FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
Provisional Specification
(See section 10 and rule 13)
NOVEL IMPROVED COMPOSITIONS FOR CANCER TRERAPY
PANACEA BIOTEC LIMITED
A company incorporated under the laws of India having their office at 104, SAMARPAN COMPLEX, NEW-LINK ROAD, CHAKALA, ANDHERI (E), MUMBAI 400099, MAHARASHTRA, INDIA
The following specification describes the invention

NOVEL IMPROVED COMPOSITIONS FOR CANCER TRERAPY
The present invention relates to novel and improved compositions of anti-cancer drugs.
The present invention relates to novel and improved compositions of anti-cancer drugs including but not limited to alkylating agents, antimetabolites, anthracenediones, natural products, hormones, antagonists, radiosensitizers, platinum coordination complexes, adrenocortical suppressants, immunosuppressive agent, substituted ureas, and COX-2 inhibitors.
The present invention specifically relates to novel and improved compositions of Taxanes, such as paclitaxel and docetaxel.
The present invention relates to novel and improved compositions of anti-cancer drugs, preferably Taxanes, their derivatives or their analogues, methods of manufacturing these formulations and methods of treating cancer patients with these compositions.
The present invention relates to novel and improved compositions of anti-cancer drugs, preferably Taxanes, such as paclitaxel and docetaxel, their derivatives or their analogues, methods of manufacturing these compositions and methods of fractionating the particles in particular size range and methods of treating cancer patients with these compositions, which provide reduced chemotherapy induced side-effects especially reduced chemotherapy-induced-alopecia.
The novel and improved compositions of anti-cancer drugs, preferably Taxanes such as paclitaxel and docetaxel, their derivatives or their analogues, are colloidal delivery systems, for cancer therapy with drastically reduced chemotherapy-induced-alopecia, prepared in a particular size range. The effective average size range of the particles of the present composition is from about 70 nm to about 200 nm, preferably is from about 80 nm to about 120 nm.
BACKGROUND OF THE INVENTION
A wide variety of anti-cancer agents have been developed till date for treatment of various types of cancers in mammals and newer and newer agents are being developed as chemotherapeutics wherein the research is aimed at developing tumor specific anti-cancer agents while increasing the potency against drug-resistant tumors.
Newer discoveries are on-going, but to-date chemotherapeutic agents such as 5-Flurouracil (5FU), Doxorubicin and the Taxanes are a mainstay of therapy for patients with a variety of cancers including ovarian, breast, lung, colon, prostate, head and neck, cervical and brain and others.
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Clinical protocols frequently would involve combining anti-cancer drugs to produce increased therapeutic efficacy. However the use of these drugs has been limited by associated toxicities, including nausea, myelosuppression, alopecia, vomiting and stomatitis and also cardio-toxicity.
From amongst all these associated toxicities mentioned above, alopecia (or hair loss) due to chemotherapy is one of the most distressing and traumatic side-effect for cancer patients as it causes depression, loss of self-confidence, and humiliation in men and women of all ages. Some patients refuse to undergo treatment because of the physical and emotional angst that results from treatment-related alopecia. Hair loss has a significant influence upon patient's psychological condition and it is a serious problem affecting the quality of life of patient's. There is thus a pressing need to provide a type of cancer treatment with drastically reduced chemotherapy-induced-alopecia.
Taxanes are anticancer cytotoxics that stabilize cellular microtubules. Taxane compounds useful in the composition and methods described herein include paclitaxel and docetaxel, as well as natural and synthetic analogs thereof, which possess anticancer or anti-angiogenic activity. Paclitaxel and docetaxel have substantial activity, and one or both of these agents are widely accepted as components of therapy for advanced breast, lung, and ovarian carcinomas.
Docetaxel is an antineoplastic agent belonging to the taxoid family. It is prepared by semi-synthesis beginning with a precursor extracted from the renewable needle biomass of yew plants. Taxotere® is sterile docetaxel injection composition, indicated for the treatment of patients with locally advanced or metastatic breast cancer after failure of prior chemotherapy. TAXOTERE in combination with doxorubicin and cyclophosphamide is indicated for the adjuvant treatment of patients with operable node-positive breast cancer.
Paclitaxel, (formerly called taxol), belonging to the taxane class of chemotherapy agents has been widely used for many years in intravenous forms for the treatment of breast and ovarian cancer or non-small cell lung carcinoma (NSCLC). Along with the tremendous potential that paclitaxel has shown as an antitumor drug, clinical problems with solubility, toxicity, poor bioavailability and development of drug resistance are sufficiently severe that the need for formulations of paclitaxel derivatives or analogues with better therapeutic efficacy and less toxicity is very clear.
Paclitaxel (Taxol®) is currently available as a solution for iv infusion in a vehicle composed of Cremophor® EL which has been shown to cause toxic effects such as life-threatening anaphylaxis. This Cremophor/Ethanol formulation of paclitaxel precipitates upon dilution with infusion fluid, and fibrous precipitates are formed in some compositions during storage for extended periods of time. Additional information regarding Cremophor formulations of paclitaxel may be found in Agharkar et al., United States Patent No. 5,504,102.
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Recently introduced Abraxane®, is a paclitaxel protein-bound particles for injectable suspension. It is an albumin-bound form of paclitaxel which breaks quickly in the liver to release free drug which then circulates in the blood to produce the initial therapeutic response, however it also manifests itself as toxic side effects, such as complete hair loss, infections due to low WBCs count, fatigue, weakness and inflammation etc. Complete hair loss, or alopecia, almost always occurs with these dosage forms of Paclitaxel. This usually involves the loss of eyebrows, eyelashes, and pubic hair, as well as scalp hair.
A number of U.S. Patent Numbers are listed against this product Abraxane®, these include, U.S. Patent Number 5,439,686; 5,498,421; 5,560,933; 5,665,382; 6,096,331; 6,506,405; 6,537,579; 6,749,868 and 6,753,006.
In accordance with the invention in the above mentioned patents there are provided compositions and methods useful for the in vivo delivery of substantially water insoluble pharmacologically active agents (such as the anticancer drug paclitaxel) in which the pharmacologically active agent is delivered in the form of suspended particles coated with protein (which acts as a stabilizing agent). In these inventions attempt has been made to provide.an improvised technique to prepare protein microspheres to deliver substantially water insoluble pharmacologically active agents in aqueous suspensions for parenteral administration that does not cause allergic reactions due to the presence of added emulsifiers and solubilizing agents as employed in prior art.
United States Patent Number 5,439,686 claims "A composition for in vivo delivery of a substantially water insoluble pharmacologically active agent, wherein said agent is a solid or liquid substantially completely contained within a polymeric shell, wherein the largest cross-sectional dimension of said shell is no greater than about 10 microns, wherein said polymeric shell comprises a biocompatible polymer which is substantially crosslinked by way of disulfide bonds, and wherein said polymeric shell containing pharmacologically active agent therein is suspended in a biocompatible aqueous liquid". The polymeric shell is a biocompatible polymer, which is specifically the crosslinked protein albumin.
Unites States Patent Number 5,560,933 claims a method of preparation for the above mentioned composition of their invention, it claims "A method for the preparation of a substantially water insoluble pharmacologically active agent for in vivo delivery, said method comprising subjecting a mixture comprising: a dispersing agent containing said pharmacologically active agent dispersed therein, and aqueous medium containing a biocompatible polymer capable of being crosslinked by disulfide bonds to sonication conditions for a time sufficient to promote crosslinking of said biocompatible polymer by disulfide bonds to produce a polymeric shell containing the pharmacologically active agent therein".
United States Patent Number 6,506,405 claims formulation of paclitaxel (Taxol) for treatment of primary tumors in a subject, which achieves high local concentration of said paclitaxel at the tumor site, the formulation being substantially free of cremophor. They claim that these formulations comprising albumin shows reduced cerebral or neurologic toxicity. These are achieved by providing lesser dose of taxol over a shortened duration
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of infusion to patients, reducing the infusion volumes and providing a more stable composition of taxol.
United States Patent Number 6,749,868 provides a drug delivery system in which part of the molecules of pharmacologically active agent are bound to the protein (eg. Human serum albumin) and are therefore immediately bioavailable upon administration to a mammal and the other part of the pharmacologically active agent is contained within nanoparticles coated by protein. The protein coated drug nanoparticles are prepared by using high shear in the absence of conventional surfactants to yield particles with a diameter of less than about 1 micron, in particular unusually small nanoparticles less than 200 nm diameter which can be sterile-filtered through a 0.22 micron filter to provide sterile solid formulations useful for intravenous injection.
None of the above patent describes a method of manufacturing a paclitaxel composition such that the composition is in particular size range to provide cancer therapy with drastically reduced chemotherapy-induced-alopecia, which is one of the most traumatic side-effects for cancer patients. The above patents which are related to the commercially available product Abraxane® provides a product which avoids causing allergic reactions by avoiding emulsifiers like Cremophor, and provides a stable, sterilized microparticular or nanoparticular delivery systems for the substantially water insoluble active agent like paclitaxel. But it fails to provide a formulation of paclitaxel devoid of or having reduced side effects like alopecia or hair loss. The product leaflet for Abraxane® mentions under PATIENT INFORMATION, Hair loss as one of the important side-effects observed in studies of patients taking Abraxane®. It says Complete hair loss, or Alopecia, almost always occurs with Abraxane®.
There is research paper publications related to study of temperature- and pH-sensitive core-shell nanoparticles of paclitaxel for intracellular delivery by Yang et al, Front Biosci. 2005 Sep 1; 10:3058-67, which describe encapsulating paclitaxel with temperature and pH-sensitive amphiphilic polymeric poly(N-isopropylacrylamide-co-acrylic acid-co-cholesteryl acrylate) (P(NIPAAm-co-AA-co-CHA)) to form nanoparticles. This research paper however does not discuss or mention the methods of manufacturing these particle compositions and fractionating the particles in a particlular size range, which is suitable to drastically reduce chemotherapy-induced-alopecia in cancer patients.
United States Patent Number 5,399,363 relates to surface modified anticancer nanoparticles, wherein the particles consists essentially of a crystalline anticancer agent having a surface modifier preferably which are nonionic and anionic surfactant adsorbed on the surface to maintain as effective average particle size of less than about 1000 nm. The use of surfactants would itself contribute towards the toxicity of the composition and this patent does not mention any report on reduction of specific chemotherapy-induced -alopecia. The use of specific range of particle size of paclitaxel nanoparticles composition containing biodegradable polymers to achieve reduction in specific chemotherapy induced side-effects like reduced alopecia is neither demonstrated nor
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predicted from '363 invention. The specific invention of '363 is to have non-crosslinked surface modifiers absorbed on the surface of a crystalline anti-cancer medicaments
United States Patent Number 6,136,846 claims a composition for delivering paclitaxel in vivo comprising paclitaxel, a solvent like ethanol or propylene glycol and a water-miscible solubilizer like esterified d-a-tocopherol acid succinate. It was noted that prior researchers formulated insoluble drug like paclitaxel using 50% cremophor and 50% dehydrated alcohol, but such formulations precipitates upon diluton with infusion fluid and is unstable on storage. Hence '846 invention has provided an improved formulation of paclitaxel by using water-miscible solubilizers other than cremophor to provide formulations with improved long term stability.
PCT Publication WO 2004/084871 relates to poly(lactic-co-glycolic acid) and poly(lactic acid) (PLA) nanoparticles that encapsulate a low molecular weight and water-soluble drug that can deliver the drug to target legion sites where the particles gradually release the drug over a prolonged period of time. Basically the invention of WO '487 relates to converting a low-molecular, water-soluble and non-peptide drug into a hydrophobic drug by interacting it with metal ion and then encapsulating the hydrophobicized drug into PLGA or PLA nanoparticles and allowing a surfactant to be adsorbed onto the surface of the particles. As mentioned in this application, the nanoparticles have a diameter of 50 to 300 nm, but this patent does not relate to or mention water-insoluble anti-cancer drugs like paclitaxel and others and does not provide a composition, which has reduced chemotherapy-induced-alopecia, which is the most distressing and traumatic side-effects for cancer patients.
United States Application No. 20060041019 claims an agent for inhibiting hair loss caused by an antitumor agent wherein the agent is a mixture of cyclic and/or straight chain poly lactic acids having a condensation degree of 3 to 20. Preferably, the mixture of cyclic and/or straight chain poly lactic acids having a condensation degree of 3 to 20 as per the inventors of the '019 application is a mixture of polylactic acids that is produced by polymerizing lactide in the presence of the compound represented by formula (3): Me-N(R.sup.l)(R.sup.2) wherein Me represents an alkali metal and R.sup.l and R.sup.2 each independently represent an aliphatic group or an aromatic group.
It has thus been seen that none of these prior known formulations have provided a method of manufacturing formulations of anti-cancer drugs like paclitaxel, docetaxel and others with reduced alopacia related side-effects. In-spite of the various attempts made earlier to provide anti- cancer compositions with improved efficacy, none of these compositions show low clinical side effects especially none has provided methods to reduce the specifically distressing side effects of alopecia or hair loss.
There is therefore a need for novel and improved compositions comprising anticancer agents and methods of treatment using these compositions to overcome the stability problems and to alleviate the various clinical side-effects of the prior known marketed formulations, most importantly reducing the treatment induced alopecia or hair loss and method of preparing the same.
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There is such a need for example drugs like 5-fluorouracil, doxorubicin, docetaxel, paclitaxel, its derivatives or its analogues,
SUMMARY OF THE INVENTION
The present invention is directed to novel and improved compositions of anti-cancer drugs, basically the poorly soluble anti-cancer agents, and its method of manufacturing and methods of treating cancer therapy with these compositions.
The important aspect of the invention is directed to colloidal delivery systems like nanoparticulate compositions comprising poorly soluble anti-cancer drugs like paclitaxel and at least one biodegradable polymers and copolymers such that the composition is having an effective average particle size of from about 70 nm to about 200 nm. Such an effective average particle size provides a composition which when administered to patients for treatment of cancer therapy, has reduced chemotherapy-induced side-effects like alopecia.
Another aspect of the invention is directed to pharmaceutical compositions comprising a nanoparticulate composition of the invention of the specified particle size range preferably comprising poorly soluble anti-cancer agents, at least one biodegradable polymer, a secondary polymer, which is temperature and pH sensitive, and a pharmaceutically acceptable carrier, as well as any desired excipients. The specified particle size range of the pharmaceutical nanoparticulate composition is in the range of about 80 nm to about 120 nm, which upon administration of the composition to a mammal increases in size to have an average particle size range of from about 120 nm to about 200 nm.
This invention further discloses a method of making such a nanoparticulate composition comprising of mixing at least one anti-cancer active agent with at least one polymer in the presence of one or more pharmaceutically acceptable carriers as well as any desired excipients to provide a composition such that the particles of the said composition have an effective average particle size of from about 80 nm to about 120 nm due to which such compositions provide reduced chemotherapy-induced-alopecia. The nanoparticles in the size range of about 80 nm to about 120 nm are selectively separated by standard techniques in the final composition.
The present invention is further directed to a method of treatment comprising administering to a mammal in need a therapeutically effective amount of a nanoparticulate drug/copolymer composition according to the invention.
Both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.
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DETAILED DESCRIPTION OF THE INVENTION
It is an object of this present invention to provide novel and improved compositions for cancer therapy.
The anti-cancer active agent useful in the present invention are preferably selected from alkylating agents, antimetabolites, anthracenediones, natural products, hormones, antagonists, radiosensitizers, platinum coordination complexes, adrenocortical suppressants, immunosuppressive agent, substituted ureas, and COX-2 inhibitors.
In the present invention (a) the alkylating agent is selected from the group consisting of
chlormethine, chlorambucile, melphalan, uramustine, mannomustine,
extramustinephoshate, mechlorethaminoxide, cyclophosphamide, ifosfamide, trifosfamide, tretamine, thiotepa, triaziquone, mitomycine, busulfan, piposulfan, piposulfam, carmustine, methyl-CCNU, lomustine, semustine, streptozotocine, mitobronitole, dacarbazine and procarbazine; or (b) the antimetabolite is selected from the group consisting of methotrexate, fluorouracil, floxuridine, tegafur, cytarabine, idoxuridine, flucytosine, mercaptopurine, thioguanine, azathioprine, tiamiprine, vidarabine, pentostatin, and puromycme; or (c) the natural product is selected from the group consisting of phenesterine, etoposide, teniposide, adriamycine, daunomycine, doctinomycin, daunorubicin, doxorubicin, mithramycin, bleomycin, actinomycin, epirubicin, mitomycin, L-asparaginase, alpha-interferon, camptothecin, taxol, and retinoic acid; or (d) the hormone or antagonist is selected from the group consisting of prednisone, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, ethinyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone, flutamide, leuprolide; or (e) the anticancer agent is selected from the group consisting of cisplatin, carboplatin, mitoxantrone, hydroxyurea, mitotane, aminoglutethimide, cyclosporine, azathioprine, sulfasalazine, methoxsalen, and thalidomide.
In addition, the anti-cancer agent can be an immunosuppressive drug, such as, for example, cyclosporine, azathioprine, sulfasalazine, methoxsalen and thalidomide. The anti-cancer agent can also be a COX-2 inhibitor.
The anti-cancer drug preferably used in the present invention is taxanes and derivatives thereof (e.g. docetaxel, paclitaxel and derivatives thereof, taxotere and derivatives thereof and the like) but does not exclude other anti-cancer drugs like (for e.g. doxorubicin, methotrexate, cisplatin, daunorubicin, adriamycin, cyclophosphamide, actinomycin, bleomycin, epirubicin, mitomycin, methotrexate, 5-fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferons, camptothecin, phenesterine, tamoxifen, piposulfan, and derivatives thereof and the like).
The preferred anti-cancer agent is poorly soluble agents chosen from derivatives of paclitaxel, docetaxel, 5-fluorouracil and doxorubicin.
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The anti-cancer agent can be used alone or in combination with one or more anti-cancer agents.
It is a further objective of the present invention to provide novel and improved compositions for cancer therapy with reduced side-effects.
We have surprisingly found that providing a composition of paclitaxel, its derivatives or its analogues such that the composition is a colloidal delivery system of the active agent paclitaxel having a particular particle size range significantly reduces the side-effects especially the chemotherapy-induced-alopecia.
Colloidal delivery systems for the active agents include liposomes, microemulsions, nanocapsules, nanospheres, microparticles and nanoparticles. These delivery systems offer the advantages of targeting, modulation of distribution and flexible formulation and have a polymer structure, which may be designed and produced in a manner that is adapted to the desired objective.
It is an object of this invention to provide nanoparticles encapsulating paclitaxel, its derivatives or its analogs and to provide methods of manufacturing nanoparticles encapsulating paclitaxel, its derivatives or analogs to achieve maximum encapsulation efficiency. It is also the object of this invention to provide a method of fractionating the nanoparticles encapsulating paclitaxel, its derivatives or analogs with a specific effective particle size distribution.
As used herein, particle size refers to a number average particle size and size distribution as measured by conventional particle size analyzers well known to those skilled in the art, such as sedimentation field flow fractionation, photon correlation spectroscopy, laser light scattering or dynamic light scattering technology and by using transmission electron microscope (TEM). By "effective particle size distribution" it is meant that all the particles in the formulation have a number average particle size in diameter from about 70 nm to about 200 nm when measured by the above-noted techniques. In particularly preferred embodiments of the invention, the effective average particle size is between about 80 nm to about 150 nm. In some embodiments of the invention, the effective average particle size is between about 80 nm to about 120 nm.
It is reported in the literature that the particles below about 70 nm diameter permeate through normal blood capillaries to skin and hair roots. Thus nanoparticles encapsulating paclitaxel of particle size above 70 nm diameter would not permeate through normal blood capillary to skin and hence to the hair roots and thus significantly reduce the loss of hairs. Particles larger than 200 nm diameters are preferentially recognized by reticuloendothelial system (RES) and removed from blood circulation.
With reference to the effective average particle size, it is preferred that at least about 90%, or at least about 95% and, more preferably, at least about 99% of the particles have a particle size of between from about 70 nm to about 200 nm when measured by the
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above-noted techniques. In particularly preferred embodiments, essentially all of the particles have a size between from about 70 nm to about 200 nm.
It is further the object of this invention to provide nanoparticles encapsulating the active ingredient, paclitaxel with a specific particle size distribution to reduce the side-effects of hair loss and to provided targeted delivery of the nanoparticles to the tumor cells.
The drug encapsulated nanoparticles in the present invention are engineered in such a way that under in-vitro conditions, at room temperature, more than about 95% of the particles or more preferably more than about 99% of the particles have size between 80 -120 nm but interestingly due to the temperature sensitiveness of the particles, when these particles are injected in-vivo, the particle size would increase to about 120 - 200 nm. Thus in-vivo the particles would always be in the range of particle size, which is prevented from permeation from normal blood capillaries to skin and hence to hair roots and also to keep them circulating in blood for longer period of time. When these particles reach the tumor site, they are easily permeated through leaky and hyperpermeable tumor microvasculature where the particles are retained (i.e enhanced permeation and retention effect) and the drug released.
This type of delivery uses biodegradable/biocompatible polymers to encapsulate the active ingredient. These bidegradable primary polymers delay the release of the anticancer active agent and maintain the nanoparticles composition in the target site for a longer period of time for therapeutic effectiveness. The biodegradable polymers are selected from the group comprising of poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) (PLGA), poly(s-caprolactone) pullulan, poly(ethylene glycol), poly (N-vinyl pyrrolidone), polystyrene, chitosan, gelatin, dextran, cyclodextrins and the like and derivatives thereof.
The compositions according to the present invention, which includes microparticles, liposomes, nanocapsules, nanospheres, and nanoparticles etc. are manufactured by the standard conventional methods used in the art.
According to this invention in order to achieve targeted release of the active ingredient at the tumor site, a temperature sensitive and outer surface modified nanoparticles are prepared by applying a temperature responsive interpolymer complex capable of showing thermal responsiveness in an aqueous solution like poly-N-acetylacrylamide or poly(N-isopropylacrylamide) or polyvinyl alcohol and poly(ethylene glycol), poly(acrylamide) or poly(methacrylamide) to the nanoparticles encapsulating the active drug paclitaxel. Such nanoparticles with hydrophilic surfaces would circulate in the blood for longer period of time and because of the thermal sensitivity of the particles i.e showing upper critical solution temperature (UCST) or lower critical solution temperature (LCST) in an aqueous solution, the particle size increases when injected in-vivo at 37°C; the particle size further increases several folds when the particles are accumulated in tumor due to difference in physiological conditions in tumor microenvironment and the encapsulated active drug is released at the tumor site.
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Thus it is an object of the present invention to provide methods for preparing such temperature sensitive and outer surface modified nanoparticles encapsulating paclitaxel for controlled and site specific-delivery at the tumor site, thus providing maximum therapeutic effect of the drug with minimum adverse effects at a lower dose of the active ingredient.
EXAMPLE
Example 1: Synthesis of PLGA Nanoparticles Encapsulating Paclitaxel:
The nanoparticles from PLGA were synthesized using double emulsion approach via w/o/w double emulsion. In a typical experiment, 100 mg of PLGA was dissolved in 2 mL dichloromethane and 10 mg paclitaxel was dissolved in 1.0 mL of absolute ethanol. Both solutions were slowly mixed together with stirring. A primary water-in-oil (w/o) emulsion was made by emulsifying 500 uL phosphate buffer saline in above solution. The primary water-in-oil emulsion was then further emulsified in poly(N-acetylacrylamide) solution to form the water-in-oil-in-water (w/o/w emulsion). The w/o/w emulsion thus made was homogenized to form the paclitaxel-loaded nanoparticles on evaporation of the solvents. The solution was then centrifuged and the nanoparticles in the size range from 80 - 120 nm size diameter ranges were selectively separated. The nanoparticles were then dispersed in sterile water and lyophilized immediately for future use.
Example 2: PLGA Coupled Covalently to Pullulan Micellar Nanometer Aggregates and Loading of Paclitaxel:
PLGA was coupled covalently to pullulan by activating PLGA with N-hydroxy succinimide. The pullulan-PLGA complex was purified using gel filtration and characterized by FTIR, H-NMR and mass spectroscopy. The hydrophobized pullulan solution was lyophilized and kept in deep freeze was future use.
100 mg of hydrophobized pullulan was dissolved in 10 mL of water and the solution vortexed to form the micelles. A paclitaxel solution prepared in ethanol was added slowly to the micellar solution and dissolved until the solution was clear indicative of drug encapsulation in micellar formulation. Drug loaded particles in range of 80 - 120 nm were preferentially separated and the solution was lyophilized.
The encapsulation efficiency or loading capacity and the release behaviour of paclitaxel from the nanoparticles were determined by standard techniques using HPLC and particle size determined using the conventional particle size analyzer.
Example 3: Coating of Nanoparticles with Thermosensitive Polymers:
Drug loaded nanoparticles were suspended in aqueous buffer (pH 4-5). To this solution, a solution of carbodi-imide was added and the resulting solution was vortexed and
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continuously stirred at room temperature for 4 hours. The nanoparticles were then separated by centrifugation (or by filtration or dialysis). An aqueous solution of the polymer (N-acetylacrylamide) was added dropwise to the nanoparticles suspension and the mixture was vortexed. The solution was then further stirred, the particles purified and lyophilized for future use.
Example 4: Fractionation of Nanoparticles in a Particular Size Range:
10.0 mg lyophilized powder of paclitaxel-loaded nanoparticles was suspended in aqueous buffer with the aid of sonication. The solution was filtered through 0.2 um Millipore filtration unit and the filtrate was subjected to asymmetrical flow field-flow fractionation using the manufacturer's standard protocol for fractionation of particles using this technique. Different fractions were collected and subjected to particle size analysis using the standard techniques to determine the particle size and size distribution.
Dated 10th day of July 2006
Si9na,ure:uM?<*W
Vivek Kathpalia Constituted Patent Agent for the Applicant
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