FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rule 13)


MICROEMULSION COMPOSITIONS FOR BIOLOGICAL/S
Intas Biopharmaceuticals Limite(i
An Indian company having its registered office at*
PlotNo:423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad-382 210
Gujarat, India

The following specification describes the invention and the manner in which it is to performed.


FIELD OF THE INVENTION
The present invention relates to a pharmaceutically acceptable, optically clear and transparent water-in-oil (w/o) microemulsion composition comprising solubilized biological and their use. The invention further describes about w/o microemulsion system where the aqueous phase droplets are dispersed in the oil phase containing different ratios of surfactants and co-surfactants. The aqueous phase consists of about 10-50% v/v of total volume where water soluble biological/s can be loaded. The said biological/s is dissolved in the aqueous phase droplets with uniform size distribution. The microemulsions made in the present invention are stable under refrigerated conditions and have acceptable organo-leptic properties. The composition described herein improves the absorption of biological/s through mucous membrane.
BACKGROUND OF THE INVENTION
Biological e.g. proteins, peptide, nucleotides, vaccines, hormones, small interfering ribonucleic acid (siRNA), Deoxyribonucleic acid (DNA), enzymes, etc. are important as they are used to cure a number of diseases including diabetes (e.g. Insulin), cancers (e.g. Interferon, monoclonal antibodies), heart attacks, strokes, cystic fibrosis (e.g. Enzymes, Blood factors), inflammation diseases (e.g. Tumor Necrosis Factors), anemia (e.g. Erythropoietin), hemophilia (e.g. Blood clotting factors) etc. Formulating these biological is usually a difficult task as these macromolecules are large and unstable and are easily modified or denatured under mild stressed conditions.
Usually biological/s is not suitable for administration through oral route as they are poorly absorbed due to their large size and polarity and charge distribution, at the same time they may undergo denaturation/enzymatic degradation in the gastrointestinal tract. As a result, most biological/s is usually available as aqueous injectable compositions which require repeated injections and frequent visits to the medical service providers. This results in patient incompliance and increased healthcare costs. There is a need to develop a delivery system for these macromolecules which can be administered through non-parenteral route. The development of such a self-administrable delivery system would enable patients to avoid invasive route of administration (Intravenous/Intramuscular/Subcutaneous) and thereby increasing the patient comfort thereby improving patient compliance.
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An emulsion is a mixture of two or more immiscible liquids. One liquid (the dispersed phase) is dispersed in the other (the continuous phase). Emulsions tend to have a cloudy appearance, because the many phase interfaces (the boundary between the phases is called the interface) scatter light that passes through the emulsion. Emulsions are unstable and thus do not form spontaneously. Energy input through shaking, stirring, homogenizing, or spray processes are needed to form an emulsion. Over time, emulsions tend to revert to the stable state of the phases comprising the emulsion.
Compared to emulsions, microemulsions are thermodynamic ally stable, isotropically clear dispersions of two immiscible liquids stabilized by interfacial films of surface-active molecules usually called surfactants. The formation of microemulsions usually involves a combination of three to five components, viz., oil, water, a surfactant, a co-surfactant and an electrolyte. The tendency towards water-in-oil (w/o) or an oil-in-water (o/w) microemulsion is dependent not only on the volume of dispersed and dispersant (or continuous) phase but also on the properties of the surfactants. Surfactants are conveniently classified on an empirical scale known as the hydrophilic-lipophilic balance (HLB) which runs from 1 to 20. In general, w/o microemulsions are formed using surfactants which have an HLB value in the range of about 3 to 8.
Microemulsions are characteristically transparent or opalescent when viewed by optical microscopic means. In the undisturbed state, they are optically isotropic when examined under polarized light The dispersed phase typically comprises particles or droplets which are normally between 5 and 200 nm in size which gives them optical transparency. These particles are usually spherical, although other structures are feasible.
The role of co-surfactant is to increase the interfacial fluidity by penetrating the surfactant film and consequently creating a disordered film due to the formation of void space among surfactant molecules. The use of a cosurfactant or a co-solvent in microemulsions is optional.
There are many advantages to the use of a microemulsion over a conventional emulsion for drug formulation. Microemulsions forms spontaneously, without the need for a high input of energy and are therefore easy to prepare and scale up for commercial applications; they have thermodynamic stability due to their small particle size and therefore have a long shelf life; they have an isotropically clear appearance and therefore its stability can be monitored by
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spectroscopic means; they have a relatively low viscosity and are therefore easy to mix, fill, transport and administer.
EP 0597007B describes about the pharmaceutically acceptable, stable, self-emulsifying (w/o) microemulsions comprising (i) a lipophilic phase comprising a medium-chain fatty acid triglyceride and a low HLB surfactant, (ii) an aqueous-based hydrophilic phase containing a water-soluble therapeutic agent, and (iii) a high HLB surfactant having improved drug-delivery characteristics. This patent describes about a self-emulsifying (w/o) microemulsion which on exposure to water or gastrointestinal fluids forms an emulsion for oral delivery of proteins and peptides. Microemulsion described in this patent comprised of aqueous phase less than 10%. However, for the microemulsion system to be successful it should solubilize therapeutically effective amount of biological/s. Unlike small drug molecules, mostly this biological/s is complex and large having high molecular weight. To solubilize, therapeutic effective quantity, usually higher aqueous phase solubilization (as much as 30-40% v/v) is required.
US6916785 disclosed about a pharmaceutical composition suitable for oral administration in the form of a homogeneous solution which on exposure to water or gastrointestinal fluids forms an emulsion having a particle size of less than 5 microns, the solution containing: (a) a pharmaceutically effective amount of a cyclosporin, in particular Cyclosporin A (b) a carrier medium which is a triglycerol monoester of a fatty acid having from 6 to 30 carbon atoms or mixtures thereof, (c) polyethylene glycol, (d) a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) greater than 10, and (e) optionally, a viscosity reducing agent, the composition being substantially free from ethanol. The preferred carrier medium is triglycerol monooleate. Examples of the viscosity reducing agent are glycerol monocaprylate and glycerol monooleate. This patent discloses about a self emulsifying formulation containing triglycerides which doesn't contain any aqueous or hydrophilic phase.
US 6761903 disclosed about an invention relates to pharmaceutical compositions and methods for improved solubilization of triglycerides and improved delivery of biological. Compositions of the present invention include a carrier, where the carrier is formed from a combination of a triglyceride and at least two surfactants, at least one of which is hydrophilic. Upon dilution with an aqueous medium, the carrier forms a clear, aqueous dispersion of the triglyceride and surfactants. This patent mainly focuses on o/w microemulsion and polysaccharide and low
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molecular drugs which can be dissolved in the oil phase. This invention is more towards triglycerides containing formulations and their drawbacks.
US5824638 discloses stable water in oil (w/o) oral insulin microemulsion, comprising continuous phase of hydrophobic material selected from group consisting of a long chain carboxylic acid or ester or alcohol dispersed in aqueous phase or having hydrophilic discontinuous phase of a long chain carboxylic acid or alcohol. This patent mainly discloses the invention about the oral delivery of insulin wherein the aqueous hydrophilic phase is present in an amount of about 5.1 to about 9.9 weight percent of the emulsion which may not be adequate for loading of therapeutically effective amount of proteins/peptides. Further, this invention discloses use of long chain carboxylic acid or ester or alcohol as hydrophobic phase which is not used in the current invention.
Indian patent application 681/MUM/2004 relates to the microemulsion compositions for transmucosal administration of proteins and peptides and process thereof. In particular this invention relates to oil in water (o/w) microemulsion compositions of peptides such as insulin and the process of making the same. This invention describes a microemulsion system where protein is present in the continuous aqueous phase and not solubilized in the dispersed phase. The composition also contains permeation enhancer and there is hardly any role of microemulsion in enhancing the absorption of biological/s through mucosal membranes.
As is seen from the available prior art, none of the formulation is having the required characteristics of the ideal microemulsion for delivering of biological/s through non-parenteral route e.g transmucosal membranes. The objective of the present invention is to develop a microemulsion formulation having high aqueous phase solubilization capacities, stable under refrigerated, room and at elevated temperature, with high loading of biological/s, acceptable organo-leptic properties, and uniform size distribution of the droplets in the aqueous phase. The further objective of the present invention is to increase the absorption of the biological/s solubilized inside the microemulsion composition through mucosal tissue.
OBJECTIVE OF THE INVENTION
The primary objective of the invention is to provide pharmaceutically acceptable, optically clear and transparent water-in-oil (w/o) microemulsion composition comprising

(a) an oily phase having a fatty acid derivatives of glycerol e.g. medium chain triglyceride and a low HLB surfactant with HLB values and in which the ratio of the medium-chain fatty acid triglyceride to the low HLB surfactant is from 1:0.01 to 1:5
(b) one or more high HLB surfactants
(c) an aqueous phase containing a water soluble biological or their combination dissolved in suitable protein stabilizing buffer with or without other excipients.
(d) a co-solvent or co-surfactant
(e) optionally with flavoring agent or taste-masking agent.
(f) optionally with mucoadhesive or permeation enhancer agent to enhance the bioavailability.
The term "medium-chain triglycerides' as used above refers to a fatty acid having 6 to 12 carbon atoms and suitably the low HLB surfactant will have an HLB value in the range of about 3 to 8, and the high HLB surfactant has an HLB value of 9 and above.
The aqueous phase is composed of water soluble biological or their combination such as proteins, peptide, nucleotide, vaccine, hormone, siRNA, DNA, enzymes etc. either alone or in combination. The said biological/s is dissolved in suitable stabilizing buffer with or without excipients before solubilization in microemulsion system.
The said biological/s is dissolved in the aqueous phase which constitutes about 10 - 50 % v/v of microemulsion. The diameter of droplets or particles of the microemulsion composition of the present invention ranges from 8-150 nm.
The microemulsions of the present invention are stable under refrigeration (5±3 °C), room (25±2°C) and at elevated temperature (40±3°C).
The microemulsion has acceptable organo-leptic properties and is safe when administered to patients.
This invention relates to a microemulsion composition and preparation comprising biological/s such as a high-molecular weight protein / peptide which inherently cannot be easily absorbed through the mucous membrane but which is incorporated in such a way into microemulsion that its absorption through the mucous membrane is improved.
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Another objective of the invention is to make biologicaL/s available in the systemic circulation by adhesion and substantial permeation of the microemulsion through the wide surface area of the buccal or sublingual or nasal mucosa.
DESCRIPTION OF THE INVENTION
The invention relates to the pharmaceutically acceptable water-in-oil (w/o) microemulsions comprise of a biological/s, process for their preparation and their use for mucosal delivery of the same.
According to the present invention, water-in-oil microemulsions comprises
(a) an oily phase having a fatty acid derivatives of glycerol e.g. medium chain triglyceride and a low HLB surfactant with HLB values between 3-8 and in which the ratio of the medium-chain fatty acid triglyceride to the low HLB surfactant is from 1:0.01 to 1:5
(b) one or more high HLB surfactants with HLB values above 9
(c) an aqueous phase containing a water soluble biological or their combination dissolved in suitable protein stabilizing buffer and other excipients.
(d) a co-solvent or co-surfactant
(e) optionally with flavoring agent or taste-masking agent.
(f) optionally with mucoadhesive or permeation enhancer agent to enhance the bioavailability.
Suitable medium chain triglycerides for use in the present invention may be of natural, semi¬synthetic or synthetic origin and may include blends of different medium-chain fatty acid triglycerides. The term "medium-chain fatty acid" as used herein refers to a fatty acid having 6 to 12 carbon atoms, preferably 8 to 10 carbon atoms which may be branched or unbranched and which may be optionally substituted e.g Glycerol Caprylate Caprate, Propylene Glycol Dicaprate, Propylene Glycol Dicaprylate/ Dicaprate, Propylene Glycol Dilaurate, Glyceryl Tricaprylate/ Caprate, Glyceryl Triacetate, Glyceryl Tricaprylate/ Caprate/ Stearate, Polyoxylglycerides. The triglyceride suitably comprises of 50 to 100% (w/w) of Caprylic (C8) and from 0 to 50% (w/w) of capric (Cio) acid triglycerides. Suitable examples include but not limited to Captex 350, Captex 355, Captex 300, Captex 850, Captex 8000, Miglyol 810, Miglyol 812, Miglyol 818, Mazol 1400, Labrafac™ Lipophile Wl 1349, Labrasol, etc.
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Suitable low HLB surfactants for use in the present invention include Glyceryl Mono and dicaprate, Glyceryl Caprylate/Caprate, Glyceryl Oleate, Glyceryl Mono oleate, Glyceryl Monostearate, Propylene Glycol Monocaprylate, Propylene Glycol Monolaurate, Sorbitan esters, Glycerol Monocaprylocaprate. Sodium Lauroyl Dilactate, Sodium Stearoyl Dilactate, a D-Tocopherol. Suitable examples include but not limited to Qipmul MCM, Span 20, Sorbitan Monooleate, Sorbitan Trioleate, Sorbitan Sesquioleate, etc.
Suitable high HLB surfactants for use in the present invention include non-ionic surfactants such as (a) polyoxyethylene fatty acid esters (b) polyoxyethylene sorbitan fatty acid esters (c) polyoxyethylene glycol long-chain alkyl ethers (d) polyoxyethylene glycol long chain alkyl esters (e) polyoxyl hydrogenated vegetable oil. For use herein, the high HLB surfactants preferably have an HLB values 9 and above. Suitable examples include but not limited to Polysorbate 20, Polysorbate 60, Polysorbate 80, Cremophor RH 40, Cremophor EL, Polaxamer 127, Polaxamer

Suitably, the blend of low and high HLB surfactants will have an HLB value in the range of 6 to
15.
Suitable co-solvents for use in the present invention include alcohols, propylene glycol, polyethylene glycol, isopropyl myristate.
Flavouring agents or taste masking agents are essential to make the formulation organo-leptically acceptable. Flavoring agents (flavors) well known to those skilled in the art can be used. These favoring agents may be chosen from synthetic flavor oils and flavoring aromatics, and/or oils, oleo resins and extracts derived from plants, leaves, flowers, fruits and so forth, and combinations thereof. Representative oil soluble flavoring agents include: spearmint oil, cinnamon oil, oil of wintergreen (methylsalicylate), peppermint oils, clove oil, bay oil, anise oil, eucalyptus oil, thyme oil, cedar leaf oil, oil of nutmeg, oil of sage, oil of bitter almonds, peanut butter flavor, chocolate flavor, rum flavor, cassia oil, cinnamon mint flavor, corn mint oil, cardamom flavor, ginger flavor, cola flavor, cherry cola flavor, etc.
As described herein "stable composition" is used to refer optical clarity / transparency i.e. no change in physical appearance of the (w/o) microemulsion by visual inspection and droplet size measured by using Malvern Zetasizer with time stored under refrigeration (5±3 °C); room (25±2
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°C) and at elevated temperature (40±3 °C). No change in physical appearance by visual inspection implies to optical clarity / transparency i.e. non-existence of haziness, cloudiness, opaqueness, sedimentation, precipitation, crystallization and phase separation, etc.
As used herein, "globule size" is used to refer to the size of droplets in the composition in diameter, as measured by conventional particle size analyzers well known to those skilled in the art, such as, photon correlation spectroscopy, laser light scattering or dynamic light scattering technology and by using transmission electron microscope (TEM) or scanning electron microscope (SEM). By "uniform droplet size distribution" it is meant that all the droplets in the formulation have an average diameter of globule in the range of 8 - 150nm when measured by above mentioned techniques. As used herein, the term "nm" refers to nanometer, size less than 1 micron, wherein micron is a unit of measure of one one-thousandth of a millimeter.
The term "biological" refers to proteins, peptide, nucleotides, vaccines, hormones, siRNA, enzymes, etc. which are derived either from natural or recombinant sources having biological activity, is soluble in the aqueous phase and has an HLB value of at least that of the high HLB surfactant used in the formulation, to ensure that the drug is preferentially dissolved in the hydrophilic rather than the lipophilic phase.
For Intas Biopharmaceuticals Limited,
Jayanthi M Executive - IPM
To
The Controller of Patents, The Patent Office, at Mumbai.
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