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"Nanocompositions Of Thymoquinone"

Abstract: This  invention  relates  to  a  novel  formulation /composition  of Thymoquinone  (THQ)  in  the  form  of  nanoemulsion/mucoadhesive Nanoemulsion/nanoparticles/mucoadhesive/nanoparticles/liposomes/ micelles/nanophytosomes having size less than 1 micron.

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Patent Information

Application #
Filing Date
04 January 2010
Publication Number
27/2011
Publication Type
INA
Invention Field
CHEMICAL
Status
Email
Parent Application

Applicants

REGISTRAR
JAMIA HAMDARD HAMDARD NAGAR, NEW DELHI-110 062

Inventors

1. SHADAB AHMAD PATHAN
JAMIA HAMDARD HAMDARD NAGAR, NEW DELHI-110 062
2. R.K.KHAR
JAMIA HAMDARD HAMDARD NAGAR, NEW DELHI-110 062
3. FARHAN JALEES AHMED
JAMIA HAMDARD HAMDARD NAGAR, NEW DELHI-110 062
4. GAURAV KUMAR JAIN
JAMIA HAMDARD HAMDARD NAGAR, NEW DELHI-110 062
5. SOHAIL AKHTAR
JAMIA HAMDARD HAMDARD NAGAR, NEW DELHI-110 062
6. S.M. ABBAS ZAIDI
JAMIA HAMDARD HAMDARD NAGAR, NEW DELHI-110 062

Specification

"NANOCOMPOSITIONS OF THYMOQUINONE". FIELD OF INVENTION This invention relates to nano compositions/formulation of Thymoquinone (THQ) and preparation thereof. BACKGROUND OF INVENTION Over the recent years, there has been growing interest in naturally occurring phytochemical compounds with their therapeutic potential, because they are relatively nontoxic, inexpensive and available in an ingestive form. More than 25% of drugs used during the last 20 years are directly derived from plants, while the other 25% are chemically altered natural products (Vuorelaa et al., 2004). The black seed (Nigella sativa, Ranunculaceae family), also known as Black Caraway Seed and "the Blessed Seed", is an annual herb that grows in countries bordering the Mediterranean Sea, Pakistan and India (Fig. 1A). Although black seed is not a significant component of the human diet, it is regarded in the Middle East as part of an overall holistic approach to health and is thus incorporated into diets and everyday lifestyles. The seed has been used as a natural remedy for more than 2000 years to promote health and treat diseases. Black seed is one of the most extensively studied plants both phytochemically and pharmacologically. Numerous studies have shown that the seeds and oil of this plant are characterised by a very low degree of toxicity (Ali & Blunden, 2003). The chemical composition of black seed is very rich and diverse. Aside from its active ingredient crystalline nigellone, black seed contains 15 amino acids, proteins, carbohydrates, both fixed oils (84% fatty acids, including linolenic and oleic) and volatile oils, alkaloids, saponins, crude fiber, as well as minerals, such as calcium, iron, sodium and potassium. Thymoquinone (TQ) is the bioactive constituent of the volatile oil of black seed (54%) andwas first extracted by El-Dakhakhany (1963). TQ has been shown to exert anti-inflammatory, anti-oxidant and anti-neoplastic effects both in vitro and in vivo. The active lipid soluble compound TQ can be found only in a crystalline triclinic form as determined by high resolution X-ray powder diffraction (Pagola et al., 2004). Several methods have been used to quantify the levels of TQ in black seed oil, such as gas chromatography (Houghton, Zarka, de las Heras, & Hoult, 1995), high performance liquid chromatography (Ghosheh, Houdi, & Crooks, 1999) and differential pulse polarography (Michelitsch & Rittmannsberger, 2003). Thymoquinone is a pharmacologically active quinone, which possesses several properties including analgesic and anti-inflammatory actions (6-7); protective effect on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampus (8) and renal ischemiareperfusion- induced oxidative damage in rats (9), anticonvulsant (10-11), antineoplastic (12-14), and the inhibition of eicosanoids generation (7). Thymoquinone demonstrated the anti-inflammatory effect in experimental asthma (15). Khanna et al. (1993) reported analgesic and CNS depressant activities of N. sativa oil, and Abdel-Fattah et al. (2000) who determined the antinociceptive effect of thymoquinone with a central mechanism through opioid receptors. Thymoquinone, is also reported to possess a strong antioxidant property (Houghton et al., 1995). Thymoquinone protects organs against oxidative damage induced by a variety of free radical generating agents including doxorubicininduced cardiotoxicity (Nagi and Mansour, 2000), carbon tetrachloride evoked hepatotoxicity (Nagi et al., 1999), nephropathy produced by cisplatin (Badary et al., 1997), autoimmune aswell as allergic encephalomyelitis (Mohamed et al., 2003;Mohamed et al., 2005) and gastric mucosal injury induced by ischemia reperfusion (El-Abhar et al., 2003). Nanoscience may be defined as the extension of existing sciences into dimensional realms much smaller than previously considered feasible, with nanotechnology representing the practical application thereof. Current nanotechnology developments have led to nanomedicine, a new field which includes many diagnostic and therapeutic applications involving nanomaterials and nanodevices (Kagan et al., 2005). It has been envisioned that nanomedical products will have a huge impact on public health care (Zajtchuk, 1999). Among the many nanoscience breakthroughs in recent years, potential applications include toxicity reduction where the focus has been on decreasing drug concentration in overdose cases, for example, the tricyclic antidepressants (Ma and Henry, 2001; Gabel and Hinkelbein, 2004; Brucculeri et al., 2005). Superior performance has been demonstrated for some approaches to detoxification therapy using nanoparticles and nanoemulsions (Morey et al., 2004; Varshney et al., 2004) relative to their macro-scale counterparts. However, interest in the application of nanotechnology to preparation of natural bioactive medicine is comparatively low, mainly for two reasons. Firstly, the modest cost of traditional medicine reduces the economic benefit of utilizing nanotechnology. Secondly, the toxicity of the traditional medicines used in the common medicinal preparations is relatively low. Hence, application of nanotechnology to commonly used traditional bioactive medicine materials may not only improve their bioactivity but also reduce the amount of the nanopharmaceuticals required. The major problems with the Thymoquinone are: (1) TQ has low bioavailability due to its low solubility in aqueous media (86µg/mL); (2) TQ is not very stable and easily decomposes; (3) It is difficult for drug crystals to disperse homogeneously because of their hydrophobicity in aqueous solution or blood; These biopharmaceutical and pharmacokinetic hurdles leads to poor oral bioavailability of TQ and hindered its development beyond preclinical stage. Nanocompositions have emerged as most promising delivery systems for such poorly bioavailable compounds and have been extensively studied in the past few years for oral delivery. The nanocarriers by virtue of their size and surface properties are taken up intact by M cells in Payer's patches of the gut associate lymphoid tissue followed by its systemic circulation. The nanocarriers can improve the oral bioavailability of poorly bioavailable drugs due to their specialized uptake mechanism. These polymeric nanocarriers are capable of preventing the gastrointestinal degradation and first pass metabolism of encapsulated drugs. Furthermore, nanoparticles are capable of sustaining drug release in plasma for longer time period, thus reduce the frequency of administration. Biodegradable and non-degradable particles have been explored for this purpose, however, biodegradable carriers made of PLGA or Chitosan have an edge over others for the established safety of these materials. OBJECTS OF INVENTION The main object of this invention is to develop novel formulation/compositions of Thymoquinone. Other object is to develop a formulation of Thymoquinone in the form of Solid and in liquid state for administration for various therapeutics purposes. Another object is to develop a formulation of Thymoquinone to provide for spontaneous formation of thermodynamically stable nanoemulsion/mucoadhesive nanoemulsion droplets having a particle size less than 1 micron. Yet another object is to develop a formulation of Thymoquinone where in the Thymoquinone will be in the form of Nanoparticle/Mucoadhesive nanoparticles. Yet another object is to develop formulation of Thymoquinone where in the Thymoquinone will be in the form of Nanoparticles made up of various Biodegradable polymers from synthetic (e.g. PLA, PLGA, PBCA, NIPAAM, PCL, Lecithin, Lipids) as well as natural origin (e.g. Chitosan, Alginates, Hylouronic acid etc.) Yet another object is to develop formulation of Thymoquinone where in the THQ is formulation in the form of Solid Lipid Nanoparticles. Yet another object is to develop formulation of Thymoquinone where in the THQ is formulation in the form of Phospholipid complex and nanosized with the hi pressure homogenizer to get nanophytosomes. Yet another object is to develop formulation of THQ in the nanoparticle form of micelles with the use of SDS surfactants. Further object is to develop a formulation of Thymoquinone that can be administered through nasal, oral, parenteral and topical routes. STATEMENT OF INVENTION This invention relates to a novel formulation/composition of Thymoquinone (THQ) in the form of nanoemulsion/mucoadhesive Nanoemulsion/nanoparticles/mucoadhesive/nanoparticles/liposomes/ micelles/nanophytosomes having size less than 1 micron. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS Fig. 1. (A) shows the black seed herb and (B) its bioactive component thymoquinone. Chemical characteristics are: (i) synonym is 2-isopropyl-5-methyl-l,4-benzoquinone (Fig. 1B), shows (ii) molecular formula C10H12O2 and (iii) MW 164.2. Fig . 2. shows TEM image of optimized nanoemulsion formulation. Fig. 3. illustrates average droplet size of optimized THQ loaded microemulsion containing oleic acid, Tween 20, diethyleneglycolmonoethyl ether (Carbitol). Fig. 4. demonstrates TEM and AFM Images of THQ Nanoparticles Fig. 5. shows [A] TEM Image of Thmoquinone Liposomes [B] Particle Size Distribution of prepared liposomes DETAILED DESCRIPTION OF INVENION The invented formulations are nanoparticles, mucoadhesive nanoparticles, nanoemulsion/mucoadhesive Nanoemulsion, Solid lipid nanoparticles, nanosized phospholipid complex, micelles, comprising THQ as a drug and remaining will be the different Excipient, biodegradable polymers, and oils having a particle size of less than l|im. The invented formulation is useful for effective delivery of THQ via nasal, oral, topical, and parenteral route for its therapeutic uses. 1. The THQ nanoparticles may be used alone, or may be coated with one or more surface-active agents ("surfactants"), polymers, adhesion promoters, or other additives or excipients. They also may be incorporated 20 into tablets or capsules or other dosage forms, or encapsulated. Many different excipients are commonly used in drug formulations. Classes of excipients include, but are not limited to, tableting aids, disintegrants, glidants, antioxidants and other preservatives, enteric 25 coatings, taste masking agents, and the like. References describing such materials are readily available to and well-known by the practitioners in the art of drug formulations. The excipients may be added during any of the steps described below for including surfactants in the 30 particles. For example, the excipient may be added during the formation of the nanoparticle; during the dispensing of the nanoparticles to form a dosage form; or during the administration of the nanoparticles. 2. The selection of the additives or excipients is determined in part by the projected route of administration. Any of the conventional routes (e.g. inhalation, oral, rectal, vaginal, topical, and parenteral, Nasal) are suitable for, and may be enhanced by, the use of the nanoparticulate drug formulations. Suitable formulations include oral formulations, aerosols, topical formulations, parenteral formulations, and implantable compositions. In particular, the nanoparticulate dug formulations are particularly suitable for delivering hydrophobic and other poorly soluble drugs, such as those in bioavailability classes II and IV, by oral or aerosol administration, thereby replacing a parenteral route of administration. Optionally the THQ nanoparticles contain a surfactant to eliminate or reduce aggregation of the particles. The surfactant adheres to the surface of the nanoparticles. Typically, a surfactant facilitates the dispersion of the nanoparticles in any or all of the initial non-solvent mixtures in which the particle is formed, the medium in which the nanoparticles are taken up for administration, and the medium (e.g. gastrointestinal fluid) into which the particle is later delivered. 3. Any surfactant may be useful in the THQ nanoparticles. Suitable surfactants include small molecule surfactants, often called detergents, and macromolecules (i.e. polymers). The surfactant may also contain a mixture of surfactants. In formulations for parenteral administration, the surfactant is preferably one that is approved by the FDA for pharmaceutical uses. In formulations for non-parenteral administration, the surfactant may be one that is approved by the FDA for use in foods or cosmetics. The surfactant may be present in any suitable amount. In preferred embodiments, effective surfactants are present as only a small weight fraction of the THQ nanoparticles, such as from 0.1% to 10% (wt of surfactant/weight of the THQ). However, larger proportions of surfactant may be needed or convenient, thus the surfactant may be present in a weight percent of 20%, 50% or up to about 100% of the weight of the collagen, particularly when the particles are small and the total surface area is accordingly large. 4. Suitable polymers include soluble and water-insoluble, and biodegradable and non-biodegradable polymers, including hydrogels, thermoplastics, and homopolymers, copolymers and blends of natural and synthetic polymers. Representative polymers which can be used include hydrophilic polymers, such as those containing carboxylic groups, including polyacrylic acid. Bioerodible polymers including polyanhydrides, poly(hydroxy acids) and polyesters, as well as blends and copolymers thereof, also can be used. Representative bioerodible poly(hydroxyl acids) and copolymers thereof which can be used include poly(lactic acid), poly(glycolic acid), poly(hydroxybutyric acid), poly(hydroxyvaleric acid), poly (caprolactone), poly(lactide-co-caprolacto- ne), and poly(lactide-co-glycolide). Polymers containing labile bonds, such as polyanhydrides and polyorthoesters, can be used optionally in a modified form with reduced hydrolytic reactivity. Positively charged hydrogels, such as chitosan, and thermoplastic polymers, such as polystyrene also can be used. Representative natural polymers which also can be used include proteins, such as zein, modified zein, casein, gelatin, gluten, serum albumin, and polysaccharides such as dextrans, polyhyaluronic acid and alginic acid. Representative synthetic polymers include polyphosphazenes, polyamides, polycarbonates, polyacrylamides, polysiloxanes, polyurethanes and copolymers thereof. Celluloses also can be used. As defined herein the term "celluloses" includes naturally occurring and synthetic celluloses, such as alkyl celluloses, cellulose ethers, cellulose esters, hydroxyalkyl celluloses and nitrocelluloses. Exemplary celluloses include ethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose triacetate and cellulose sulfate sodium salt. Polymers of acrylic and methacrylic acids or esters and copolymers thereof can be used. Representative polymers which can be used include poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly (methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). Other polymers which can be used include polyalkylenes such as polyethylene and polypropylene; polyarylalkylenes such as polystyrene; poly(alkylene glycols), such as poly(ethylene glycol); poly(alkylene oxides), such as poly(ethylene oxide); and poly(alkylene terephthalates), such as poly(ethylene terephthalate). Additionally, polyvinyl polymers can be used, which, as defined herein includes polyvinyl alcohols, polyvinyl ethers, polyvinyl esters and polyvinyl halides. Exemplary polyvinyl polymers include polyvinyl acetate), polyvinyl phenol and polyvinylpyrrolidone. Polymers which alter viscosity as a function of temperature or shear or other physical forces also may be used. Poly(oxyalkylene) polymers and copolymers such as poly(ethylene oxide)-poly (propylene oxide) (PEO-PPO) or poly(ethylene oxide)-poly(butylene oxide) (PEO-PBO) copolymers, and copolymers and blends of these polymers with polymers such as poly(alpha-hydroxy acids), including but not limited to lactic, glycolic and hydroxybutyric acids, polycaprolactones, and polyvalerolactones, can be synthesized or commercially obtained. For example, polyoxyalkylene copolymers are described in U.S. Pat. Nos. 3,829,506; 3,535,307; 3,036,118; 2,979,578; 2,677,700; and 2,675,619. Term "Nanocomposites" is meant including, but not limited to, 5 whatever follows the word nanoparticles, polymeric nanoparticles, mucroadhesive nanoparticles, mucoadhesive polymeric nanoparticles, nanoemulsions, polymeric nanoemulsions, Liposomes, polymeric micelles, solid lipid nanoparticles, nanophytosomes. Thus, use of the term "Nanocomposites" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. Aspects of the invention will now be described in greater detail by reference to the following non-limiting examples. Examples THQ Nanoemulsion/Mucoadhesive Nanoemulsion: THQ loaded Nanoemulsion/Mucoadhesive Nanoemulsion were prepared by aqueous titration method. Optimized amount of THQ (Oily phase) and Smix were mixed in a definite ratio of %v/v with vigorous shaking. The obtained mixture is titrated against aqueous phase/ or Mucoadhesive polymeric aqueous phase, visually transparent solution is obtained that is the indicative of Nanoemulsion formation, which is further evaluated by characterization. The, optimized THQ loaded nanoemulsion was characterized on the basis of characteristics parameters of nanoemulsion. The nanoemulsion was colloidal dispersions having average droplet size ranging from 22.0 to 78.55 nm. A value of polydispersity index (PI), which is a measure of uniformity of droplet size within the formulation, was also calculated. The NE formulation exhibited a narrow size distribution (PI= 0.247). The results of dynamic light scattering (DLS) measurements were in agreement with the droplet size measured by TEM. As a result of viscosity measurement, Viscosity of the nanoemulsion formulation was very low as expected for o/w emulsion (28.55 ± 2.03mP). Refractive index is the net value of the components of nanoemulsion and indicates isotropic nature of formulation. The mean value of the refractive index for the nanoemulsion was found to be 1.409. The pH value for the optimized THQ loaded nanoemulsion formulation was recorded 5.6± 0.219, which is favorable for intranasal delivery because the pH of the nasal mucosa is in the range of 5.5 to 6.The zeta potential of the invented formulation was found to be -35±0.49 to +32±0.61 which THQ Loaded Chitosan nanoparticles THQ-loaded chitosan nanoparticles were prepared by the precipitation method (Yan et ah, 2005). In brief, the chitosan was dissolved in an aqueous solution of acetic acid (1%, v/v) at a concentration of 0.2% w/v. THQ was dissolved in 1.0 mL of acetone and this was then added to the polymer solution. A solution of tripolyphosphate (0.2%, w/v) was subsequently added dropwise during vigorous stirring at 1500 rpm and concurrent bath sonication leading to the formation of nanoparticles. Chitosan-sodium alginate nanoparticles Chitosan-sodium alginate nanoparticles were prepared by the modified complex coacervation method as reported by Calvo et al. Briefly, an aqueous solution of polysaccharide chitosan (CS, polycation) was mixed with an aqueous solution of sodium alginate (ALG, polyanion) under controlled magnetic stirring resulting in the formation of nanoparticles under mild conditions because of ionic interactions. ALG was dissolved in distilled water at various concentrations. CS was dissolved in aqueous acetic acid solution (2% w/v) at various concentrations under stirring. About 50 mL of ALG solution was sprayed into 50 mL of CS solution containing the drug and 0.50% surfactant under continuous magnetic stirring (Remi 2MLH, REMI Corp., India) at 1000 ± 25 rpm for 60 min. The formation of the nanoparticles could be observed as the appearance of turbidity and opalescent suspension formation. The rate of the ALG solution spraying to CS solution was controlled (approximately 0.5 g min-1) so as to avoid the formation of microparticles or precipitate. An aliquot of the nanoparticles suspension was centrifuged (REMI high speed, cooling centrifuge, REMI Corp., India) at 18,000 rpm for 30 min. at 4°C and the drug content in supernatant was measured by validated UV spectrophotometric. Poly (butylcyanoacrylate) nanoparticles Poly(butylcyanoacrylate) (PBCA) nanoparticles were prepared by anionic polymerisation of Isobutylcyanoacrylate (IBCA, n-butyl-2-cyanoacrylate) monomer in acidic media as described. About 50 mL solution of IBCA monomer in acetone was sprayed into 50 mL acidic solution of drug in 0.1N HCl containing 1% surfactant under continuous magnetic stirring (Remi 2MLH, REMI Corp., India) at 800 ± 25 rpm. After 3 h, the reaction was finalized by neutralization with 0.1N NaOH. The formation of the nanoparticles could be observed as the appearance of bluish tinge or light turbidity and opalescent suspension formation. The rate of the monomer solution spraying to acidic solution of drug was controlled (approximately 1.0 g min-1) so as to avoid the formation of microparticles or precipitate. An aliquot of the nanoparticles suspension was centrifuged (REMI high speed, cooling centrifuge, REMI Corp., India) at 18,000 rpm for 30min. Poly (lactide-co-glycolide) nanoparticles Poly (lactide-co-glycolide) (PLGA) nanoparticles were prepared by nano-precipitation or emulsion-solvent evaporation method. The nature of the organic solvent, the nature of the aqueous phase (water or buffer) and the concentration of the PVA was varied. The solvent, which gave smallest particle size of PLGA nanoparticles, was acetone. PLGA was first dissolved in 10 mL of acetone and sprayed at controlled rate (approximately 1.0 g min-1) into 20 mL of an aqueous phase containing drug and 2% PVA under continuous magnetic stirring (Remi 2MLH, REMI Corp., India) at 1000 ± 25 rpm. PLGA nanoparticles formed instantaneously as observed by the appearance of slight turbidity and formation of opalescent suspension. The organic solvent was finally removed by evaporation under vacuum at 40°C, until a final volume of 20 ml was reached. An aliquot of the nanoparticles suspension was centrifuged (REMI high speed, cooling centrifuge, REMI Corp., India) at 18,000 rpm for 30 min. at 4°C and the drug content in supernatant was measured by validated UV spectropho tome trie method. Three kinds of phenomena were observed: solution, aggregates and opalescent suspension. The zone of opalescent suspension was further examined as nanoparticles. Turbidity of the nanoparticulate suspensions formed were measured after dilution with DDW (1 mL diluted to 10 mL) on Shimadzu 1601 UV-Visible spectrophotometer (Shimadzu, Japan) at 420 nm, using DDW as the reference. For the selection of the working concentration range of independent variables for different manufacturing process of polymeric nanoparticles, the acceptable turbidity limit for nanoparticulate formulations was decided to be between 0.10-0.20. This range was selected based on the examination of these samples under Transmission Electron Microscope, which showed the average particle size of these nanoparticles to be varying between 150 nm to 500 nm. Preparation of Thymoquinone Solid Lipid Nanoparticles (THQ-SLN) The THQ, stearic acid and soybean lecithin were weighed with electric balance (BP-121S, sartorius Ltd., Germany) precisely and were dissolved in absolute alcohol in water bath at 70 °C. An aqueous phase was prepared by dissolving glycerine in distilled water. The resultant organic solution was rapidly injected through an injection needle into the stirred aqueous phase (80 °C). The resulting suspension was stirred at 80 °C for 2 h continually. The THQ-SLN original suspension was then ultrasonicated for 300 s using Ultrahomogenizer (JY 9211, Ningbo Scientz Biotechnology Co. Ltd.). The resulting dispersion was then allowed to cool at room temperature and was filtered through a millipore filter (0.45µm) in order to remove any titanic granules from the probe. Samples were kept at 4 °C. Thymoquinone Nanoparticles prepared by Hi pressure homogenization: The preparation of THQ nanoparticles was carried out by nanosuspension using various optimized conditions including selection of surfactants, setting of time and rate of homogenization, and adjustment of solvent volume evaporation in the formulation, to finally obtain uniform particles and avoid clustering of particles. Briefly, the THQ (2 g) and Pluronic F68 (PF68) (1 g) were dissolved in 120 ml of ethanol. PF68 was selected over Tween or polyvinyl alcohol as surfactant because the latter types produced particles aggregation and precipitation which was not observed in the case of PF68. The solution was then quickly injected into 280 ml aqueous solution containing PF68, and then highly homogenized by using the pressure homogenizer at 5000-25,000 rpm for 5cycles, which permitted the formation of nanospheres with size

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1 13-DEL-2010-GPA-(30-12-2010).pdf 2010-12-30
2 13-DEL-2010-Form-1-(30-12-2010).pdf 2010-12-30
3 13-DEL-2010-Correspondence-Others-(30-12-2010).pdf 2010-12-30
4 13-del-2010-form-5.pdf 2011-08-21
5 13-del-2010-form-2.pdf 2011-08-21
6 13-del-2010-form-1.pdf 2011-08-21
7 13-del-2010-drawings.pdf 2011-08-21
8 13-DEL-2010-Drawings (11-1-2010).pdf 2011-08-21
9 13-del-2010-description (complete).pdf 2011-08-21
10 13-del-2010-correspondence-others.pdf 2011-08-21
11 13-DEL-2010-Correspondence-Others (11-1-2010).pdf 2011-08-21
12 13-del-2010-claims.pdf 2011-08-21
13 13-del-2010-abstract.pdf 2011-08-21