Nanotechnolqgy Approach for Immunomodulatory Drug

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Complete SpecificationFORM 2

THE PATENTS ACT, 1970(39 of 1970)&THE PATENT RULES, 2003

COMPLETE SPECIFICATION (See section 10 and rule 13)1. TITLE OF THE INVENTION: Nanotechnology approach for immunomodulatory drug.2.APPLICANT(S)(a) NAME :Shah Divyen Mukeshbhai(b) NATIONALITY :Indian(c) ADDRESS : 21-VIP Socienty; B/H- Utkarsh Vidyalaya; P.O.Vidut nagar, Vadodara-390015,Gujarat, India(a) NAME : Dr. Londhe Vaishali Y.(b) NATIONALITY: Indian(c) ADDRESS :F1-702, Vijay Nagar Society, Swami Nityanand Marg, Andheri (E),

Mumbai 400069,Maharashtra, India3.PREAMBLE TO THE DESCRITION COMPLETEThe following specification describes invention4. DESCRIPTION (Description shall start from next page)5. CLAIMS (9 claims)6. DATE AND SIGNATURE (to be given on the last page of specification)7. ABSTRACT OF THE INVENTION (to be given along with complete specification onthe separate page)Note:*Repeat boxes in case of more than one entry*To be signed by the applicant(s)or the authorized registered patent agent*Name of the applicant should be given in full, family name in the beginning*Complete address of the applicant should be given stating with postal index no. /code, state and country*Strike out the column which is/are not applicable

COMPLETE SPECIFICATIONNANOTECHNOLQGY APPROACH FOR IMMUNOMODULATORY DRUGFollowing is the general description of the present invention:Technical Field of the Invention:This invention relates application of nanotechnology for delivering immunomodulatoryagent for improvement of immunity. The nanoparticles are prepared in aqueous phase

and thus the method is suitable for organic sensitive compounds, too. The presentinvestigation relates to water soluble nanoparticles for delivering animmunomodulatory agent and a preparing method thereof, more precisely, the watersoluble nanoparticles for delivering immunomodulatory agent for targeting tolymphatic system.Background and Prior art of the Invention:Lack of immunity can cause many diseases like tuberculosis, flu and many infections.Vaccines are the only reliable option which is specific for specific antigen, and thusvaccines for many diseases are still under development. Thus there is a need for the

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development of non-specific immunomodulatory agent which can preventimmunosuppression after chronic pretreatment, which can be useful in majordiseases/infections which causes prevention of immunosuppression. The traditionalmedication is failing to target medications to lymphatic system, which has causedrelapse of diseases like AIDS, filaria, anthrax, extra pulmonary tuberculosis, etc.because the virus/bacteria are still alive in lymphatic system. Thus there is a greatneed of medication which can target drugs to lymphatic system. One of the easiest wayis to target Peyer's patches.Nanoparticulate drug delivery system is good option for many drugs because they notonly reduces dose and toxicity but also increases bioavailability with site specificity.Nanoparticles are small enough to enter to lymphatic vessels and lymph nodes whengiven via oral route, intramuscular injection, direct injection to organ/tumor,subcutaneous injection, etc. When the nanoparticles are given in to body organ, theycan move to lymphatic vessels which later on traps to lymph nodes or movecompletely through lymphatic system and return to blood at thoracic duct.Microparticles of size less than 10 m can be located to Peyer's patch, but cannot be

COMPLETE SPECIFICATION

absorbed. Particles less than 1 m in size can be absorbed easily by Peyer's patch. Ithas been observed that particles less than 150 nm can be absorbed by Peyer's patchand can go to systemic circulation.In Indian Ayurveda system, there are lots of plants used for immuno-maintenance or toenhance immunity, but there use is limited due to high heavy metal content, lack ofscientific evidence and unknown mechanism action. The synthesized drugs are havingknown mechanism of action with sufficient scientific evidence for their application.Chitosan is obtained from deacetylation of chitin. The degree of deacetylation andmolecular weight of chitosan can have impact on many properties of nanoparticles.Chitosan generates positive charge in dilute acidic medium. Chitosan is biocompatible,biodegradable and mucoadhesive in nature. It is generally recognized as safe (GRAS

as per USFDA). Thus, chitosan is expected to be a relevant material for biomedicalindustry.OBJECTIVES AND SUMMARY OF THE INVENTION:Objective of the present invention is to develop nanoparticles in aqueous phase forimmunomodulatory activity. The nanoparticles were prepared by interaction ofnegative charge generating agent and positive charge generating agent.BRIEF DESCRIPTION OF DRAWING:The present investigation is best understood with reference to the accompanyingdrawings, which are:Fig. 1 is photograph the morphology of present investigation taken by TEM(Transmission Electron Microscopy),Fig. 2 is set of graphs showing the results of heat analysis for positive charge

generating agent (Fig. 2(a)); immunomodulatory agent (Fig. 2(b)); negative chargegenerating agent (Fig. 2(c)); drug entrapped nanoparticle (Fig. 2(d)) and placebonanoparticles (Fig. 2(e)),

COMPLETE SPECIFICATIONFig. 3 is set of graphs showing the FTIR spectrums of positive charge generating agent(Fig. 3(a)); immunomodulatory agent (Fig. 3(b)); negative charge generating agent (Fig.3(c)); drug entrapped nanoparticle (Fig. 3(d)) and placebo nanoparticles (Fig. 3(e)),Fig. 4 is uptake study on caco-2 cell line,




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Fig. 5 is Effect of drug loaded nanoparticles on blood cells of rat for cyclophosphamideinduced myelosuppressionFig. 6 is Effect of drug loaded nanoparticles on %neutrophil adhesionFig. 7 is Effect of drug loaded nanoparticles on DTH response using SRBC as anantigen in ratsFig. 8 is Effect drug loaded nanoparticles on antibody titres to antigenically challengedratDETAILED DESCRIPTION OF THE INVENTION:Chitosan ((14)-2-amino-2-deoxy--D-glucan), used here is a linear polyamine with ahigh ratio of glucosamine to acetyl-glucosamine units is natural mucoadhesivecationic polymer which is obtained by partial deacetylation of chitin. The degree ofacetylation of chitosan is between 60-95% with a molecular weight of about 40kDa to80kDa.Chitosan solution in the present invention is a solution formed by dissolving chitosanin a solvent. The solvent can be any which can dissolve chitosan but is not limited toacetic acid, succinic acid, malic acid and formic acid. The concentration of acetic acidcan be about 1% to 5% v/v. The concentration of chitosan in chitosan solution is about0.5% to 4% w/v.

The negatively charged solution is formed by dissolving the polyanion is aqueousphase. The negative charge generating agent is any poly anion which can generatenegative charge but is not limited to sodium tri poly phosphate (STPP), dextran sulfateand sodium alginate. The concentration of negative charge generating agent is about0.5% to 2% w/v.The nanoparticles is prepared by gradual addition of negative charge generatingsolution to chitosan solution under stirrer with rotation speed up to 3000 rpm for about10 min to 50 min.

COMPLETE SPECIFICATIONA more precise method of preparation of chitosan nanoparticles for delivering

immunomodulatory agent is given below:(a) Use chitosan solution with pH range of about 3 to about 6.(b) Use a negative charge generating agent solution in which immunomodulatory agentis dissolve with a pH range of about 7 to 11.The negative charge generating solution with immunomodulatory agent is addedgradually to chitosan solution under stirrer. The ratio of chitosan: negative chargegenerating agent is maintained between 2:1 to 10:1.The chitosan nanoparticles obtained from the present investigation have an averageparticle diameter of about 100 nm to 400 nm, preferably about 100 nm to 200 nm. Thegiven process can reproduce nanoparticles in aqueous phase, without the need oforganic phase, in a mild condition. Therefore, the nanoparticles obtained from aboveprocess are safe to use for many organic sensitive compound, DNA, RNA and many

more agent for pharmacological application.The following example is given as reference:EXAMPLE-1:Preparation of chitosan nanoparticles:Chitosan is obtained from India sea foods. Prepare a chitosan solution of 1-2 mg/ml in1% acetic acid and adjust the pH below 6.5. Prepare a Sodium tri poly phosphate(STPP) solution and adjust the pH to 8. Add STPP solution to chitosan solutiongradually with stirrer for 30 min. The chitosan/STPP ratio was maintained between 2:1to 4:1.




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EXAMPLE - 2:

Preparation of drug loaded chitosan nanoparticles:

COMPLETE SPECIFICATIONChitosan is obtained from India sea foods. Prepare a chitosan solution of 1-2 mg/ml in1% acetic acid and adjust the pH to 4.00.2. Prepare Sodium tri poly phosphate (STPP)solution in which immunomodulatory agent (drug) is pre dissolved and adjust the pH to9. Add drug-STPP solution to chitosan solution gradually with stirrer for 30 min. Thechitosan/STPP ratio was maintained to 4:1.EXPERIMENTAL EXAMPLE - 1:Morphological observation of drug loaded nanoparticles:Transmission Electron Microscopy was used for characterization of morphology ofNanoparticles. A drop of sample solution was placed on top of the copper grids andwas dried by sodium vapor lamp, then coated with carbon. TEM pictures were takenwith a CM 12 Philips transmission electron microscope (Philips, Amsterdam,Netherlands). Fig. 1 shows circular spherical- shaped nanoparticles with a dark, denseand solid structure indicating absence of cavity in nanoparticles. The Chitosan-STPPcomplex membrane is very dense, which prevents the majority of the electron beam

passing through it, resulting in the dark, solid and dense structures of the nanoparticleobserved. Thus nanoparticles obtained are useful as drug carrier because they offerexcellent absorption and distribution.EXPERIMENTAL EXAMPLE - 2:Photon correlation spectroscopy (PCS) measurementPCS was measured with zetasizer-nano series (Malvern Instrument, UK) for thedetermination of particle size and surface charge. The particle size was found to besimilar as that with TEM. The surface charge was found to be in positive range becausethe chitosan was in higher amount compare to the negatively charge forming agent.EXPERIMENTAL EXAMPLE - 3:Differential Scanning calorimetry (DSC) measurement:

COMPLETE SPECIFICATIONDSC experiments were carried out to characterize the physical state of drug inChitosan nanoparticles. The heating rate was 10 C/min, where nitrogen served aspurge gas and the system was cooled down by liquid nitrogen in the range between 0-300C. The DSC (DSC-2000, Dupont, USA) instrument was calibrated for temperatureusing octadecane and indium. Furthermore, for enthalpy calibration, indium was sealedin aluminum pans with a sealed empty pan as a reference. The observed thermogramsof each compound are given in figure 2. The endothermic peak of Chitosan is seen at125.42C (The peak varies between 110-140C according to degree of deacetylation),STPP at 128.62 C and 213.49C, and Chitosan-STPP (placebo) nanoparticles at 128.15C. Drug shows endothermic peak at 239.02C and exothermic peak at 266.85C.

However, the drug loaded nanoparticles shows endothermic peak at 179.20C, in whichthe characteristic peak of drug and Chitosan-STPP nanoparticles were absent whichindicates that drug is completely dispersed molecularly in Chitosan-STPP interaction.EXPERIMENTAL EXAMPLE - 4:Measurement of FTIR:To identify the synthesis of chitosan nanoparticle in above experiment-2, FTIR wascarried out. Chitosan (Fig. 3(a)) is showing peaks at 3130.39, 2363.16, 1655.09, 1401.05,1116.36 cm-1 which are due to NH2 scissoring, C-OH bending and C-C-C bending.Other characteristic peak of Chitosan are seen at 754.42, 656.08, 603.60 cm-1. STPP




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(Fig. 3(b)) shows characteristic peaks at 3631.23,3150.37,2326.03, 1678.26, 1401.22,1210.77, 1127.61 (P=0 bending), 904.71, 718.81, 658.13, 617.81, 509.78 cm-1. But innanoparticles without drug (Fig. 3(c)), the peak shifts to 3134.46 from 3130.39 cm-1which is more wider, indicating hydrogen bonding. The amino group absorption alsoshifts to 1633.97 from 1655.09 cm-1 and a new peak appears at 1197.04 cm-1, which isan indication that these groups interacted with STPP creating ionic bonds. Theseinteractions reduce Chitosan solubility which causes Chitosan separation from thesolution in the form of nanoparticles. Drug (Fig. 3(d)) shows characteristic peaks at3138.43, 1577.87, 1527.47 (secondary amide band), 1402.79, 1208.04 (C=S stretching),941.95, 841.01, 740.68, 696.53(secondary N-H bending), 648.21, 535.86 cm-1. But whendrug is entrapped in to nanoparticles (Fig. 3(e)), many characteristic peaks of itdisappears indicating strong interaction

COMPLETE SPECIFICATIONbetween drug and nanoparticle matrix. The peak at 1527.47, 941.95, 841.01 and 535.86disappears in drug loaded nanoparticles which were seen in drug. The peak of drug at1577.87 shifts to 1589.45cm-1, 1208.04 shifts to 1205.82, 740.68 shifts to 757.02, and696.53 shifts to 701.23 in drug loaded nanoparticle. The peak of placebo nanoparticles

at 1116.69 shifts to 1127.74 cm-1 in drug loaded nanoparticles, while the peak at1197.04 disappears in drug loaded nanoparticles. These change in peak shows stronginteraction of drug with nanoparticle matrix.EXPERIMENTAL EXAMPLE - 5:Uptake study:Isothiocynate group of FITC readily reacts with the primary amine group of theChitosan polymer. Chitosan solution (2mg/ml) is prepared in 0.1 M acetic acid. 5 ml ofFITC solution (1 mg/ml in ethanol) is added slowly to Chitosan solution undercontinuous stirring at room temperature in dark. The reaction between theisothiocyanate group of FITC and the amino group of the D-glucosamine residue wasallowed to proceed for 10 hours. Then the pH of the solution is raised to 10 by 0.1 M

NaOH leading to precipitation of FITC labeled Chitosan. FITC labeled Chitosanprecipitates are centrifuged and washed with ethanol/water (70/30) until nofluorescence is detected in supernatant.The FITC labeled Chitosan polymer is dissolved in 0.1 M acetic acid. 4 ml of drug-STPPsolution is added to 10 ml of FITC-Chitosan solution under magnetic stirring.The monolayer of caco-2 cells were incubated with 100 g/ml of FITC labelednanoparticle suspension. After the incubation, cells were washed with PBS (pH 7.4)and observed under a fluorescent microscope (Olympus BX61, Japan) and imageswere captured. (Fig. 4)EXPERIMENTAL EXAMPLE - 6:Cyclophosphamide induced myelosuppression:Cyclophosphamide is immunosuppressant in nature. The formulation was able to

restore the bone marrow suppression. Various hematological parameters like WBC,RBC, Hemoglobin,

COMPLETE SPECIFICATIONPlatelet count and Lymphocyte were accessed by automated hematology analyzer(Sysmex, Japan). (Fig. 5)EXPERIMENTAL EXAMPLE - 7:Neutrophil adhesion test:Neutrophils are unable to divide further, have limited protein synthesis capacity, are




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having main role in body defense by direct or indirect role in phagocytosis, exocytosis,intracellular and extracellular killing and chemotaxis. Total leukocyte cell count anddifferential leukocyte cell count were accessed and the nanoparticle treated animalsshowed higher % neutrophil adhesion indicating increasing immunity of body. (Fig. 6)EXPERIMENTAL EXAMPLE - 8:Delayed type hypersensitivity test:The cell-mediated immune response of ding loaded nanoparticles was assessed byDTH reaction, i.e. foot pad inflammation. The cell mediated immunity of nanoparticletreated animals is increased due to fasten recovery in the inflammation due to antigen(Sheep red blood cells-SRBC). (Fig. 7)EXPERIMENTAL EXAMPLE - 9:Heamagglutination litre:The humoral immunity involves interactions of B cells with the antigen and theirsubsequent proliferation and differentiation in to antibody secreting plasma cells. Theresponse of humoral mediated immunity is via neutralization of antigen or eliminationof antigen which may involve complement activation and opsonization. Theaugmentation of the humoral immune response to SRBCs by drug loadednanoparticles. as evidenced by increase in the antibody titre in rats (Fig. 8) indicated

the enhanced responsiveness of T and B lymphocyte subsets, involved in the antibodysynthesis. The high values of haemagglutinating antibody titre indicate thatimmunostimulation was achieved through humoral immunity.

COMPLETE SPECIFICATIONWe claim1. A nanoparticulate drug delivery for delivering an immunomodulatory agent which isprepared by mild ionic gelation method.2. A nanoparticulate drug delivery for delivering an immunomodulatory agent as inclaim 1, where in the ratio of positive charge generating agent to negative chargegenerating agent is between 1:2 to 1:20.

3. A nanoparticulate drug delivery for delivering an immunomodulatory agent as inclaim 1, where in the pH of positive charge generating agent is below 7 and thenegative charge generating agent is above 7.4. The nanoparticles are administered in a composition selected from the groupconsisting of suspension, gel, spray or powdered form viaoral/parenteral/intramuscular/intra nasal route for mucosal delivery ofimmunomodulatory agent.5. The size of nanoparticles is between 20 to 400 nm and zeta-potential is between +2 to+55 mv.6. The nanoparticles conjugated with ligand for a predetermined target or agentcausing change in lipophillicity resulting in escape through reticulo endothelial system(RES).

7. A method of increasing the efficiency of therapeutic agent/ vaccine to a subject in aneed of treatment with that agent; there by increasing the efficacy of therapeutic agent,decreasing side effect and dose in the subject.8. The nanoparticles are targeting peyer's patch; there by if a detectable moiety isattached, will facilitate diagnostic imaging for peyer's patch.9. The nanoparticles given by any route are maintaining immunity during bothimmunosuppressed and immunostimulant condition.

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Nanotechnolqgy Approach for Immunomodulatory Drug

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