FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE SPECIFICATION [See section 10]
Process of preparing stable phospholipid formulation in injectable form using aqueous solvent by formation of bile salt conjugates
Nabros Pharma Pvt. Ltd.
401/501, Ashwamegh Complex
Near Mithakhali Under Bridge, Navrangpura, Ahemedabad. Gujarat
(India).
An Indian Company and
Misra Ambikanandan,
Professor and Head, Pharmacy Department,Faculty of Technology and Engineering, M.S.University of Baroda.Vadodara.Gujarat-390 001, India,
An Indian national
The following specification particularly describes the invention and the manner in which it is to be performed.


Title of the invention:
Process of preparing stable phospholipid formulation in injectable form using aqueous solvent by formation of bile salt conjugates
Background art:
Phospholipids are natural fats and produce certain essential fatty acids upon cleavage in the body. The resultant fatty acid lowers the cholesterol level and increase HDL level which in turn acts as heart protective. The compounds are also widely used in the diseases such as liver disorders, cirrhosis, degenerating neurological disorders and liver tonic. Phospholipids, due to highly lipophilic nature are insoluble in water and soluble in non polar solvents such as alcohols and chloroform. The present invention describes formulation of phospholipids in injectable form using aqueous solvent. The major challenge exists is solubilization of lipid in aqueous vehicle to obtain stabilized injectable dosage form. Bile salts are known to form conjugates with certain fatty acids and phospholipids. This invention describes the" formulation and optimization aspect of formation of bile salt conjugates of phospholipids derived from vegetable source and its stabilization.
Summary of the invention:
According to this invention there is a process of preparing stable phospholipid formulation in injectable form using aqueous solvent by formation of bile salt conjugates comprising the steps of
i. charging the bite salt and phospholipids in s.s. vessel and adding 90% w/w

of sterile water,
ii. heating the resultant mixture at 40 to 45°C and stirring for 30 minutes, iii.adding benzyl alcohol and stirring for 10 minutes, iv. making up the volume using water for injection, v. filtering the solution twice using 0.22 micron membrane filter. In this process the lipids are converted to form a complex using bile salts and the salt formation is optimized for concentration of bile acids and the process parameters.The optimization of bile salt conjugate stabilizes and yields the finished product which ensures minimal free concentration of either bile salts or phospholipids.
The finished product has the phospholipids: bile salt ratio as 1 .0.10 and forms a stable complex which has minimal free concentration of bile salt and phospholipids.
In this process the formed complex is highly unstable and oxidizes rapidly to form bile salts and saturated fatty acids,inorder to prevent oxidation nitrogen blanket are provided at various stages.The nitrogen level maintained in the solution is 1.50 mg/ml and less and the head space nitrogen blanket is Nl T 99% v/v.The nitrogen used is a special purified IOIAR 2 grade which may stabilize the preparation.
The finished product is further stabilized by selection and optimization of borosilicate glass ampoule, where in the glass ampoule is chloride and barium free which prevents the precipitation and cleavage of the formed complex due to increase in the alkalinity of the product. Detail description of the invention:
I. The phospholipids are insoluble in polar solvents/vehicles such as water and may limit its application in form of injectable preparation. The lipids are converted to form a complex using bile salts. The salt formation was optimized for concentration of bile acids and the process parameters.

2. The optimization of bile salt conjugate will stabilize and yield the finished product which ensures minimal free concentration of either bile salts or phospholipids. The phospholipids: bile salt ratio 1 :0.10 forms a stable complex which has minimal free concentration of bile salt and phospholipids. 3,The;formed complex is highly unstable and may oxidize rapidly to form bile salts and saturated fatty acids. The process has been designed to prevent oxidation by providing nitrogen blanket at various stages. The nitrogen level maintained in the solution was 1.50 mg/ml and less. The head space nitrogen blanket was NL T 99% vivo.
4. The entrapment of nitrogen in the head space atmosphere as claimed in 3 will further enhance the stabilization of the finished product. The nitrogen used was a special purified IOLAR 2 grade which may stabilize the preparation.
5. The finished product is further stabilized by selection and optimization of borosilicate glass ampoule. The glass ampoules was cWoride and barium free which prevents the precipitation and cleavage of the formed complex due to increase in the alkalinity of the product.
6. The process for formation and stabilization as depicted above yielded a product suitable for injectable form of phospholipids.

Composition of the present invention
Example 1: Injection bv 1:0.05 bile salt conjugate ratio:

S.No. Ingredients Quantity in mg! 5 mL
1. Phospholipids 250.0
2. Bile salt 12.50
3. Benzyl alcohol 0.50
4. Purified water Q.S.
Example 2:
Injection bv 1:0.10 bile salt conjugate ratio:

S.No. f—
5. Ingredients Quantity in mg!5 ml

Phospholipids 250.0
6. Bile salt 25.0
7. Benzyl alcohol 0.50
8. Purified water Q.S.

Manufacturing procedure for the present invention:
Example 1:
Injection bv 1:0.05 bile salt conjugate ratio:
1. Charge bile salt and phospholipids in s.s. Vessel and add 90% w/w of
sterile
water for injection.
2. Heat the resultant mixture at 40 to 45°C and stir for 30 minutes.
3. Add benzyl alcohol and stir for 10 minutes.
4. Make up the volume using water for injection.
5. Filter the solution twice using 0.22 micron membrane filter.
6. Send the bulk solution for analysis to Q.C. department.
7. Upon Q.C. approval fill the solution in USP type I glass ampoules.
8. Send the samples for analysis and upon release, pack as per the current packing specifications.
Example 2:
Injection bv 1:0.10 bile salt conjugate ratio:
1. Charge bile salt and phospholipids in s.s. vessel and add 90% w/w of sterile water for injection.
2. Heat the resultant mixture at 40 to 45°C and stir for 30 minutes.
3. Add benzyl alcohol and stir for 10 minutes.
4. Make up the volume using water for injection.
5. Filter the solution twice using 0.22 micron membrane filter.
6. Send the bulk solution for analysis to Q.C. department.
7. Upon Q.C. approval fill the solution in USP type 1 glass ampoules.
8. Send the samples for analysis "and upon, release, pack as per the current

in a 5ml amber color glass ampoule.
02. Identification Positive for essential phospholipids I.H.S.
03. Extractable volume 5.0 ml to 5.2 ml I.H.S.
04. PH 7.5-9.5 I.H.S.
05. Particulate matter To comply the test. I.H.S.
06. Test
for
sterility To comply the test. I.H.S.
07. Test for pyrogen To comply the test. I.H.S.
DESCRIPTION :
A clear yellow colored solution free from particulate matter filled in 5 ml amber color glass ampoule.
IDENTIFICATION: Essential phospholipids
It gives tests for phosphate.
Transfer 5 ml solution content to platinum crucible. Ignite first under the low

name
and then at high temperature till all carbon is burnt off. Dissolve the residue
in 5 ml 1 N Sulphuric acid and filter to 1 ml of the filtrate add 1 ml nitric acid and
ammonium molybdate solution.
Yellow precipitates are produced.
Identification of Riboflavine
Fill the contents of the ampoule in a test tube. Under UV light a vivid
ycllt'w.green fluorescence is observed.
EXTRACTABLE VOLUME:
Five containers containing approximately the same volume are selected for
volume estimation. The containers are then brought to a temperature of 20
degree centigrade plus minus(+- )2 degree centigrade. This step is essential
since temperature has an effect on quantum of volume. Now using a syringe
with a capacity not exceeding twice the volume to be measured and fitted
with a needle, the contents of the each of the five containers maintained at a
temperature of 20 plus minus(+- )2 degree centigrade are withdrawn as
completely as possible & transferred to a graduated cylinder of capacity
ranging from 10 ml (depending upon the contents / container).
The average content of the five containers should not be less than the
labeled volume per container and preferably within 110% of the claim but in
any event should not be more than 115% of the label claim.
PH: 7.5 - 9.5

packing specifications.
The main advantages of the invention are
1. The phospholipids are insoluble in polar solvents/vehicles such as water and may limit its application in form of injectable preparation. The lipids are converted to form a complex using bile salts.
2. The optimization of bile salt conjugate will stabilize and yield the finished product which ensures minimal free concentration of either bile salts or phospholipids.
3. The formed complex as claimed in 1 and 2, is highly unstable and may oxidize rapidly to form bile salts and saturated fatty acids. The process has been designed to prevent oxidation by providing nitrogen blanket at various stages.
4. 'The entrapment of nitrogen in the head space atmosphere -s Haimed in 3 will further enhance the stabilization of the finished product.
5. The finished product as claimed in :, and 4, is further stabilized by
selection and optimization of borosilicate glass ampoule.
QUALITY CONTROL PROCEDURE

SR. NO. TEST SPECIFICATION REFERENCE
01 Description A Clear, yellow color solution free from particulate matter filled I.H.S.

TEST FOR STERILITY:
The test is carried out using' Membrane filtration Method'. This method needs exceptional skill and special knowledge. The test must be carried out under conditions designed to avoid accidental contamination of the product during the test.
The working conditions in which the test is performed should be monitored regularly by sampling the air and surfaces of the working area and by carrying out control tests.
(a) APPARATUS:
The units consists of a closed reservoir and a receptacle between which a
properly supported membrane of 0.45 u porosity, diameter of app. 47 mm.
Assemble and sterilize the entire unit with the membrane in place prior to
use.
(b)DILUTING FLUID
Dissolve 1 gm of bacteriological peptone in water to make 1000 ml. Filter to
clarify, adjust to pH 7.1 plus minus 0.2, disperse into flasks in 100ml
quantities and sterilize at 121 degree Centigrade for 18-20 minutes.
(c) MEDIUM FOR AEROBIC AND ANAEROBIC/. BACTERIA:
FLUID THROUGH THYOGLYCOLATE MEDIUM

L - cystine 0.500 gm
Sodium Chloride 2.5gm


Dextrose 5.5GM
Granular Agar (less than 15% moisture by weight) 750GM
Yeast extract (water soluble) 5.0 gm
Pancreatic digest of CASEIN 15.00gm
Sodium
thioglycolate or Thioglycollic acid ,5gm or .3ml
Resazurin (1.10% fresh solution) 1.000ml
Distilled water 1000ml
Final pH after sterilisation 7.1 plus minus .2

Mix the ingredients other than the thioglycollate and the resazurin, in the order given above in a mortar, with through grinding. Stir in some heated water, transfer to a suitable container, add the remainder of the water-bath. Add the sodium thioglycollate, then I N sodium hydroxide so that the pH of the completed and sterilizes medium will be 7. I +- 0.2. Reheat the solution, but do not boil, filter (if necessary) through a moistened filter paper and add the resazurin solution. Distribute into suitable vessels and sterilize by autoclaving for 18 to 20 minutes at 121 degree (15 lbs psi, approximately 1.0 Kg/square cm). Cool promptly to 25° and store at 20- 30 degree centigrade, avoiding excessive light. If more than 30 percent of the uppermost portion of the medium has changed to a pinkish color it is unsuitable for use. Medium more than three weeks old should not be used.
(d) MEDIUM FOR FUNGI AND AEROBIC BACTERIA:
SOYABEAN CASEIN DIGEST MEDIUM

Pancreatic digest of casein 17.000 gm
Papaic digest of Soyabean meal 3.000 gm
Sodium Chloride 5.000 gm
Dibasic Potassium Phosphate 2.500 gm
Dextrose 2.500 gm

Final pH after sterilizations 7.3+-0.2
Dissolve the solids in the water, warming slightly to effects solution. Cool to room temperature and add, if necessary, sufficient 0.1 N sodium hydroxide to give a final pH after sterilization of between 7.1 and 7.5. Filter if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at 121 degree for about twenty minutes.
(e) METHOD OF TEST:
Aseptically transfer a small quantity of diluting fluid on the membrane and
filter it.
Take 20 ampoules of Diclofenac Sod. Injection and cleanse the exterior
surface with a suitable antimicrobial agent.
Transfer the entire quantity of solution from each of the twenty ampoules
aseptically on to the membrane. Draw the solution rapidly through the filter
with the aid of vacuum.
Wash the membrane by filtering through it three successive quantities each
of approximately 100 ml of sterile diluting fluid.
After filtration, aseptically remove the membrane from the holder, cut the
membrane in half. Aseptically immerse one half of the membrane in 100 ml
of soyabean-casein digest
medium, and incubate at 20° to 25° C for seven days. Similarly, immerse the
other half of the membrane in 100 ml of fluid thioglycollate medium and
incubate at 30° to 35° C for seven days.
f) OBSERVATIONS & INTERPRETATION OF RESULTS:
At intervals during the incubation period and at its conclusion, examine the
media for macroscopic evidence of microbial growth. If no evidence of growth
is found, the Diclofenac Sodium Injection being examined passes the test for

sterility.
If evidence of microbial growth is found, reserve the containers showing this and perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found, the preparation being examined passes the test for sterility. If evidence of microbial growth is readily distinguishable from those growing in the containers reserved in the first test the preparation being examined fails the test for sterility. If they are readily distinguishable from those growing in the containers reserved in the first test perform a second re-test using twice the number of samples. If no evidence of microbial growth is found in the second re-test, the preparation being examined passes the test for sterility. If evidence of growth of any micro-organism is found in the second retest the preparation being examined fails the test for sterility.
PARTICULATE MATTER:
Note: BP has not specifics any protocol of test for particulate matter in small volume parental. However, Diclofenac Sodium Injection when examined under normal daylight with unaided eye should be practically free from particulate matter.
ASSAY:
Diclofenac Sodium: Weigh accurately 50 mg of Diclofenac Sodium working standard and dissolve in 2 ml of alcohol and make up to 100 ml with 0.2 N sodium Hydroxide Solution. Dilute 2 ml of this solution to 100 ml with 0.2N Sodium Hydroxide Solution.
Test Solution: Dilute 2 ml or Diclofenac Sodium Injection to 100 ml with 0.2N Sodium Hydroxide Solution. Dilute 2 ml of this solution to 100 ml with 0.2 N

sodium hydroxide solution so as to get a dilution of approximately 10 meg
per ml.
Measure the absorbance of standard as well as test solution at 276 nm and
calculate the quantity in mg of Diclofenac Sodium per ml taking by the
formula:
In which WS is the weight 0.5 WS (AT/AS) of Dicofenac Sodium working
standard in mg. AT & AS is the absorbance of test solution and standard
solution.
ASSAY:
Estimation of Essential phosphoipids
ampoules is determined by means of HPLC; High Performance Liquid
Chromatography, according to the method external standard by means of UV
detection.
To state the accuracy another HPLC method is used (appendix 3-3.3)
The result (precision appendix 1-1.1) of (3-sn-phosphatidyl) choline and
lysophosphatidylcholine of both analytical methods do not differ significantly,
so that the accuracy of the methods is guaranteed (appendix 4-4.1)
The linearity of the method for (3-sn-phosphatidyl) choline see appendix 5-
5.1
Materials
1) (3-sn-phosphatidyl) choline from soybeans, standard substance, laboratory
samples with a content of 93% of (3-sn-phosphatidyl) choline. (If the content in the standard substance, laboratory sample differs from the stated content (93%) the detected content is corrected to 93% by means of factor f).
2) Lysophsophatidyl choline from soybeans. Standard substance, laboratory
sample with exact content in lysophosphatidyl choline.

3) N-Hexane, HPLC quality
4) Propanol-2 HPLC quality
5) Water HPLC quality
6) Methanol, HPLC quality
7) Tert Butyl Methyl ether, HPLC quality X) Millex HV 0.45 urn Filter Unit
Procedure
Standard solution:
Weight accurately approx 100 mg (3-sn-phosphatidyl) choline from soybeans (1) and approx. 30 mg lysophosphatidyl choline (2) and dissolve in a 100-ml volumetric flask with methanol (6), filing upto the calibration mark at 20 degree C. After filtration through a Millex HV filter (8) use a sample of this solution for analysis. Test solution:
Pipettee the quantity of injectible solution of this invention until the expected quantity of (3-sn-phosphatidyl) choline from soybeans in the test is identical with that in the standard. Then dissolve this quantity in a 100 ml volumetric flask with a mixture of methanol (6) and tert. butyl methyl ether (7) and fill up to the calibration mark at 20 degree C. After filtration through a Millex HV filter (8) use a sample of this solution for analysis.
Apparatus / Apparatus Conditions:
Merk/
Hitachi - HPLC equipment consisting of:
D2500 Chromato Integrator
Merck T 6300 Column Thermostat
Merck/Hitachi L4000 UY Detector

Merck / Hitachi L6200 Intelligent Pump
Merck/ Hitachi 655 A-40 Autosampler
Erma ERC-3512 Degaser
or correspondent HPLC equipment.
Column Kromasil Si 5A. length 125 mm. 04 mm i.D.
Precolumn LiChrosorb SI 60-5 A, length 20 mm, 0. 4 mm i.D.

Flow : 1ml/min
Oven temperature : 40 degree C
Injection volume :10μl
Wave length : 205 nm
Separate by the following mixture of solvents:
Propanol-2 (4) In-hexane (3) /water (5)
105 g 22 g , 32 g (m/m)
Evaluation
The quantitative determination is performed according to the method external standard. For this purpose inject one after the other exactly the same quantity of test and standard into the separation column. Calculate by comparing the standard peak areas with the test peak areas with respect to the initial weight.
FpxExfx 100
% (3-sn-phosphatidyl)choline =
FsxP Fp = peak area of test solution Fs= peak area of standard solution E = initial weight lysophosphatidylcholine - standard in mg. P = initial weight test in mg f= lysophosphatiuylcholine content in standard.
\ *
Determination of benzal alcohol
General
Benzyl alcohol is separated by gas chromatographyon a Carbo-Wax column.
Ethylene
Glycol is added as internal standard. Ethanol serves as solvent.
Benzyl alcohol is identified at the same time by comparison of the retention
times of standard and test sample.
Reagents
(1) Ethanol, p.a
(2) Ethylene glycol as internal standard
(3) Benzyl alcohol, DAB 10 Procedure
(a) Calibration solution:
The calibration solution contains approx. 80 mg of ethylene glycol (2) (amount = AE), accurately weighed, and 80 mg of benzyl alcohol (3) (amount = AB), accurately weighed, in 25.00 ml ethanol (I). Sequence of the peaks and retention times:
ethylene glycol (IS) 200
benzyl alcohol 510
(b) Test solution
Pipette approx. 40 mg ethylene glycol (2) (amount = AS), accurately weighed, and 1.00 ml of the test sample into a 10 ml volumetric flask. GC conditions Apparatus: Carlo Erba
Column: CP Sil 8CB 50 m diameter 0.32 mm

Injector: 250 degree C
Detector: FID 2500C
carrier gase:H2 2.0 bar
combustible gases: Hydrogen 30 ml/min
AAir 300 ml/min Make-up gas: 1.0 bar Oven: 80°C 2 min 20°C/min. 200 degree c 210°C5min
Calculation
CF=( AB x areas) / Area x AE
(AS x CF x area x 5)/(1 ml sample x areas) = mg benzyl alcohol / 5 ml
CF = correction factor
IS=ethylene glycol
I = benzyl alcohol