Title of Invention	STABLE INJECTABLE OIL-IN-WATER NANOEMULSION
Abstract	The present invention provides an injectable oil-in-water nanoemulsion having average particle size less than 200nm comprising: water insoluble drugs in the concentration of at least 0.01% w/v; oils selected from one or more vegetable oils such as Soya oil, olive oil; MCT oil or mixture thereof, in a concentration of 1% w/v to 30% w/v; Phospholipid in the concentration of 0.1 to 5% w/v; Pegylated phospholipid in the concentration of 0.01 - 10% w/v; other excipients such as isotonicity agents, buffer, antioxidant, stabilizer.

Title of Invention

STABLE INJECTABLE OIL-IN-WATER NANOEMULSION

Abstract

The present invention provides an injectable oil-in-water nanoemulsion having average particle size less than 200nm comprising: water insoluble drugs in the concentration of at least 0.01% w/v; oils selected from one or more vegetable oils such as Soya oil, olive oil; MCT oil or mixture thereof, in a concentration of 1% w/v to 30% w/v; Phospholipid in the concentration of 0.1 to 5% w/v; Pegylated phospholipid in the concentration of 0.01 - 10% w/v; other excipients such as isotonicity agents, buffer, antioxidant, stabilizer.

Full Text	FORM 2 THE PATENTS ACT, 1970 (39 OF 1970) AND THE PATENT RULES, 2003 PROVISIONAL SPECIFICATION (See section 10; rule 13) Title of the Invention: "Stable Injectable oil-in-water Nanoemulsion Applicants): (a) Name BHARAT SERUMS AND VACCINES LTD. (b) Nationality An Indian company incorporated under the Companies Act 1956. (c) Address 17 Floor, Hoechst House, Nariman Point, Munibai - 400 021. Maharashtra, India. The present invention relates to oil in water emulsion containing pegylated phospholipid for parenteral adrninistration of taxoid drugs or other water insoluble drugs. Background and prior art: Docetaxel is commercially available in the form of an injection concentrate under brand name Taxotere. Taxotere is formulated in polysorbate 80 as solubiliser. Taxotere injection comprises two compartment formulations that require two-step dilution before infusion. The first step involves dilution with content of diluent vial (13% ethanol in water for injection) and the second step involves further dilution with diluents such as Dextrose Injection or normal saline etc. for parenteral administration. Polysorbate 80 causes severe hypersensitivity reaction and fluid retention, hence patients require pre-medications. Thus the marketed formulation has serious manufacturing, handling and dosing limitations. Further Polysorbate 80 can not be used with PVC delivery apparatus because of its tendency to leach diethyl hexyl phthalate, which is highly toxic. To avoid these difficulties of mixing two solution before injection following inventions have been reported-US 5478860 describes a stable micro-emulsion for hydrophobic compounds comprising mixture of oil, phospholipid, pegylated lipids. A typical process comprises adding drug to oil, solubilising pegylated lipid and EPC in chloroform, adding this chloroform solution to oil containing drug, admixing well. Chloroform is removed under vacuum to form a film. Film is hydrated with HEPES buffered saline solution (HBSS) followed by addition separately prepared -2- EPC vesicles to obtain drug to oil weight ratio 1:15. Then the mixture was passed through microfluidizer to decrease the size below 1 OOnm. US 2006/0067952A1 describes injectable oil in water emulsion of taxoid drugs, comprising phospholipids and vegetable oils, which has to be diluted with aqueous fluid before administration. However, the emulsion is not a stable product and it is preserved as a lyophilized powder and needs reconstitution before use. A typical process comprises mixing drug, soya oil, MCT oil, egg lecithin and sufficient amount of ethanol to form clear solution. The solution is dried under vacuum until residual ethanol is less than 2.0% by weight. Aqueous phase is prepared by dissolving glycerin and glycine in water and aqueous phase as added to oil phase under higher shear mixer to obtain crude emulsion. pH was adjusted to about 6.5 and the emulsion is passed through microfluidiser and the resulting emulsion is filtered through sterile 0.2u filter. However, US 2006/0067952Al dose not give a stable product therefore lyophilisation and reconstitution is required before use. WO2008/042841A2 describes pre-concentrate composition comprising docetaxel containing co-solvent like ethanol and propylene glycol phospholipids, and pegylated phospholipids, suitable for parenteral administration to treat neoplasm conditions upon dilution with aqueous fluids. This pre-concentrate is a non¬aqueous solution and forms emulsion on dilution. However when used in larger doses it is harmful due to toxicity of solvents such as ethanol. WO2008/042841A2 contains co-solvent which is harmful when given in larger doses. -3- Object of Invention: Object of the present invention is to make formulation which is devoid of hypersensitivity reaction and fluid retention there-by avoiding pre-medications. Another object of the present invention is to avoid co-solvents like ethanol in the formulation thereby eliminating adverse effects causes by the cosolvents. Yet another object of the present invention is to develop a process to make a stable liquid emulsion so that lyophilisation and reconstitution before administration is avoided. Summary of the Invention: Accordingly, the present invention provides an injectable oil-in-water nanoemulsion having average particle size less than 200nm comprising: water insoluble drugs in the concentration of at least 0.01% w/v; oils selected from one or more vegetable oils such as Soya oil, olive oil; MCT oil or mixture thereof, in a concentration of 1% w/v to 30% w/v; Phospholipid in the concentration of 0.1 to 5% w/v; Pegylated phospholipid in the concentration of 0.01 - 10% w/v; other excipients such as isotonicity agents, buffer, antioxidant, stabilizer. In another embodiment of the present invention the process for manufacturing of the above composition comprises following steps, solubilising or dispersing the drug in the oil phase at a temperature of 60 - 70°C; dispersing Phospholipids, Pegylated phospholipid and other excipients in aqueous phase; transferring oil phase to aqueous phase under stirring to form coarse emulsion; homogenization of coarse emulsion to get globule size below 200nm; sterilization of Emulsion by filtration through 0.2u. sterilizing grade filter under aseptic conditions. -4- Detail description of the invention: 1. Nanoemulsion - The definition of emulsions by the International Union of Pure and Applied Chemistry (IUPAC) states: "In an emulsion, liquid droplets and/or liquid crystals are dispersed in a liquid". Obviously, microemulsions are excluded from this definition if the word "dispersed" is interpreted as non-equilibrium and opposite to "solubilized", term that can be applied to microemulsions and micellar systems. Therefore, there is a fundamental difference between microemulsions and nano-emulsions. Microemulsions are equilibrium systems (i.e. thermodynamically stable), while nano-emulsions are non-equilibrium system with a spontaneous tendency to separate into the constituent phases. According to this invention Nano-emulsions are emulsions (non-equilibrium systems) with a small droplet size (in the nanometer range, e.g. 20-200 nm). Nanoemulsions are not to be mistaken with the classic "microemulsions", which are thermodynamically stable and are often referred to as "self-emulsifying systems". Microemulsions are formed when the surface tension is reduced to nearly zero and is only achieved by particular surfactants, combinations or particular packing of the adsorbed layer with surfactant and co-surfactant. These exhibit a very low viscosity and basically comprise swollen micelles with solubilized oil (and drugs). Microemulsion systems are transparent (optically isotropic), but upon dilution they can form conventional emulsion systems Oil includes vegetable oils & medium chain triglycerides (MCT's). For examples vegetable oil includes, but are not limited to soyabean oil, corn oil, safflower oil, olive oil, etc. vegetable oils are typically long chain triglyceride (usually 14 to about 22 carbons in length). -5- MCT oil either can naturally derived or synthetic. MCT are made from free fatty acid usually about 8 to about 12 carbon lengths. Representatives are commercially available as "Miglyol 840, MIGLYOL 812, CRODAMOL GTCC-PN, NEOBEE M-5 oil. The amount of oil, by weight, generally used in this invention of about 3% to about 7%. Suitable emulsifier includes phospholipids. Phospholipids are trimester of glycerol with two fatty acid & one phosphate ion. Examples of Phospholipids includes but not limited to phosphatidylcholine, lecithin, and more & mixture of phospholipids. The amount of oil, by weight, generally used in this invention of about 1% to about 3%. The ratio of oil to phospholipids in the nanoemulsion of the present invention is from about 7:1 to about 1:1 (w/w). A phospholipid -PEG conjugate for this invention is PEG-phosphatidyl ethanolamine. The PEG chain in such phospholipid preferable has molecular weight in the range of 2000 to 5000. Preferred ratio of pegylated phospholipid to non-pegylated phospholipids used in the invention is about 1:1 to about 1:3 on weight basis. The composition of present invention may optionally contain additives such as acidifier, alkalinizer, buffer, stabilizer, tonicity modifying agents and other biocompatible materials. Such agents are generally present in aqueous phase of emulsion which helps in stabilizing the emulsion. .£. . Examples of acidifier but are not limited to hydrochloric acid, citric acid, acetic acid, etc. Examples of alkalinniser includes sodium hydroxide, sodium citrate etc. Other biocompatibles materials includes but are not limited to sucrose, mannitol, trehalose, albumin, sorbitol, glecerol, glycine etc. EXAMPLES: The invention will now be illustrated with the help of examples. Examples are for illustrations purpose only and do not restrict the scope the invention. Example 1: Equipments used Water bath. Ultra Turrax IKA stirrer, Niro Saovi Homogenize!. Formula Ingredients Quantity Docetaxel trihydrate 214.0 mg MCT oil 10.0 gm Egg Lecithin 2.4 gm DSPE PEG 2000 1.0 gm Glycerol 4.50 gm Water For injection q.s to 200 ml 0.05NHC1 Solution q.s to adjust the pH -7- Procedure Oil phase: 1. Required Quantity of Docetaxel Trihydrate weighed in required quantity of MCT oil. 2. Sonicated above mixture for 10 min and heated to get clear oily colorless liquid. Aqueous Phase 3. Weighed quantity of Glycerol was mixed with required quantity of Water For injection at Room Temperature (20°C±5°C). 4. Required quantity of DSPE PEG-2000 was solubilized in above solution obtained in Step 3. 5. Required quantity of Egg Lecithin is then dispersed in the aqueous solution obtained at Step 4. Coarse Emulsion 6. Oily phase is transferred to aqueous phase under high speed stirring (on Ultra Turax IKA stirrer) to obtain coarse emulsion. Homogenization 7. Coarse emulsion obtained was immediately homogenized at higher pressure to get desired particle size distribution. -8- 8. Adjusted the pH of above emulsion. 9. Emulsion is then filtered through 0.2u filter, filled in vials and sealed under nitrogen purging. Example 2: Composition and process is same as Example 1 except that in Example 2 DSPE PEG-2000 was not used and homogenization is carried at higher pressure for longer period. Table 1 -Observations of Example 1, Example 2, Example 6, Example 9, Example 10 Results Tests Example 1 Example 2 Example 6 Example 8 Example 9 Example 10 Appearance White Opaque Liquid White Opaque Liquid White Opaque Liquid White Opaque Liquid White Opaque Liquid White Opaque Liquid pH Aq. Phase 5.65 6.33 6.59 6.69 7.8 6.6 Coarse 5.82 5.98 6.11 7.01 7.2 6.15 Final Homogenisation 5.68 5.96 6.08 6.64 6.34 5.79 After pH adjustment 4.88 4.63 4.94 4.50 4.68 4.75 Particle Size (nm) Coarse 212.0 283.4 164.7 201.6 180.0 256.2 Final Homogenisation 95.9 142.2 94.8 101.8 99.0 106.8 After pH adjustment 99.2 140.7 93.0 102.4 99.7 104.1 -9- Stability Result: Example No. Time Temperature Conditions 2-8°C 25°C 40°C 1 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid 24 hrs White Opaque Liquid White Opaque Liquid White Opaque Liquid 2 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid 24 hrs White opaque liquid with settling of drug White opaque liquid with settling of drug White opaque liquid with settling of drug 6 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid 24 hrs White Opaque Liquid White Opaque Liquid White Opaque Liquid 8 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid 24 hrs White Opaque Liquid White Opaque Liquid White Opaque Liquid 9 Initial. White Opaque Liquid White Opaque Liquid White Opaque Liquid 24 hrs White opaque liquid with settling of drug White opaque liquid with settling of drug White opaque liquid with settling of drug 10 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid 24 hrs White Opaque Liquid White Opaque Liquid White Opaque Liquid Conclusion With increasing homogenization time for emulsion without pegylated phospholipids it is not possible to reduce the average particle size below 140nm. Emulsion without pegylated phospholipids shows settling of drug after 24hrs where as emulsion product prepared incorporating pegylated phospholipids dose not show any settling of drug at all storage conditions studied. Example 3: Acute Toxicity Study for composition product of Example 1 A) Single dose Acute Toxicity in Mice Animal : Mice Species : Swiss albino No. of animals per group : 10 Dose : 150mg/kg -10- Sample % mortality after 14 days Example 1 50% Taxotere 100% B) Single Dose Acute Toxicity in Rat Sample Mortality l0mg/kg 30mg/kg 50mg/kg Example 1 0/6 0/6 2/6 Taxotere 0/6 2/6 5/6 Example 4: Toxicity study for composition product of Example 1 Animal Species Dose Dosage schedule Mice Swiss albino 10,22, 33, 50mg/kg q4d X 3 (0, 4, 8 days) Sample Dose % mortality after 14 days Example 1 l0 mg/kg 0% 22 mg/kg 0% 33 mg/kg 0% 50 mg/kg 40% Taxotere l0 mg/kg 0% 22 mg/kg 10% 33 mg/kg 20% 50mg/kg 70% Example 5: Comparative Single dose pharmacokinetic in Rat Composition of Example 1 is used and Taxotere is used as a comparator. Animal : Rat Species : Wistar Dose : l0mg/kg -11 - Time (hrs) Plasma concentration (ng/mL) Taxotere Example 1 0.083 1374.15 4070.84 0.5 445.41 564.21 4 166.29 221.33 8 59.11 191.82 24 68.49 53.74 Cmax and AUC with composition of Example 1 were found to be higher than that obtained with Taxotere. Example 6: The process and quantities of excipients are same as Example 1 except in stead of Docetaxel trihydrate, Docetaxel anhydrous was used. Observations and stability results are given in Table 1. Conclusion This example shows emulsion with docetaxel anhydrous shows similar stability profile as docetaxel trihydrate. Example 7: In-vitro Plasma Study of products of Example 1 and Example 2 Procedure 1. 0.2 ml of Docetaxel emulsion mixed in 0.9 ml of Human plasma in eppendorff tube. 2. Particle size of mixture is analyzed. 3. The mixed sample is incubated at 37°C for 24 hr. 4. Particle size of incubated sample is analyzed. -12- Observations Example No. Initial particle size After Incubation at 37°C for 24 Hr Example 1 105.1 nm 106.2 nm 105.1nm 103.9 nm Example 2 140 nm 1.32micron 140 nm 1.47micron Nanoemulsion prepared with pegylated phospholipid is stable in plasma where as emulsion prepared without pegylated phospholipid is not physically stable. Example 8: Nanoemulsion prepared using mixture of vegetable oil and MCT oil Equipments used Water bath, Ultra Turrax IKA stirrer, Niro Saovi Homogenizer. Formula Ingredients Quantity Docetaxel trihydrate 107'mg MCT oil 2.5 gm Soya Oil 2.5 gm Egg Lecithin 1.2 gm DSPE PEG 2000 500mg Glycerol 2.25 gm Water For injection q.s to l00 ml 0.05N HC1 Solution q.s to adjust pH Procedure Same as Example 1 Observations and stability results are given in Table 1. -13- Example 9 - Prepared as per US 2006\0067952Al Equipments used Water bath, Ultra Turrax IKA stirrer, Niro Saovi Homogenizer. Ingredients Qty/Batch Docetaxel Trihydrate 53.5' mg MCT oil 1.5gm Soya oil 1.5 gm Ethanol 2.0 ml Egg Lecithin 3.1 gm Glycine 50 mg Glycerol 2.25 gm Water For injection q.sto 100 ml 0.05N HCI Solution q.s to adjust pH Procedure Same as Example 1 Observations and stability results are given in Table 1. This example shows the essentiality of pegylated phospholipids for the stability of the formulation. Example 10 - Formulation prepared with DPPC as surfactant instead of egg lecithin Equipments used Water bath, Ultra Turrax IKA stirrer, Niro Saovi Homogenizer. Ingredients Qty/Batch Docetaxel Trihydrate 108.0Tmg MCT oil 5.0 gm DPPC 1.0 gm DSPE PEG 2000 1.0 gm Glycerol 2.25 gm Water For injection q.sto 100 ml 0.05N HCI Solution q.s to adjust pH -14- Procedure Same as Example 1 Instead of egg lecithin DPPC was dispersed in aqueous phase. Observations and stability results are given in Table 1. Advantages of the invention: 1. The compositions of the present invention are free from ethanol and surfactant Polysorbate-80. Therefore composition of present invention is devoid of hypersensitivity reaction and fluid retention characteristics of these ingredients. 2. No pre-medication required to overcome hypersensitivity reactions experienced with currently marketed preparation. 3. Shows higher Cmax and AUC may lead to better efficacy at equivalent doses or if the dosage is reduced then it may lead to less toxic effects of the drug. 4. The process of preparation is free from any solvent and co-solvent like ethanol, propylene glycol and chloroform. 5. Process gives stable nanoemulsion which gives Enhanced Permeability Retention (EPR) effect. Dated this 17th day of July 2008. To The Controller of Patents, The Patent Office, At Mumbai. - 15-

Full Text

FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See section 10; rule 13)

Title of the Invention:
"Stable Injectable oil-in-water Nanoemulsion
Applicants):
(a) Name
BHARAT SERUMS AND VACCINES LTD.

(b) Nationality

An Indian company incorporated under the Companies Act 1956.

(c) Address

17 Floor, Hoechst House, Nariman Point, Munibai - 400 021. Maharashtra, India.

The present invention relates to oil in water emulsion containing pegylated phospholipid for parenteral adrninistration of taxoid drugs or other water insoluble drugs.
Background and prior art:
Docetaxel is commercially available in the form of an injection concentrate under brand name Taxotere. Taxotere is formulated in polysorbate 80 as solubiliser. Taxotere injection comprises two compartment formulations that require two-step dilution before infusion. The first step involves dilution with content of diluent vial (13% ethanol in water for injection) and the second step involves further dilution with diluents such as Dextrose Injection or normal saline etc. for parenteral administration.
Polysorbate 80 causes severe hypersensitivity reaction and fluid retention, hence patients require pre-medications. Thus the marketed formulation has serious manufacturing, handling and dosing limitations.
Further Polysorbate 80 can not be used with PVC delivery apparatus because of its tendency to leach diethyl hexyl phthalate, which is highly toxic.
To avoid these difficulties of mixing two solution before injection following inventions have been reported-US 5478860 describes a stable micro-emulsion for hydrophobic compounds comprising mixture of oil, phospholipid, pegylated lipids. A typical process comprises adding drug to oil, solubilising pegylated lipid and EPC in chloroform, adding this chloroform solution to oil containing drug, admixing well. Chloroform is removed under vacuum to form a film. Film is hydrated with HEPES buffered saline solution (HBSS) followed by addition separately prepared
-2-

EPC vesicles to obtain drug to oil weight ratio 1:15. Then the mixture was passed through microfluidizer to decrease the size below 1 OOnm.
US 2006/0067952A1 describes injectable oil in water emulsion of taxoid drugs, comprising phospholipids and vegetable oils, which has to be diluted with aqueous fluid before administration. However, the emulsion is not a stable product and it is preserved as a lyophilized powder and needs reconstitution before use.
A typical process comprises mixing drug, soya oil, MCT oil, egg lecithin and sufficient amount of ethanol to form clear solution. The solution is dried under vacuum until residual ethanol is less than 2.0% by weight. Aqueous phase is prepared by dissolving glycerin and glycine in water and aqueous phase as added to oil phase under higher shear mixer to obtain crude emulsion. pH was adjusted to about 6.5 and the emulsion is passed through microfluidiser and the resulting emulsion is filtered through sterile 0.2u filter.
However, US 2006/0067952Al dose not give a stable product therefore lyophilisation and reconstitution is required before use.
WO2008/042841A2 describes pre-concentrate composition comprising docetaxel containing co-solvent like ethanol and propylene glycol phospholipids, and pegylated phospholipids, suitable for parenteral administration to treat neoplasm conditions upon dilution with aqueous fluids. This pre-concentrate is a non¬aqueous solution and forms emulsion on dilution. However when used in larger doses it is harmful due to toxicity of solvents such as ethanol.
WO2008/042841A2 contains co-solvent which is harmful when given in larger doses.
-3-

Object of Invention:
Object of the present invention is to make formulation which is devoid of hypersensitivity reaction and fluid retention there-by avoiding pre-medications. Another object of the present invention is to avoid co-solvents like ethanol in the formulation thereby eliminating adverse effects causes by the cosolvents.
Yet another object of the present invention is to develop a process to make a stable liquid emulsion so that lyophilisation and reconstitution before administration is avoided.
Summary of the Invention:
Accordingly, the present invention provides an injectable oil-in-water nanoemulsion having average particle size less than 200nm comprising: water insoluble drugs in the concentration of at least 0.01% w/v; oils selected from one or more vegetable oils such as Soya oil, olive oil; MCT oil or mixture thereof, in a concentration of 1% w/v to 30% w/v; Phospholipid in the concentration of 0.1 to 5% w/v; Pegylated phospholipid in the concentration of 0.01 - 10% w/v; other excipients such as isotonicity agents, buffer, antioxidant, stabilizer.
In another embodiment of the present invention the process for manufacturing of the above composition comprises following steps, solubilising or dispersing the drug in the oil phase at a temperature of 60 - 70°C; dispersing Phospholipids, Pegylated phospholipid and other excipients in aqueous phase; transferring oil phase to aqueous phase under stirring to form coarse emulsion; homogenization of coarse emulsion to get globule size below 200nm; sterilization of Emulsion by filtration through 0.2u. sterilizing grade filter under aseptic conditions.
-4-

Detail description of the invention:
1. Nanoemulsion - The definition of emulsions by the International Union of Pure and Applied Chemistry (IUPAC) states: "In an emulsion, liquid droplets and/or liquid crystals are dispersed in a liquid". Obviously, microemulsions are excluded from this definition if the word "dispersed" is interpreted as non-equilibrium and opposite to "solubilized", term that can be applied to microemulsions and micellar systems. Therefore, there is a fundamental difference between microemulsions and nano-emulsions. Microemulsions are equilibrium systems (i.e. thermodynamically stable), while nano-emulsions are non-equilibrium system with a spontaneous tendency to separate into the constituent phases.
According to this invention Nano-emulsions are emulsions (non-equilibrium systems) with a small droplet size (in the nanometer range, e.g. 20-200 nm).
Nanoemulsions are not to be mistaken with the classic "microemulsions", which are thermodynamically stable and are often referred to as "self-emulsifying systems". Microemulsions are formed when the surface tension is reduced to nearly zero and is only achieved by particular surfactants, combinations or particular packing of the adsorbed layer with surfactant and co-surfactant. These exhibit a very low viscosity and basically comprise swollen micelles with solubilized oil (and drugs). Microemulsion systems are transparent (optically isotropic), but upon dilution they can form conventional emulsion systems
Oil includes vegetable oils & medium chain triglycerides (MCT's). For examples vegetable oil includes, but are not limited to soyabean oil, corn oil, safflower oil, olive oil, etc. vegetable oils are typically long chain triglyceride (usually 14 to about 22 carbons in length).
-5-

MCT oil either can naturally derived or synthetic. MCT are made from free fatty acid usually about 8 to about 12 carbon lengths. Representatives are commercially available as "Miglyol 840, MIGLYOL 812, CRODAMOL GTCC-PN, NEOBEE M-5 oil.
The amount of oil, by weight, generally used in this invention of about 3% to about 7%.
Suitable emulsifier includes phospholipids. Phospholipids are trimester of glycerol with two fatty acid & one phosphate ion. Examples of Phospholipids includes but not limited to phosphatidylcholine, lecithin, and more & mixture of phospholipids.
The amount of oil, by weight, generally used in this invention of about 1% to about 3%.
The ratio of oil to phospholipids in the nanoemulsion of the present invention is from about 7:1 to about 1:1 (w/w).
A phospholipid -PEG conjugate for this invention is PEG-phosphatidyl ethanolamine. The PEG chain in such phospholipid preferable has molecular weight in the range of 2000 to 5000. Preferred ratio of pegylated phospholipid to non-pegylated phospholipids used in the invention is about 1:1 to about 1:3 on weight basis.
The composition of present invention may optionally contain additives such as acidifier, alkalinizer, buffer, stabilizer, tonicity modifying agents and other biocompatible materials. Such agents are generally present in aqueous phase of emulsion which helps in stabilizing the emulsion.
.£. .

Examples of acidifier but are not limited to hydrochloric acid, citric acid, acetic acid, etc.
Examples of alkalinniser includes sodium hydroxide, sodium citrate etc.
Other biocompatibles materials includes but are not limited to sucrose, mannitol, trehalose, albumin, sorbitol, glecerol, glycine etc.
EXAMPLES:
The invention will now be illustrated with the help of examples.
Examples are for illustrations purpose only and do not restrict the scope the invention.
Example 1:
Equipments used
Water bath. Ultra Turrax IKA stirrer, Niro Saovi Homogenize!.
Formula

Ingredients Quantity
Docetaxel trihydrate 214.0 mg
MCT oil 10.0 gm
Egg Lecithin 2.4 gm
DSPE PEG 2000 1.0 gm
Glycerol 4.50 gm
Water For injection q.s to 200 ml
0.05NHC1 Solution q.s to adjust the pH
-7-

Procedure
Oil phase:
1. Required Quantity of Docetaxel Trihydrate weighed in required quantity of MCT oil.
2. Sonicated above mixture for 10 min and heated to get clear oily colorless
liquid.
Aqueous Phase
3. Weighed quantity of Glycerol was mixed with required quantity of Water For injection at Room Temperature (20°C±5°C).
4. Required quantity of DSPE PEG-2000 was solubilized in above solution obtained in Step 3.
5. Required quantity of Egg Lecithin is then dispersed in the aqueous solution obtained at Step 4.
Coarse Emulsion
6. Oily phase is transferred to aqueous phase under high speed stirring (on
Ultra Turax IKA stirrer) to obtain coarse emulsion.
Homogenization
7. Coarse emulsion obtained was immediately homogenized at higher
pressure to get desired particle size distribution.
-8-

8. Adjusted the pH of above emulsion.
9. Emulsion is then filtered through 0.2u filter, filled in vials and sealed under nitrogen purging.
Example 2:
Composition and process is same as Example 1 except that in Example 2 DSPE PEG-2000 was not used and homogenization is carried at higher pressure for longer period.
Table 1 -Observations of Example 1, Example 2, Example 6, Example 9, Example 10

Results
Tests Example 1 Example
2 Example 6 Example 8 Example 9 Example 10
Appearance White
Opaque
Liquid White
Opaque
Liquid White
Opaque
Liquid White
Opaque
Liquid White
Opaque
Liquid White
Opaque
Liquid
pH Aq. Phase 5.65 6.33 6.59 6.69 7.8 6.6

Coarse 5.82 5.98 6.11 7.01 7.2 6.15

Final
Homogenisation 5.68 5.96 6.08 6.64 6.34 5.79

After pH adjustment 4.88 4.63 4.94 4.50 4.68 4.75
Particle
Size
(nm) Coarse 212.0 283.4 164.7 201.6 180.0 256.2

Final Homogenisation 95.9 142.2 94.8 101.8 99.0 106.8

After pH adjustment 99.2 140.7 93.0 102.4 99.7 104.1
-9-

Stability Result:

Example
No. Time Temperature Conditions

2-8°C 25°C 40°C
1 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid

24 hrs White Opaque Liquid White Opaque Liquid White Opaque Liquid
2 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid

24 hrs White opaque liquid with settling of drug White opaque liquid with settling of drug White opaque liquid with settling of drug
6 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid

24 hrs White Opaque Liquid White Opaque Liquid White Opaque Liquid
8 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid

24 hrs White Opaque Liquid White Opaque Liquid White Opaque Liquid
9 Initial. White Opaque Liquid White Opaque Liquid White Opaque Liquid

24 hrs White opaque liquid with settling of drug White opaque liquid with settling of drug White opaque liquid with settling of drug
10 Initial White Opaque Liquid White Opaque Liquid White Opaque Liquid

24 hrs White Opaque Liquid White Opaque Liquid White Opaque Liquid
Conclusion
With increasing homogenization time for emulsion without pegylated phospholipids it is not possible to reduce the average particle size below 140nm.
Emulsion without pegylated phospholipids shows settling of drug after 24hrs where as emulsion product prepared incorporating pegylated phospholipids dose not show any settling of drug at all storage conditions studied.
Example 3: Acute Toxicity Study for composition product of Example 1
A) Single dose Acute Toxicity in Mice
Animal : Mice
Species : Swiss albino
No. of animals per group : 10
Dose : 150mg/kg
-10-

Sample % mortality after 14 days
Example 1 50%
Taxotere 100%
B) Single Dose Acute Toxicity in Rat

Sample Mortality

l0mg/kg 30mg/kg 50mg/kg
Example 1 0/6 0/6 2/6
Taxotere 0/6 2/6 5/6
Example 4: Toxicity study for composition product of Example 1

Animal
Species
Dose
Dosage schedule

Mice
Swiss albino 10,22, 33, 50mg/kg q4d X 3 (0, 4, 8 days)

Sample Dose % mortality after 14 days
Example 1 l0 mg/kg 0%

22 mg/kg 0%

33 mg/kg 0%

50 mg/kg 40%
Taxotere l0 mg/kg 0%

22 mg/kg 10%

33 mg/kg 20%

50mg/kg 70%
Example 5: Comparative Single dose pharmacokinetic in Rat
Composition of Example 1 is used and Taxotere is used as a comparator.

Animal : Rat
Species : Wistar
Dose : l0mg/kg
-11 -

Time (hrs) Plasma concentration (ng/mL)

Taxotere Example 1
0.083 1374.15 4070.84
0.5 445.41 564.21
4 166.29 221.33
8 59.11 191.82
24 68.49 53.74
Cmax and AUC with composition of Example 1 were found to be higher than that obtained with Taxotere.
Example 6:
The process and quantities of excipients are same as Example 1 except in stead of Docetaxel trihydrate, Docetaxel anhydrous was used.
Observations and stability results are given in Table 1.
Conclusion
This example shows emulsion with docetaxel anhydrous shows similar stability profile as docetaxel trihydrate.
Example 7: In-vitro Plasma Study of products of Example 1 and Example 2
Procedure
1. 0.2 ml of Docetaxel emulsion mixed in 0.9 ml of Human plasma in eppendorff tube.
2. Particle size of mixture is analyzed.
3. The mixed sample is incubated at 37°C for 24 hr.
4. Particle size of incubated sample is analyzed.
-12-

Observations

Example No. Initial particle size After Incubation at 37°C for 24 Hr
Example 1 105.1 nm 106.2 nm

105.1nm 103.9 nm
Example 2 140 nm 1.32micron

140 nm 1.47micron
Nanoemulsion prepared with pegylated phospholipid is stable in plasma where as emulsion prepared without pegylated phospholipid is not physically stable.
Example 8: Nanoemulsion prepared using mixture of vegetable oil and MCT oil
Equipments used
Water bath, Ultra Turrax IKA stirrer, Niro Saovi Homogenizer.
Formula

Ingredients Quantity
Docetaxel trihydrate 107'mg
MCT oil 2.5 gm
Soya Oil 2.5 gm
Egg Lecithin 1.2 gm
DSPE PEG 2000 500mg
Glycerol 2.25 gm
Water For injection q.s to l00 ml
0.05N HC1 Solution q.s to adjust pH
Procedure
Same as Example 1
Observations and stability results are given in Table 1.
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Example 9 - Prepared as per US 2006\0067952Al
Equipments used
Water bath, Ultra Turrax IKA stirrer, Niro Saovi Homogenizer.

Ingredients Qty/Batch
Docetaxel Trihydrate 53.5' mg
MCT oil 1.5gm
Soya oil 1.5 gm
Ethanol 2.0 ml
Egg Lecithin 3.1 gm
Glycine 50 mg
Glycerol 2.25 gm
Water For injection q.sto 100 ml
0.05N HCI Solution q.s to adjust pH
Procedure
Same as Example 1
Observations and stability results are given in Table 1.
This example shows the essentiality of pegylated phospholipids for the stability of
the formulation.
Example 10 - Formulation prepared with DPPC as surfactant instead of egg
lecithin
Equipments used
Water bath, Ultra Turrax IKA stirrer, Niro Saovi Homogenizer.

Ingredients Qty/Batch
Docetaxel Trihydrate 108.0Tmg
MCT oil 5.0 gm
DPPC 1.0 gm
DSPE PEG 2000 1.0 gm
Glycerol 2.25 gm
Water For injection q.sto 100 ml
0.05N HCI Solution q.s to adjust pH
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Procedure
Same as Example 1
Instead of egg lecithin DPPC was dispersed in aqueous phase.
Observations and stability results are given in Table 1.
Advantages of the invention:
1. The compositions of the present invention are free from ethanol and
surfactant Polysorbate-80. Therefore composition of present invention is
devoid of hypersensitivity reaction and fluid retention characteristics of
these ingredients.
2. No pre-medication required to overcome hypersensitivity reactions
experienced with currently marketed preparation.
3. Shows higher Cmax and AUC may lead to better efficacy at equivalent
doses or if the dosage is reduced then it may lead to less toxic effects of
the drug.
4. The process of preparation is free from any solvent and co-solvent like
ethanol, propylene glycol and chloroform.
5. Process gives stable nanoemulsion which gives Enhanced Permeability
Retention (EPR) effect.
Dated this 17th day of July 2008.

To
The Controller of Patents,
The Patent Office,
At Mumbai.
- 15-

STABLE INJECTABLE OIL-IN-WATER NANOEMULSION

Documents:

Inventors:

PCT Conventions: