated phenol groups are highly prone to react with oxygen hence process of tigecycline preparation occurs under nitrogen blanket. The temperature of water for injection used to prepare a solution of tigecycline is about 2° C to 10° C.

The composition of present invention containing tigecycline, carbohydrate, suitable anti-oxidant agent in water for injection and adjust pH with buffering agent are expected to possess greater stability on reconstitution. Because of antioxidant agent chances of oxidative degradation are minimized. The carbohydrate is used in present composition acts against the epimer formation of tigecycline. Carbohydrate includes lactose, mannose, sucrose, glucose, monosaccharide and disaccharide e.g. aldose. For present composition lactose is most preferable. Lactose used in present composition is having different forms such as lactose anhydrous and lactose monohydrate. The composition of invention includes solution of tigecycline, carbohydrate, suitable anti-oxidant agent and buffering agent. The suitable carbohydrate used for this invention is lactose monohydrate. Lactose monohydrate (carbohydrate used in the present composition) minimizes the rate of epimer formation in the present invention.
Suitable anti-oxidant agent used in the present composition reduces the oxidative degradation of tigecycline. An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Anti-oxidant reduces the oxidative degradation by reacting with free radicals. Examples of suitable antioxidant include citric acid, ascorbic acid, thiols and sodium metabisulphite. Sodium metabisulphite is most preferred.
Buffering agent used in invention capable of adjusting the pH of a tigecycline, carbohydrate and anti-oxidant solution to between 4.0 - 5.0, 5.0 - 8.0 and 6.0-8.0. The examples of buffering agents can be used such as lactic acid, citric acid, acetic acid, 0.1 N HCL, succinates and phosphoric acid. The most suitable buffering agent is 0.1N HCL. The tigecycline reconstitute powder and solution analyzed by UV- spectroscopy method.

The present invention may be prepared by a number of methods. Some of those methods are described below.
Method I: Tigecycline is dissolved in water for injection under nitrogen at temperature of about 2° C to 8° C. The pH of initial solution is between 6.0 - 7.0 (7.03). The dissolved oxygen level should be less then 1ppm. Suitable carbohydrate and anti-oxidant is dissolved in the solution and adjust the pH of the final solution with buffering agent. Orange color of solution formed. Then the solution is fill in vial and lyophilized to form a powder.
Method II: Take freshly collected Water for injection and maintain the temperature up to 2° C to 8° C. Start nitrogen bubbling. Add anti-oxidant and carbohydrate slowly at constant stirring to dissolve completely. Then add slowly tigecycline to the aqueous solution with constant stirring under nitrogen bubbling. Adjust the pH of the solution between 6.0 - 8.0. Make up the volume of the solution with sterile water for injection. Orange color of solution formed. Fill in the vial and lyophilized the solution to dryness to form cake,
The method used for this invention is method 11 as mentioned above. Carbohydrate is used to minimize the rate of epimerization and antioxidant used to reduce the oxidative pathway in the formulation. In method II, due to the addition of antioxidant and carbohydrate in the 1st step (see flow cliart), the present composition less prone to undergo degradation.
The compositions of the invention are prepared for single-dosage use. The vial and stoppers used for lyophilization process should be of approved pharmaceutical grade. The size of vials used for the preparation is 5 ml. The vial and stoppers should be cleaned and sterilized. The Storage temperature of
8

tigecycline powder for injection vials is about 20° C - 25° C. The tigecycline reconstitute powder for injection can be reconstitute by adding sufficient diluent to achieved desired concentration of tigecycline. Diluent used for reconstitution can be any pharmaceutical diluent such as 0.9% saline solution, Lactated Ringer's solution for injection, dextrose solutions and water for injection.
The pH observed after reconstitution of lyophilized cake is between 6.0 - 8.0 precisely, 6.87. The tigecycline reconstitution solution can be analyzed by UV-spectroscopy method.

MANUFACTURING PROCESS FLOW CHART

Water For Injection IP

Collection of batch quantity of WF1 and Cooling between 2°C to 8°C

Continuous nitrogen purging for required time

Check purging time and
dissolved oxygen in water

Sodium Mctabisulphitc IP

Add dispensed quantity to WFI with continuous stirring and nifrGgen purging.

Lactose Monohydrate IP

Add dispensed quantity to solution with continuous stirring and nitrogen purging

Tigecyclin

Add dispensed quantity to solution with continuous stirring and

Check weight of
Tigecyclin
Check for complete
dissolution

Water For Injection IP

Add cool WFI and make up the required volume.

Continue stirring and nitrogen purging for

Check final pH. Sample for final

Procedure For Lyophilization:

Pre-cooling: Shelf set point (°C):

NO
is°r

Pre-cooling

Stage - ^ Final T (°C) Ramp Duration (mins) Soak Duration (mins) Pressure ■OiBar)
Ramp and Soak -1 -10.0 30 120 N.A.
Ramp and Soak -2 -45.0 60 300 N.A.
Primary Drying

Stage j Final T (OQJL Ramp Duration
(mins) | Soak Duration (mins) *:-_ Pressure (nBar)
Ramp and Soak -1 -25.0 60 120 400
Ramp and Soak -2 -18.0 30 420 300
Ramp and Soak -3 -10.0 60 240 300
Ramp and Soak -4 0.0 60 300 200
Ramp and Soak -5 10.0 60 180 100

Primary Pressure Rise test (N.A)

Stage ( " Maximum
increase - of P (uBar) - Duration (min)/ Test times Interval (mins) |:
Primary Pressure Rise test 20 1 4 3
Secondary Drying

Stage Final T(°C)1 Rairtjr Duration
(mins): Soak ■>£ Duration (mins) Pressure : (MBar)
Ramp and Soak -1 10.0 60 120 100
Ramp and Soak -2 20.0 30 120 50
Ramp and Soak -3 30.0 60 300 30
Secondary Pressure Rise test V

Stage .- e Maximum ■% increase of P (u£ar) Duration (min) Test times
*
t - Interval (mins)
Secondary Pressure Rise test 10.0 1 3 30
Condenser cooling V

Stage - *■::" Final Temperature (°C) *= Soak Duration (mins),
Condenser cooling -40 12

Chamber Evacuation V

Stage ?■■ 'j\larm: PI ~":-Set point (u^Bar) Alarm: P2 .r Set point (uBarjl^ Continue evacuation
under
P2 (Sec)
Chamber Evacuation 200 800 15
Vacuum break and stoppering - Manual - No Shelf cooling for unloading - No
YIELD STATEMENT UPTO OPTICAL INSPECTION

Sr.No Step-wise Theoretical yield Permissible Yield
01 % Yield after Filling and half Bunging 100% NLT98.0%
02 % Yield after Lyophilization & Sealing 100% NLT97.0%

SUMMARY OF INVENTION:
The present invention is related to a composition and manufacturing process for tigecycline injection in bulk manufacturing oi lyophilized powder by controlling the degradation. Due to the tendency of tigecycline to degrade, the bulk manufacturing of tigecycline are prepared under low-temperature and less oxygen conditions to minimize the degradation.
It has been observed that tigecycline oxidative degradation could be minimized when the pH is lowered. But at low pH epimerization of tigecycline can be occurred which causes another degradation. At low pH, epimerization appears as the major degradation pathway.
This invention provides the manufacture of tigecycline reconstitute powder for injection with very less degradants by controlling the temperature and level of dissolved oxygen in bulk solution.
The prepare tigecycline reconstitute powder for injection by using solutions of water for injection (WFI) and tigecycline on a 70L to 90L batch size or greater than that.
This invention is providing a manufacturing process of tigecycline in water for injection (WFI) in a concentration of to 50 mg/3ml wherein the oxygen level in the water is very less by controlling water flushing.
Tigecycline reconstitutes with saline for intravenous administration to a patient. However, tigecycline should be used as early as possible after dilution because tigecycline undergoes rapid degradation via oxidation.

The temperature of water for injection used to prepare a solution of tigecycline is about 2° C to 8° C.
The composition of present invention containing tigecycline in water for injection, carbohydrate, suitable anti-oxidant agent and buffering agent, are expected to possess greater stability time on reconstitution.
The manufacturing process of present invention comprises of following steps: Prepare aqueous solution at a concentration of 50 mg/3ml, tigecycline with Water for injection (WFI), under controlled temperature at about 2° C to 10° C. Dissolve carbohydrate and suitable anti-oxidant agent in aqueous solution of tigecycline and WFI. Filing of the vial with the above preparation. Followed by lyophilization process.
The whole process mentioned above occurs under nitrogen blanket The tigecycline reconstitute powder and solution analyzed by UV- spectroscopy method.

We claim:
1. The process for manufacturing a tigecycline composition containing solution of anti-oxidant, which reduces the oxidative degradation, and carbohydrate used to reduce epimerization of tigecycline to form aqueous solution and lyophilized the solution to dryness to form cake.
2. The process of claim 1 wherein said carbohydrate suitable for reducing epimerization is selected from lactose, mannose, sucrose, glucose, monosaccharide and disaccharide e.g. aldose.
3. The process of claim 1 and claim 2 wherein said carbohydrate suitable for reducing epimerization is selected from lactose moiety having different forms such as lactose anhydrous and lactose monohydrate.
4. The process of claim 1, claim 2 and claim 3 wherein said carbohydrate suitable for reducing epimerization is lactose monohydrate.
5. The process of claim 1 wherein said the suitable anti-oxidant agent for reducing oxidative degradation is selected from citric acid, ascorbic acid, thiols and sodium metabisulphite.
6. The process of claim 1 and claim 5 wherein said the suitable anti-oxidant for reducing oxidative degradation is sodium metabisulphite.
7. The process of claim 1 wherein said the pH of the solution is in the range of 5.0 - 8.0

8. The process of claim 1 and claim 7 wherein said the preferable pH of the
solution is between 6.0 - 8.0
9. The process of anyone of claims 1 to 8 wherein said the buffer is acidic
10. The process of claim 1 and claim 9 wherein said the acid is 0.1N HCL
11. The process of claim 1 wherein said the tigecycline reconstituted solution
can be analyzed by UV-spectroscopy method.
12. The process of claim 1 wherein said the lyophilized cake obtained in
orange color.
13. A composition containing tigecycline, carbohydrate, suitable anti-oxidant
agent and buffering agent.
14. The composition of claim 13 wherein said carbohydrate includes lactose,
mannose, sucrose, glucose, monosaccharide and disaccharide e.g. aldose.
15. The composition of claim 13 and claim 14 wherein said the carbohydrate is
from disaccharide i.e. lactose
16. The composition of claim 13, claim 14, and claim 15 wherein said Lactose
is having different forms such as lactose anhydrous and lactose
monohydrate.
17. The composition of claim 16 wherein said carbohydrate is lactose
monohydrate
17

18. The composition of claim 13 wherein said the suitable anti-oxidant agent
include citric acid, ascorbic acid, thiols and sodium metabisulphite.
19. The composition of claiml3 and claim 18 wherein said the anti-oxidant is
used to reduce the oxidative degradation.
20. The composition of claim 13, claim 18 and claim 19 wherein said the
suitable anti-oxidant is sodium metabisulphite.
21. The composition of claim 13 wherein said the pH of the composition is
between 5.0 - 8.0
22. The composition of claim 13 and claim 21 wherein said the pH of the
composition is between 6.0 - 8.0
23. The composition of anyone of claims 13 to 22 wherein said the buffer is
acidic
24. The composition of claim 13 and claim 23 wherein said the acid is 0.1N
HCL