Title of Invention	"A PROCESS FOR PREPARATION OF NANOSIZE BISPHOSPHONATE PARTICLES"
Abstract	The present invention relates used as an antidote for heavy metal poisoning and pharmaceutical formulations for pulmonary administration of the nanosize bisphosphonates. The present invention also relates to nanosize bisphosphonate particles prepared by the process of present invention and the formulations thereof. More particularly, the invention relates to bisphosphonate formulation that can be administered into lungs through pulmonary delivery as a dry powder inhaler or through nebulizer which has a sustained drug delivery in lungs for over a period of 24 hours.

Title of Invention

"A PROCESS FOR PREPARATION OF NANOSIZE BISPHOSPHONATE PARTICLES"

Abstract

The present invention relates used as an antidote for heavy metal poisoning and pharmaceutical formulations for pulmonary administration of the nanosize bisphosphonates. The present invention also relates to nanosize bisphosphonate particles prepared by the process of present invention and the formulations thereof. More particularly, the invention relates to bisphosphonate formulation that can be administered into lungs through pulmonary delivery as a dry powder inhaler or through nebulizer which has a sustained drug delivery in lungs for over a period of 24 hours.

Full Text	Field of Invention The present invention comprises a process for preparation of nanosize bisphosphonate particles used as an antidote for heavy metal poisoning and pharmaceutical formulations for pulmonary administration of the nanosize bisphosphonates. The present invention also relates to nanosize bisphosphonate particles prepared by the process of present invention and the formulations thereof. Background of invention Heavy metal poisoning is toxic accumulation of heavy metals in the soft tissues of the body. Heavy metals are chemical elements that have a specific gravity at least five times that of water. There are 23 metals which are categorize as "heavy metals' that include antimony, arsenic, bismuth, cadmium, cerium, chromium, cobalt, copper, gallium, gold, iron, lead, manganese, mercury, nickel, platinum, silver, tellurium, thallium, tin, uranium, vanadium, and zinc (Glanze 1996). Interestingly, small amounts of these elements such as zinc, copper, chromium, iron, and manganese are common in our environment and required by the body which are necessary for good health, but large amounts of any of them may cause acute or chronic toxicity (poisoning). Heavy metals become toxic when they are not metabolized by the body and thereafter accumulate in the soft tissues. Once in the body, they compete with and displace essential minerals such as zinc, copper, magnesium, and calcium and interfere with organ system function. They also bind to the proteins resulting in impaired enzymatic activity which leads to damaging of many organs through out the body. Heavy metals may enter the human body through food, water, air, or absorption through the skin when they come in contact with humans in agriculture and in manufacturing, pharmaceutical, industrial, or residential settings. Industrial exposure accounts for a common route of exposure for adults. Children may be poisoned as a result of playing in contaminated soil. Heavy metal toxicity can result in damaged or reduced mental and central nervous function, lower energy levels, and damage to blood composition, lungs, kidneys, liver, and other vital organs. Long-term exposure may result in slowly progressing physical, muscular, and neurological degenerative processes that mimic Alzheimer's disease, Parkinson's disease, muscular dystrophy and multiple sclerosis. Allergies are not uncommon and repeated long-term contact with some metals or their compounds may even cause cancer (International Occupational Safety and Health Information Center 1999) by directly and indirectly damaging DNA. The usual target of these heavy metal compounds is lung though arsenic has a unique association with skin cancer that has been recognized for many years. Symptoms vary depending on the nature and the quantity of the heavy metal ingested. Patients may complain of nausea, vomiting, diarrhea, stomach pain, headache, sweating, and a metallic taste in the mouth. Depending on the metal, there might be blue-black lines in the gum tissues. In severe cases, patients exhibit obvious impairment of cognitive, motor and language skills. Table 1 illustrates the different types of heavy metals and their source and effects on the human metabolism. (Table Removed ) Uranium is 92nd element of the periodic table having 15 isotopes, 2 of them 235-U and 238-U are considered the primordial substances of the universe having half lives of 7.1 X 10 8 and 4.49 X 10 9 years respectively. It is well known that uranium is toxic to humans as well as animals and can be absorbed into the body by several routes. The inhalation pathway of the internal contamination with uranium is the most important route of entry to the extracellular fluid via broncho-alveolar tree. Soluble components of uranium absorbed from the pulmonary tree are deposited in the skeleton for several weeks, with a biological half-life in the lungs of 120 days. A considerable longer pulmonary retention of 1470 days is expected in the case of inhalation of uranium oxides. Fatal cases of uranium poisoning by the respiratory route have been described in humans with nephrotoxic syndrome, necrosis in the proximal convulated tubule and moderate degree of inflammatory and fibrotic changes resulting in scarred kidney. The principle aim in the therapeutic management of patients with internally deposited heavy metals is to prevent the absorption from the cite of entry and eliminate heavy metals from the blood stream and the target organs. The methods of treatments are based on various aspects that include: a) Treatment of patient contaminated by ingestion of heavy metals to reduce intestinal absorption: It is important to reduce the gastro-intestinal absorption of heavy metals to reduce their entry in the systemic circulation and its deposition in the target organs. The most commonly used methods include gastric lavage, emetics like apomorphine or oral preparations of ipecac, laxatives and purgatives. But numerous side effects are associated with these methods which include nausea, tachipnea, hypotension, dyspnea, syncopal attacks and exanthema which require professional medical attention. b) Treatment of patients contaminated by inhalation of heavy metals to reduce systemic absorption: The use of mucolytic substances like pancreatic bornase, Triton, tween 90 and F68 which have effect on the mucopolysaccharide and nucleoproteins in the respiratory tract tree, enable the elimination of heavy metals by expectoration. However, these substances have not been found satisfactory for practical use. c) Treatment of patients to reduce internal contamination by ingestion or inhalation of heavy metal to reduce entry into the target organs: Various complexing agents based on the ability of a ligand to form non-ionize ring complex with inorganic ions which subsequently are eliminated by kidney have been used for removal of these heavy metals. Among the numerous complexing agents which have been tested in the clinical trials, only few appear of practical concern in Ethylene Diamine Tetraacetic Acid (EDTA) has been used in experiments on animals and in human medicine for the treatment of poisoning with inorganic substances like lead, zinc, copper, chromium, manganese and nickel poisoning and contamination with transuranic acid. But the parenteral use of sodium EDTA leads to hypocalcemia. Diethyl Triaminic Pentacetic Acid (DTPA) is also a chelating agent which is used as calcium and zinc salts in case of human contamination with trans uranium elements. Both Ca-DTPA and Zn-DTPA are useful in elimination of soluble uranium salts such as nitrate or chlorides but have a low efficiency in poorly soluble compounds such as oxides. Also it is known that a constant infusion of Ca-DTPA can lead to severe toxic effects in experimental animals leading to death after several days. Other agents used in internal contamination with heavy metals include desferoxamine (DFOA), biscarboxy-1 methyl amino diethyl ether (BAETA), sulphonated tetrameric catechole amines (LICAM-C and LICAM-S), but each of them has a limited use because of their toxicity. Thus despite the advances in discovery, newer chelating agents or antidotes are required that can neutralize the toxic inhaled substances at entry point only. In pharmacology, bisphosphonates (also called diphosphonates) are a class of drugs that prevent the loss of bone mass, used to treat osteoporosis and similar diseases. Bisphosphonates are used clinically for the treatment of osteoporosis, osteitis deformans (Paget's disease of the bone), bone metastasis (with or without hypercalcemia), multiple myleoma, and other conditions that feature bone fragility. High-potency intravenous bisphosphonates have shown to modify progression of skeletal metastasis in several forms of cancer, especially breast cancer. US Patent No. 6,264,922 relates to "Nebulized aerosols containing nanopartiele dispersions" and has disclosed an aerosol comprising droplets of an aqueous dispersion of nanoparticles, said nanoparticles comprise insoluble therapeutic or diagnostic agent particles having a surface modifier thereof. It has also disclosed a method for making the aerosol and methods for treatment and diagnosis using the aerosol. US Patent No. 6,743,414 titled "Inhalation administration of biophosphonates" relates to the treatment and prevention of bone diseases in humans, including osteoporosis in postmenopausal women, Paget's Disease and hypercalcemia by administration of bisphosphonate in an inhalation form. It also discloses a pharmaceutical inhalation compositions suitable for the treatment and prevention of bone diseases. The process of present invention is more advantageous as compared to the above referred US patent document and has higher efficacy as pulmonary delivery of bisphosphonate nanoparticles formed in lungs have a retention period of more than 24 hours and thus it has a sustained effect in therapeutic dose as compared to the bisphosphonate formulations of prior art. Also the formulations disclosed in the prior arts help in the treatment of osteoporosis and deliver the drug from lungs to systemic circulation. While the formulation in the present invention uses low dose of bisphosphonate compound for heavy metal poisoning for local effect. The delivery of bisphosphonates via inhalation route can reduce the toxic effect of the heavy metals due to its chelating property. Thus the chelated metal will not be available for systemic absorption and will be excreted through feces. There are several processes known in the art for the formation of nanoparticles of bisphosphonates which are as following: a) Wet micronization by ball milling and high pressure homogenization It involves crushing of large sized particles into smaller ones by means of various form of millings. In this technique, particles are composed of a predominantly crystalline drug with surfactants or stabilizing agents which prevent crystal growth of drug during size reduction. Typically 1-50% of the surfactant is required to stabilize the nanosuspension with mean size between 120-300nm. The potential problems with this technique are the difficulty to decrease the size below certain limits, potential contamination with grinding media and adverse effects of the high shear and temperature on the chemical stability of the particles. b) Spray drying It is a one step process that converts a liquid feed into a dried particulate form via following stages: - atomization of the feed solution to form a spray; spray air contact; - drying of spray; and separation of the dried products from the gas stream. The resulting material usually produced is amorphous in nature. This method has been used to produce solid drug particles for inhalation. The main drawbacks of this process are chemical degradation, loss of biological activity of thermo labile compounds and high cost of the process. c) Spray freeze drying technique It involves the atomization of an aqueous drug solution via a two fluid or an ultrasonic nozzle into a spray chamber filed with cryogenic liquid (liquid nitrogen) or halocarbon refrigerant such as chlorofluorocarbon or fluorocarbon. Modification of spray freeze drying process involves simultaneous atomization and freezing of the drug solution into the cryogenic medium by mixing it with liquefied gas or super-critical fluid such as CO2. The main problem associated with this technique is high processing cost. d) Particle formation from liquid dispersed systems Various techniques have been developed on the principle of liquid dispersed systems which are as follows: 1) Emulsion based methods - It involves the preparation of double or triple emulsions with subsequent removal of the oil phase (a volatile organic solvent) through evaporation, non-solvent (antisolvent) extraction, or solvent dilution. The drawback of this process is the manufacturing complexity of the process which is carefully monitored and controlled. Another problem is the difficulty to completely remove all residual organic solvents from the polymer matrix. 2) Emulsion polymerization method - It includes emulsification of the monomer in the non-solvent phase with surfactant molecule that lead to the formation of monomer-swollen micelles and stabilized monomer droplets. The polymerization reaction takes place in the presence of initiator which provide energy to create free reactive monomers in continuous phase which further collide with unreacted monomers to initiate polymerization reaction. 3) Coacervation process - It involves the phenomenon of phase separation in oil between two liquid phases (differing only in composition of solute species eg. polymer rich and solvent rich) induced by change in temperature, solution composition or pH. It typically includes the hardening of the dispersed phase to yield solid particles. 4) Multiple emulsion technique - This technique is widely employed for the encapsulation of the high molecular weight molecule such as peptides and proteins inside polyester nanoparticles. The preformed polymer is dissolved in a volatile organic solvent (e.g chloroform, methylene chloride), and the organic solution is dispersed in an aqueous phase in presence of surfactants (poloxomer, polysorbate and SDS) or a stabilizer such as PVA. Evaporation of solvent is achieved by continuous emulsification under magnetic stirring resulting in precipitation of polymer and finally formation of nanospheres. 5) Supercritical fluid crystallization - Supercritical fluids (SFs) are those gases and liquid at temperatures and pressures above their critical points (TC - critical temperature; PC - critical pressure). Carbon dioxide is the most widely used supercritical fluid because of its low critical temperature, which makes it particularly suitable for heat sensitive materials such as biologicals. In addition, it is non flammable, non-toxic, inexpensive, recyclable and environment friendly. But the main disadvantages of this process are its sophistication and high cost inputs involved in generating particles with desired size and morphology. In comparison to the above mentioned processes, the process of the present invention provides nanosized particles which can be prepared by antisolvent precipitation process which is a simpler technique and involves low cost incur. Thus an advantage of this manufacturing process according to the invention is that the bisphosphonate formulation has more 50% wt of drug substance. It needs no mechanical energy for crushing or grinding that can create irregular edges. Consequently, precipitation is carried out naturally that result in absence of areas of varying energy. Object of the Invention An object of the present invention is to provide method for preparation of pharmaceutically active bisphosphonate nanoparticles, the method comprising mixing the bisphosphonate compound with a solvent and subsequently antisolvents. In another object the invention provides nanosize bisphosphonate particles for treatment of heavy metal poisoning prepared by the process of present invention. In another object, bisphosphonate formulation comprising nanosized bisphosphonate particle is provided wherein the formulation is administered into lungs through pulmonary delivery as a dry powder inhaler or through a nebulizer. In still another object, the bisphosphonate based formulation provides sustained drug delivery in lungs for over a period of 24 hours. In yet another object, the bisphosphonate based formulations for treatment of heavy metal poisoning uses low dose of bisphosphonate compound in the range of 0.1 to 0.5 mg for local effect. Summary of the Invention The present invention comprises a method of preparation of bisphosphonate nanoparticles and formulations thereof for treatment of heavy metal poisoning comprising nanoparticles of pharmaceutically active bisphosphonate compound. More particularly, the invention relates to bisphosphonate formulation which can be administered into lungs through pulmonary delivery as a dry powder inhaler or through nebulizer which has a sustained drug delivery in lungs for over a period of 24 hours. Brief description of the Drawings Figure 1: Effect of different stabilizers on particle size of alendronate sodium particles. Figure 2: Effect of different grades of poloxomer on particles size of alendronate sodium particles. Figure 3: Effect of different poloxomer concentration on the particle size of alendronate sodium. Figure 4: Effect of different antisolvents on particle size of alendronate sodium particles. Figure 5: Lung targeting images of bisphosphonate nebulizer showing 80-90% respiratory fraction. Description of invention The present invention provides a novel process for preparation of bisphosphonate nanoparticles. The present invention also provides a formulation of bisphosphonates and their salts like sodium via inhalation route for the treatment of heavy metal poisoning and methods for the pulmonary administration of bisphosphonates as an antidote for heavy metal poisoning. As inhalation route is most important route for heavy metal poisoning preferably uranium poisoning, pulmonary administration of bisphosphonates offers a number of advantages for trapping inhaled toxic substances at entry point while limiting its access to systemic circulation. For this purpose a pharmaceutical composition comprising nanosized particles of bisphosphonate having particle size in the range of 50 to 400 nm and preferably 50 to 300 nm suitable for respiratory and pulmonary administration. The process for preparation of nanosize bisphosphonate particles, the process comprises: (i) dissolving a bisphosphonate compound in distilled water, (ii) filtering solution of step (i) through a micropore size filter, (iii) adding filtrate of step (ii) to a precipitation medium, the precipitation medium comprising an antisolvent, and a stabilizer to precipitate the bisphosphonate particles; (iv) drying particles obtained in step (iii) at high temperature and high speed stirring to obtain dried powder of nanosize bisphosphonate particles. Stabilizers may serve as bulking agent when it is required to deliver small amount of bisphosphonate to the patient. In particular, these stabilizers also function to improve the physical and chemical stability of the bisphosphonate in order to rninimize the degree of aggregation, provide ease of administration via inhalation and to target the resultant nanosized particles of bisphosphonate to deep lungs. The amount of stabilizer is in the range of 1 to 20 % w/w of bisphophonate compound. The statbilizers are selected from a group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, cellulose esters, cellulose ethers, caseinates, poloxomers, casein, sodium alginate, polyethylene glycol graft copolymers, starch, chitosan, agar, pectin, sugar, gelatin, gum arabics, phospholipids and dextrins. In an embodiment of the present invention, antisolvents used in the precipitation medium preferably have a solubility of less than 0.1g/100ml of bisphosphonate compound. The antisolvent is selected from a group consisting of alcohols, ketones and ethers, preferably isopropanol, ethanol and acetonitrile, most preferably acetonitrile. The amount of bisphosphonate may range from 0.5 to 2% w/v of antisolvent The concentration of bisphosphonate compound in the distilled water solution can range from 0.5 to 5 % w/v. In an embodiment the filter in step (ii) is a micropore filter having a pore size of 0.22 micron. In an embodiment, the bisphosphonate solution is added to the precipitation medium with stirring at a speed of 400 to 800 rpm. In an another embodiment the particles obtained in step (iii) of the process are dried at a temperature ranging from 60 to 100 degree C at high stirring speed of 1500 to 2500 rpm. In an embodiment, pharmaceutically active amount of bisphosphonate nanoparticles are mixed with a water alcohol mixture to prepare bisphosphonate based formulation for pulmonary delivery through the nebulizer. The alcohol concentration relative to water is in range of 0.1-50 %, preferably 10-30%. In an another embodiment the bisphosphonate compound is selected from a group comprising alendronate, residronate, methylene diphosphonate (MDP) and ethylene hydroxyl diphosphonate (EHDP) and their sodium salts. The present invention also provides bisphosphonate nanoparticles obtained by: (i) dissolving a bisphosphonate compound into distilled water, (ii) filtering solution of step (i) through a micropore size filter, (iii) adding filtrate of step (ii) to a precipitation medium, the precipitation medium comprising an antisolvent, and a stabilizer to precipitate the bisphosphonate particles; (iv) drying particles obtained in step (iii) at high temperature and high speed stirring to obtain dried powder of nanosize bisphosphonate particles. The present invention al provides . bisphosphonate based formulation for treatment of heavy metal poisoning comprising micro or nanoparticles of a pharmaceutically active bisphosphonate compound prepared by the process of present invention. These bisphosphonate based formulation provides sustained drug delivery in lungs for over a period of 24 hours. The bisphosphonate based formulations for treatment of heavy metal poisoning uses low dose of bisphosphonate compound in the range of 0.1 to 0.5 mg for local effect. The following examples are given by the way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. Example 1 Process of preparation of alendronate sodium nanoparticles by antisolvent precipitation process 10 mg/ml alendronate sodium solution (1 mL) was prepared in HPLC grade water and passed through 0.22µ millipore filter. In 10 ml acetonitrile, 10% w/w Lutrol F68 (1 mg) was added as a stabilizer and this organic phase was maintained at 25°C temperature. The organic phase was placed on magnetic stirrer at 700 rpm speed and alendronate sodium solution was added drop-wise into organic phase. Upon complete addition of alendronate sodium solution, particles were allowed to dry at high temperature (80°C) with high speed stirring (2000 rpm). The particle size districution of alendronate sodium nanoparticles after drying was in the range of 50-300nm. The average particle size varied from 200 -300 nm. Example 2 Effect of different stabilizers on particle size of alendronate sodium nanoparticles The effect of different stabilizers on the particle size of alendronate sodium nanoparticles was investigated as shown in Figure 1. It was observed that the particle size of nanoparticles is significantly influenced by the type of stabilizer. Smallest size was achieved with polyvinyl alcohol, while the other stabilizers produced large size particles. When HPMC was used as stabilizer, large aggregated particles were formed of 1300 nm, independently of concentration and process parameters. Similar results were observed for alginate and chitosan. A slight decrease in particle size from several microns to nanometers was achieved when PEG was used as stabilizer (irrespective of molecular weight). Example 3 Effect of different grades of poloxomers on the particle size of alendronate sodium nanoparticles The effect of different grades of poloxomers on the particle size of alendronate sodium nanoparticles was studied as shown in Figure 2. Formulations were prepared by different poloxomers and their effect on particle size was investigated. Poloxomers are non-ionic poly (ethylene oxide) (PEO)-poly (propylene oxide) (PPO) co polymers that are widely used as surfactant, emulsifying agent, dispersing agent and in vivo absorption enhancer. It was observed that best size was achieved with poloxomer F68 (176 nm) as compared to F108 (921 nm) and F127 (765 nm). Example 4 Effect of different poloxomers concentration on the particle size of alendronate sodium nanoparticles Preliminary studies were carried out in order to select a narrow concentration range of poloxomer Lutrol F68 to achieve desired size. The results showed that minimum 1% concentration of poloxomer Lutrol F68 was required to produce stable alendronate nanoparticles as shown in figure 3. Large aggregates were obtained when concentration was increased to 20%. Above 20% concentration, sticky mass was obtained that showed poor flow properties. The optimized particles (176 nm) were obtained at 10% w/w poloxomer concentration. Example 5 Effect of different antisolvents on the particle size of alendronate sodium nanoparticles The effect of different antisolvents on the particle size of alendronate sodium nanoparticles was studied as shown in Figure 4. It was observed that large sized and aggregated particles were formed when ethanol, acetone and IPA were used as a precipitation medium. Minimum particle size of 313 nm was attained when precipitation was carried out in acetonitrile. However no precipitation took place when methanol was selected as antisolvent. Example 6 Effect of different drying conditions on the particle size of alendronate sodium nanoparticles The effect of different drying conditions on the particle size of alendronate sodium nanoparticles was investigated as shown in Table 2. Different techniques such as spray drying, freeze drying, rotary evaporator, vacuum drying and heating on high speed magnetic stirrer were used to study the effect of drying condition on the particle size. It was observed that particles of micronic size range were formed by using spray and freeze drying methods whereas no particles were collected by vacuum drying. Minimal particle size of Table 2: Effect of different drying techniques on the particle size of alendronate sodium particles. (Table Removed ) Example 7 Preparation of alendronate sodium formulation for their use by pulmonary route 1 g of alendronate sodium nanoparticles were dissolved in 70:30 water ethanol mixture. 2ml of this solution was nebulized by using standard nebulization apparatus to produce wet aerosols of nanoparticles for their delivery by pulmonary route. Figure 5 illustrates lung targeting images of bisphosphonate nebulizer showing 80-90% respiratory fraction. We Claim: 1. A process for preparation of nanosize bisphosphonate particles, the process comprises: (i) dissolving a bisphosphonate compound into distilled water, (ii) filtering solution of step (i) through a micropore size filter; (iii) adding filtrate of step (ii) to a precipitation medium, the precipitation medium comprising an antisolvent, and a stabilizer to precipitate the bisphosphonate particles; (iv) drying particles obtained in step (iii) at high temperature and high speed stirring to obtain dried powder of nanosize bisphosphonate particles. 2. The process as claimed in claim 1, wherein concentration of bisphosphonate compound in the distilled water solution can range from 0.5 to 5 % w/v. 3. The process as claimed in claim 1, wherein the antisolvent has a solubility of less than 0.1g/100ml of bisphosphonate compound. 4. The process as claimed in claim 1 or 3, wherein the antisolvent is selected from a group comprising of alcohols, ketones and ethers and preferably isopropanol, ethanol and acetonitrile. 5. The process as claimed in claim 1, wherein the stabilizer is selected from a group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, cellulose esters, cellulose ethers, caseinates, poloxomers, casein, sodium alginate, polyethylene glycol graft copolymers, starch, chitosan, agar, pectin, sugar, gelatin, gum arabics, phospholipids and dextrins. 6. The process as claimed in claim 1 or 5, wherein the amount of stabilizer is in the range of 1 to 20 % w/w of bisphophonate compound. 7. The process as claimed in claim 1, wherein the amount of stabilizer is in the range of 1 to 20 % w/w of precipitation medium. 8. The process as claimed in claim 1, wherein the bisphosphonate solution is added to the precipitation medium in step (iii) with stirring at a speed of 400 to 800 rpm and the particles dried in step (iv) at a temperature ranging from 60 to 100 degree C at stirring speed of 1500 to 2500 rpm. 9. The process as claimed in claim 1, wherein the bisphosphonate compound is selected from a group comprising alendronate, residronate, methylene diphosphonate (MDP) and ethylene hydroxyl diphosphonate (EHDP) and their sodium salts. 10. Bisphosphonate nanoparticles obtained by: (i) dissolving a bisphosphonate compound into distilled water; (ii) filtering solution of step (i) through a micropore size filter; (iii) adding filtrate of step (ii) to a precipitation medium, the precipitation medium comprising an antisolvent, and a stabilizer to precipitate the bisphosphonate particles; (iv) drying particles obtained in step (iii) at high temperature and high speed stirring to obtain dried powder of nanosize bisphosphonate particles. 11. A bisphosphonate formulation prepared by mixing bisphosphonate particles of claim 1 with a water alcohol mixture, wherein the alcohol concentration relative to water is in range of 0.1-50 %, preferably 10-30%.

Full Text

Field of Invention
The present invention comprises a process for preparation of nanosize bisphosphonate particles used as an antidote for heavy metal poisoning and pharmaceutical formulations for pulmonary administration of the nanosize bisphosphonates. The present invention also relates to nanosize bisphosphonate particles prepared by the process of present invention and the formulations thereof.
Background of invention
Heavy metal poisoning is toxic accumulation of heavy metals in the soft tissues of the body. Heavy metals are chemical elements that have a specific gravity at least five times that of water. There are 23 metals which are categorize as "heavy metals' that include antimony, arsenic, bismuth, cadmium, cerium, chromium, cobalt, copper, gallium, gold, iron, lead, manganese, mercury, nickel, platinum, silver, tellurium, thallium, tin, uranium, vanadium, and zinc (Glanze 1996).
Interestingly, small amounts of these elements such as zinc, copper, chromium, iron, and manganese are common in our environment and required by the body which are necessary for good health, but large amounts of any of them may cause acute or chronic toxicity (poisoning).
Heavy metals become toxic when they are not metabolized by the body and thereafter accumulate in the soft tissues. Once in the body, they compete with and displace essential minerals such as zinc, copper, magnesium, and calcium and interfere with organ system function. They also bind to the proteins resulting in impaired enzymatic activity which leads to damaging of many organs through out the body. Heavy metals may enter the human body through food, water, air, or absorption through the skin when they come in contact with humans in agriculture and in manufacturing, pharmaceutical, industrial, or residential settings. Industrial exposure accounts for a common route of exposure for adults. Children may be poisoned as a result of playing in contaminated soil. Heavy metal toxicity can result in damaged or reduced mental and central nervous function, lower energy levels, and damage to blood composition, lungs, kidneys, liver, and other vital organs. Long-term exposure may result in slowly progressing physical, muscular, and neurological degenerative processes that mimic Alzheimer's disease, Parkinson's disease, muscular dystrophy and multiple sclerosis. Allergies are not uncommon and repeated long-term contact with some metals or their compounds may even cause cancer (International
Occupational Safety and Health Information Center 1999) by directly and indirectly damaging DNA. The usual target of these heavy metal compounds is lung though arsenic has a unique association with skin cancer that has been recognized for many years.
Symptoms vary depending on the nature and the quantity of the heavy metal ingested. Patients may complain of nausea, vomiting, diarrhea, stomach pain, headache, sweating, and a metallic taste in the mouth. Depending on the metal, there might be blue-black lines in the gum tissues. In severe cases, patients exhibit obvious impairment of cognitive, motor and language skills.
Table 1 illustrates the different types of heavy metals and their source and effects on the human metabolism.
(Table Removed )
Uranium is 92nd element of the periodic table having 15 isotopes, 2 of them 235-U and 238-U are considered the primordial substances of the universe having half lives of 7.1 X 10 8 and 4.49 X 10 9 years respectively. It is well known that uranium is toxic to humans as well as animals and can be absorbed into the body by several routes. The inhalation pathway of the internal contamination with uranium is the most important route of entry to the extracellular fluid via broncho-alveolar tree. Soluble components of uranium absorbed from the pulmonary tree are deposited in the skeleton for several weeks, with a biological half-life in the lungs of 120 days. A considerable longer pulmonary retention of 1470 days is expected in the case of inhalation of uranium oxides. Fatal cases of uranium poisoning by the respiratory route have been described in humans with nephrotoxic syndrome, necrosis in the proximal convulated tubule and moderate degree of inflammatory and fibrotic changes resulting in scarred kidney.
The principle aim in the therapeutic management of patients with internally deposited heavy metals is to prevent the absorption from the cite of entry and eliminate heavy metals from the blood stream and the target organs. The methods of treatments are based on various aspects that include:
a) Treatment of patient contaminated by ingestion of heavy metals to reduce intestinal
absorption:
It is important to reduce the gastro-intestinal absorption of heavy metals to reduce their entry in the systemic circulation and its deposition in the target organs. The most commonly used methods include gastric lavage, emetics like apomorphine or oral preparations of ipecac, laxatives and purgatives. But numerous side effects are associated with these methods which include nausea, tachipnea, hypotension, dyspnea, syncopal attacks and exanthema which require professional medical attention.
b) Treatment of patients contaminated by inhalation of heavy metals to reduce systemic
absorption:
The use of mucolytic substances like pancreatic bornase, Triton, tween 90 and F68 which have effect on the mucopolysaccharide and nucleoproteins in the respiratory tract tree, enable the elimination of heavy metals by expectoration. However, these substances have not been found satisfactory for practical use.
c) Treatment of patients to reduce internal contamination by ingestion or inhalation of heavy
metal to reduce entry into the target organs:
Various complexing agents based on the ability of a ligand to form non-ionize ring complex with inorganic ions which subsequently are eliminated by kidney have been used for removal of these heavy metals. Among the numerous complexing agents which have been tested in the clinical trials, only few appear of practical concern in Ethylene Diamine Tetraacetic Acid (EDTA) has been used in experiments on animals and in human medicine for the treatment of poisoning with inorganic substances like lead, zinc, copper, chromium, manganese and nickel poisoning and contamination with transuranic acid. But the parenteral use of sodium EDTA leads to hypocalcemia. Diethyl Triaminic Pentacetic Acid (DTPA) is also a chelating agent which is used as calcium and zinc salts in case of human contamination with trans uranium elements. Both Ca-DTPA and Zn-DTPA are useful in
elimination of soluble uranium salts such as nitrate or chlorides but have a low efficiency in poorly soluble compounds such as oxides. Also it is known that a constant infusion of Ca-DTPA can lead to severe toxic effects in experimental animals leading to death after several days.
Other agents used in internal contamination with heavy metals include desferoxamine (DFOA), biscarboxy-1 methyl amino diethyl ether (BAETA), sulphonated tetrameric catechole amines (LICAM-C and LICAM-S), but each of them has a limited use because of their toxicity. Thus despite the advances in discovery, newer chelating agents or antidotes are required that can neutralize the toxic inhaled substances at entry point only.
In pharmacology, bisphosphonates (also called diphosphonates) are a class of drugs that prevent the loss of bone mass, used to treat osteoporosis and similar diseases. Bisphosphonates are used clinically for the treatment of osteoporosis, osteitis deformans (Paget's disease of the bone), bone metastasis (with or without hypercalcemia), multiple myleoma, and other conditions that feature bone fragility. High-potency intravenous bisphosphonates have shown to modify progression of skeletal metastasis in several forms of cancer, especially breast cancer.
US Patent No. 6,264,922 relates to "Nebulized aerosols containing nanopartiele dispersions" and has disclosed an aerosol comprising droplets of an aqueous dispersion of nanoparticles, said nanoparticles comprise insoluble therapeutic or diagnostic agent particles having a surface modifier thereof. It has also disclosed a method for making the aerosol and methods for treatment and diagnosis using the aerosol.
US Patent No. 6,743,414 titled "Inhalation administration of biophosphonates" relates to the treatment and prevention of bone diseases in humans, including osteoporosis in postmenopausal women, Paget's Disease and hypercalcemia by administration of bisphosphonate in an inhalation form. It also discloses a pharmaceutical inhalation compositions suitable for the treatment and prevention of bone diseases.
The process of present invention is more advantageous as compared to the above referred US patent document and has higher efficacy as pulmonary delivery of bisphosphonate nanoparticles formed in
lungs have a retention period of more than 24 hours and thus it has a sustained effect in therapeutic dose as compared to the bisphosphonate formulations of prior art. Also the formulations disclosed in the prior arts help in the treatment of osteoporosis and deliver the drug from lungs to systemic circulation. While the formulation in the present invention uses low dose of bisphosphonate compound for heavy metal poisoning for local effect.
The delivery of bisphosphonates via inhalation route can reduce the toxic effect of the heavy metals due to its chelating property. Thus the chelated metal will not be available for systemic absorption and will be excreted through feces.
There are several processes known in the art for the formation of nanoparticles of bisphosphonates which are as following:
a) Wet micronization by ball milling and high pressure homogenization
It involves crushing of large sized particles into smaller ones by means of various form of millings. In this technique, particles are composed of a predominantly crystalline drug with surfactants or stabilizing agents which prevent crystal growth of drug during size reduction. Typically 1-50% of the surfactant is required to stabilize the nanosuspension with mean size between 120-300nm. The potential problems with this technique are the difficulty to decrease the size below certain limits, potential contamination with grinding media and adverse effects of the high shear and temperature on the chemical stability of the particles.
b) Spray drying
It is a one step process that converts a liquid feed into a dried particulate form via following stages:
- atomization of the feed solution to form a spray; spray air contact;
- drying of spray; and
separation of the dried products from the gas stream. The resulting material usually produced is amorphous in nature. This method has been used to produce
solid drug particles for inhalation. The main drawbacks of this process are chemical degradation, loss of biological activity of thermo labile compounds and high cost of the process.
c) Spray freeze drying technique
It involves the atomization of an aqueous drug solution via a two fluid or an ultrasonic nozzle into a spray chamber filed with cryogenic liquid (liquid nitrogen) or halocarbon refrigerant such as chlorofluorocarbon or fluorocarbon. Modification of spray freeze drying process involves simultaneous atomization and freezing of the drug solution into the cryogenic medium by mixing it with liquefied gas or super-critical fluid such as CO2. The main problem associated with this technique is high processing cost.
d) Particle formation from liquid dispersed systems
Various techniques have been developed on the principle of liquid dispersed systems which are as follows:
1) Emulsion based methods - It involves the preparation of double or triple emulsions with subsequent removal of the oil phase (a volatile organic solvent) through evaporation, non-solvent (antisolvent) extraction, or solvent dilution. The drawback of this process is the manufacturing complexity of the process which is carefully monitored and controlled. Another problem is the difficulty to completely remove all residual organic solvents from the polymer matrix.
2) Emulsion polymerization method - It includes emulsification of the monomer in the non-solvent phase with surfactant molecule that lead to the formation of monomer-swollen micelles and stabilized monomer droplets. The polymerization reaction takes place in the presence of initiator which provide energy to create free reactive monomers in continuous phase which further collide with unreacted monomers to initiate polymerization reaction.
3) Coacervation process - It involves the phenomenon of phase separation in oil between two liquid
phases (differing only in composition of solute species eg. polymer rich and solvent rich) induced by
change in temperature, solution composition or pH. It typically includes the hardening of the dispersed
phase to yield solid particles.
4) Multiple emulsion technique - This technique is widely employed for the encapsulation of the
high molecular weight molecule such as peptides and proteins inside polyester nanoparticles. The
preformed polymer is dissolved in a volatile organic solvent (e.g chloroform, methylene chloride), and
the organic solution is dispersed in an aqueous phase in presence of surfactants (poloxomer, polysorbate
and SDS) or a stabilizer such as PVA. Evaporation of solvent is achieved by continuous emulsification
under magnetic stirring resulting in precipitation of polymer and finally formation of nanospheres.
5) Supercritical fluid crystallization - Supercritical fluids (SFs) are those gases and liquid at
temperatures and pressures above their critical points (TC - critical temperature; PC - critical pressure).
Carbon dioxide is the most widely used supercritical fluid because of its low critical temperature, which
makes it particularly suitable for heat sensitive materials such as biologicals. In addition, it is non
flammable, non-toxic, inexpensive, recyclable and environment friendly. But the main disadvantages of
this process are its sophistication and high cost inputs involved in generating particles with desired size
and morphology.
In comparison to the above mentioned processes, the process of the present invention provides nanosized particles which can be prepared by antisolvent precipitation process which is a simpler technique and involves low cost incur.
Thus an advantage of this manufacturing process according to the invention is that the bisphosphonate formulation has more 50% wt of drug substance. It needs no mechanical energy for crushing or grinding that can create irregular edges. Consequently, precipitation is carried out naturally that result in absence of areas of varying energy.
Object of the Invention
An object of the present invention is to provide method for preparation of pharmaceutically active bisphosphonate nanoparticles, the method comprising mixing the bisphosphonate compound with a solvent and subsequently antisolvents.
In another object the invention provides nanosize bisphosphonate particles for treatment of heavy metal poisoning prepared by the process of present invention.
In another object, bisphosphonate formulation comprising nanosized bisphosphonate particle is provided wherein the formulation is administered into lungs through pulmonary delivery as a dry powder inhaler or through a nebulizer.
In still another object, the bisphosphonate based formulation provides sustained drug delivery in lungs for over a period of 24 hours.
In yet another object, the bisphosphonate based formulations for treatment of heavy metal poisoning uses low dose of bisphosphonate compound in the range of 0.1 to 0.5 mg for local effect.
Summary of the Invention
The present invention comprises a method of preparation of bisphosphonate nanoparticles and formulations thereof for treatment of heavy metal poisoning comprising nanoparticles of pharmaceutically active bisphosphonate compound. More particularly, the invention relates to bisphosphonate formulation which can be administered into lungs through pulmonary delivery as a dry powder inhaler or through nebulizer which has a sustained drug delivery in lungs for over a period of 24 hours.
Brief description of the Drawings
Figure 1: Effect of different stabilizers on particle size of alendronate sodium particles.
Figure 2: Effect of different grades of poloxomer on particles size of alendronate sodium particles. Figure 3: Effect of different poloxomer concentration on the particle size of alendronate sodium. Figure 4: Effect of different antisolvents on particle size of alendronate sodium particles.
Figure 5: Lung targeting images of bisphosphonate nebulizer showing 80-90% respiratory fraction.
Description of invention
The present invention provides a novel process for preparation of bisphosphonate nanoparticles. The present invention also provides a formulation of bisphosphonates and their salts like sodium via inhalation route for the treatment of heavy metal poisoning and methods for the pulmonary administration of bisphosphonates as an antidote for heavy metal poisoning.
As inhalation route is most important route for heavy metal poisoning preferably uranium poisoning, pulmonary administration of bisphosphonates offers a number of advantages for trapping inhaled toxic substances at entry point while limiting its access to systemic circulation. For this purpose a pharmaceutical composition comprising nanosized particles of bisphosphonate having particle size in the range of 50 to 400 nm and preferably 50 to 300 nm suitable for respiratory and pulmonary administration.
The process for preparation of nanosize bisphosphonate particles, the process comprises: (i) dissolving a bisphosphonate compound in distilled water, (ii) filtering solution of step (i) through a micropore size filter, (iii) adding filtrate of step (ii) to a precipitation medium, the precipitation medium comprising an antisolvent, and a stabilizer to precipitate the bisphosphonate particles; (iv) drying particles obtained in step (iii) at high temperature and high speed stirring to obtain dried powder of nanosize bisphosphonate particles.
Stabilizers may serve as bulking agent when it is required to deliver small amount of bisphosphonate to the patient. In particular, these stabilizers also function to improve the physical and chemical stability of the bisphosphonate in order to rninimize the degree of aggregation, provide ease of administration via inhalation and to target the resultant nanosized particles of bisphosphonate to deep lungs. The amount of stabilizer is in the range of 1 to 20 % w/w of bisphophonate compound. The statbilizers are selected from a group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, cellulose esters, cellulose ethers, caseinates, poloxomers, casein, sodium alginate, polyethylene glycol graft copolymers, starch, chitosan,
agar, pectin, sugar, gelatin, gum arabics, phospholipids and dextrins.
In an embodiment of the present invention, antisolvents used in the precipitation medium preferably have a solubility of less than 0.1g/100ml of bisphosphonate compound. The antisolvent is selected from a group consisting of alcohols, ketones and ethers, preferably isopropanol, ethanol and acetonitrile, most preferably acetonitrile. The amount of bisphosphonate may range from 0.5 to 2% w/v of antisolvent
The concentration of bisphosphonate compound in the distilled water solution can range from 0.5 to 5 % w/v.
In an embodiment the filter in step (ii) is a micropore filter having a pore size of 0.22 micron.
In an embodiment, the bisphosphonate solution is added to the precipitation medium with stirring at a
speed of 400 to 800 rpm.
In an another embodiment the particles obtained in step (iii) of the process are dried at a temperature ranging from 60 to 100 degree C at high stirring speed of 1500 to 2500 rpm.
In an embodiment, pharmaceutically active amount of bisphosphonate nanoparticles are mixed with a water alcohol mixture to prepare bisphosphonate based formulation for pulmonary delivery through the nebulizer. The alcohol concentration relative to water is in range of 0.1-50 %, preferably 10-30%.
In an another embodiment the bisphosphonate compound is selected from a group comprising alendronate, residronate, methylene diphosphonate (MDP) and ethylene hydroxyl diphosphonate (EHDP) and their sodium salts.
The present invention also provides bisphosphonate nanoparticles obtained by: (i) dissolving a bisphosphonate compound into distilled water, (ii) filtering solution of step (i) through a micropore size filter, (iii) adding filtrate of step (ii) to a precipitation medium, the precipitation medium
comprising an antisolvent, and a stabilizer to precipitate the bisphosphonate particles;
(iv) drying particles obtained in step (iii) at high temperature and high speed stirring to
obtain dried powder of nanosize bisphosphonate particles.
The present invention al provides . bisphosphonate based formulation for treatment of heavy metal poisoning comprising micro or nanoparticles of a pharmaceutically active bisphosphonate compound prepared by the process of present invention. These bisphosphonate based formulation provides sustained drug delivery in lungs for over a period of 24 hours.
The bisphosphonate based formulations for treatment of heavy metal poisoning uses low dose of bisphosphonate compound in the range of 0.1 to 0.5 mg for local effect.
The following examples are given by the way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Example 1
Process of preparation of alendronate sodium nanoparticles by antisolvent precipitation process 10 mg/ml alendronate sodium solution (1 mL) was prepared in HPLC grade water and passed through 0.22µ millipore filter. In 10 ml acetonitrile, 10% w/w Lutrol F68 (1 mg) was added as a stabilizer and this organic phase was maintained at 25°C temperature. The organic phase was placed on magnetic stirrer at 700 rpm speed and alendronate sodium solution was added drop-wise into organic phase. Upon complete addition of alendronate sodium solution, particles were allowed to dry at high temperature (80°C) with high speed stirring (2000 rpm). The particle size districution of alendronate sodium nanoparticles after drying was in the range of 50-300nm. The average particle size varied from 200 -300 nm.
Example 2
Effect of different stabilizers on particle size of alendronate sodium nanoparticles
The effect of different stabilizers on the particle size of alendronate sodium nanoparticles was
investigated as shown in Figure 1. It was observed that the particle size of nanoparticles is significantly
influenced by the type of stabilizer. Smallest size was achieved with polyvinyl alcohol, while the other stabilizers produced large size particles. When HPMC was used as stabilizer, large aggregated particles were formed of 1300 nm, independently of concentration and process parameters. Similar results were observed for alginate and chitosan. A slight decrease in particle size from several microns to nanometers was achieved when PEG was used as stabilizer (irrespective of molecular weight).
Example 3
Effect of different grades of poloxomers on the particle size of alendronate sodium nanoparticles The effect of different grades of poloxomers on the particle size of alendronate sodium nanoparticles was studied as shown in Figure 2. Formulations were prepared by different poloxomers and their effect on particle size was investigated. Poloxomers are non-ionic poly (ethylene oxide) (PEO)-poly (propylene oxide) (PPO) co polymers that are widely used as surfactant, emulsifying agent, dispersing agent and in vivo absorption enhancer. It was observed that best size was achieved with poloxomer F68 (176 nm) as compared to F108 (921 nm) and F127 (765 nm).
Example 4
Effect of different poloxomers concentration on the particle size of alendronate sodium nanoparticles Preliminary studies were carried out in order to select a narrow concentration range of poloxomer Lutrol F68 to achieve desired size. The results showed that minimum 1% concentration of poloxomer Lutrol F68 was required to produce stable alendronate nanoparticles as shown in figure 3. Large aggregates were obtained when concentration was increased to 20%. Above 20% concentration, sticky mass was obtained that showed poor flow properties. The optimized particles (176 nm) were obtained at 10% w/w poloxomer concentration.
Example 5
Effect of different antisolvents on the particle size of alendronate sodium nanoparticles The effect of different antisolvents on the particle size of alendronate sodium nanoparticles was studied as shown in Figure 4. It was observed that large sized and aggregated particles were formed when ethanol, acetone and IPA were used as a precipitation medium. Minimum particle size of 313 nm was
attained when precipitation was carried out in acetonitrile. However no precipitation took place when methanol was selected as antisolvent.
Example 6
Effect of different drying conditions on the particle size of alendronate sodium nanoparticles The effect of different drying conditions on the particle size of alendronate sodium nanoparticles was investigated as shown in Table 2. Different techniques such as spray drying, freeze drying, rotary evaporator, vacuum drying and heating on high speed magnetic stirrer were used to study the effect of drying condition on the particle size. It was observed that particles of micronic size range were formed by using spray and freeze drying methods whereas no particles were collected by vacuum drying. Minimal particle size of Table 2: Effect of different drying techniques on the particle size of alendronate sodium particles.
(Table Removed )
Example 7
Preparation of alendronate sodium formulation for their use by pulmonary route 1 g of alendronate sodium nanoparticles were dissolved in 70:30 water ethanol mixture. 2ml of this solution was nebulized by using standard nebulization apparatus to produce wet aerosols of nanoparticles for their delivery by pulmonary route. Figure 5 illustrates lung targeting images of bisphosphonate nebulizer showing 80-90% respiratory fraction.

We Claim:
1. A process for preparation of nanosize bisphosphonate particles, the process comprises:
(i) dissolving a bisphosphonate compound into distilled water,
(ii) filtering solution of step (i) through a micropore size filter;
(iii) adding filtrate of step (ii) to a precipitation medium, the precipitation medium
comprising an antisolvent, and a stabilizer to precipitate the bisphosphonate particles;
(iv) drying particles obtained in step (iii) at high temperature and high speed stirring to
obtain dried powder of nanosize bisphosphonate particles.
2. The process as claimed in claim 1, wherein concentration of bisphosphonate compound in the distilled water solution can range from 0.5 to 5 % w/v.
3. The process as claimed in claim 1, wherein the antisolvent has a solubility of less than 0.1g/100ml of bisphosphonate compound.
4. The process as claimed in claim 1 or 3, wherein the antisolvent is selected from a group comprising of alcohols, ketones and ethers and preferably isopropanol, ethanol and acetonitrile.
5. The process as claimed in claim 1, wherein the stabilizer is selected from a group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, cellulose esters, cellulose ethers, caseinates, poloxomers, casein, sodium alginate, polyethylene glycol graft copolymers, starch, chitosan, agar, pectin, sugar, gelatin, gum arabics, phospholipids and dextrins.
6. The process as claimed in claim 1 or 5, wherein the amount of stabilizer is in the range of 1 to 20 % w/w of bisphophonate compound.
7. The process as claimed in claim 1, wherein the amount of stabilizer is in the range of 1 to 20 % w/w of precipitation medium.
8. The process as claimed in claim 1, wherein the bisphosphonate solution is added to the precipitation medium in step (iii) with stirring at a speed of 400 to 800 rpm and the particles dried in step (iv) at a temperature ranging from 60 to 100 degree C at stirring speed of 1500 to 2500 rpm.
9. The process as claimed in claim 1, wherein the bisphosphonate compound is selected from a group comprising alendronate, residronate, methylene diphosphonate (MDP) and ethylene hydroxyl diphosphonate (EHDP) and their sodium salts.
10. Bisphosphonate nanoparticles obtained by:
(i) dissolving a bisphosphonate compound into distilled water;
(ii) filtering solution of step (i) through a micropore size filter;
(iii) adding filtrate of step (ii) to a precipitation medium, the precipitation medium
comprising an antisolvent, and a stabilizer to precipitate the bisphosphonate particles;
(iv) drying particles obtained in step (iii) at high temperature and high speed stirring to
obtain dried powder of nanosize bisphosphonate particles.
11. A bisphosphonate formulation prepared by mixing bisphosphonate particles of claim 1 with a
water alcohol mixture, wherein the alcohol concentration relative to water is in range of 0.1-50
%, preferably 10-30%.

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Patent Number

270668

Indian Patent Application Number

1339/DEL/2010

PG Journal Number

02/2016

Publication Date

08-Jan-2016

Grant Date

07-Jan-2016

Date of Filing

09-Jun-2010

Name of Patentee

DIRECTOR GENERAL, DEFENCE RESEARCH & DEVELOPMENT ORGANISATION.

Applicant Address

DRDO, MINISTRY OF DEFENCE, ROOM NO. 348, B-WING, DRDO BHAVAN, RAJAJI MARG, NEW DELHI-110011 (INDIA)

Inventors:

#	Inventor's Name	Inventor's Address
1	BHATNAGAR, ASEEM	INMAS, BRIG. S.K. MAZUMDAR MARG, TIMARPUR, NEW DELHI
2	SULTANA SHAHEEN	JAMIA HAMDARD, HAMDARD NAGAR, NEW DELHI
3	AHMAD, FARHAN JALEES	JAMIA HAMDARD, HAMDARD NAGAR, NEW DELHI
4	MITTAL, GAURAV	INMAS, BRIG. S.K. MAZUMDAR MARG, TIMARPUR, NEW DELHI
5	TALEGAONKAR, SUSHMA	JAMIA HAMDARD, HAMDARD NAGAR, NEW DELHI
6	SULTANA, SARWAT	JAMIA HAMDARD, HAMDARD NAGAR, NEW DELHI
7	SINGH, THAKURI	INMAS, BRIG. S.K. MAZUMDAR MARG, TIMARPUR, NEW DELHI
8	NEERAJ' KUMAR	INMAS, BRIG. S.K.MAZUMDAR MARG, TIMARPUR, NEW DELHI
9	RASHID ALI	INMAS, BRIG. S.K. MAZUMDAR MARG, TIMARPUR, NEW DELHI

PCT International Classification Number

A61K31/662;

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA