Title of Invention	"A PROCESS FOR THE PREPARATION OF ALGINATE NANOPARTICLES"
Abstract	A process for the preparation of alginate nanoparticles having one or more drugs comprising singularly or combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated therein comprising of preparing an aqueous solution of said drugs or drugs along with natural polymer alginate in Distilled Water/Normal Saline/Phosphate Buffer Saline, (DW/NS/PBS), and 0.2:l-10v/v of an organic solvent such as methanol is added to enhance the solubility of hydrophobic drugs, characterized by natural olymer; mixing . calcium chloride with drug/alginate solution in 18:1-22:1 v/v ratio, with a molarity of calcium chloride maintained between 15-20M; mixing chitosan solution with drug/alginate solution of step (ii) in 8:1-12:1 v/v ratio with an alginate/chitosan ratio maintained between 4:1-7:1 w/w; allowing the nanoparticles to mature for 1-18 h recovering the nanoparticles by centrifugation at 19,000-37,000 rpm, at 4°C-30°C for 30 min-45 min.

Title of Invention

"A PROCESS FOR THE PREPARATION OF ALGINATE NANOPARTICLES"

Abstract

A process for the preparation of alginate nanoparticles having one or more drugs comprising singularly or combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated therein comprising of preparing an aqueous solution of said drugs or drugs along with natural polymer alginate in Distilled Water/Normal Saline/Phosphate Buffer Saline, (DW/NS/PBS), and 0.2:l-10v/v of an organic solvent such as methanol is added to enhance the solubility of hydrophobic drugs, characterized by natural olymer; mixing . calcium chloride with drug/alginate solution in 18:1-22:1 v/v ratio, with a molarity of calcium chloride maintained between 15-20M; mixing chitosan solution with drug/alginate solution of step (ii) in 8:1-12:1 v/v ratio with an alginate/chitosan ratio maintained between 4:1-7:1 w/w; allowing the nanoparticles to mature for 1-18 h recovering the nanoparticles by centrifugation at 19,000-37,000 rpm, at 4°C-30°C for 30 min-45 min.

Full Text	Field of Invention This invention relates to natural polymer nanoparticles having one or more tuberculosis drugs encapsulated therein, which can be administered orally, and to a process for the preparation thereof. In particular this invention relates to sodium alginate nanoparticle having one or more of drugs encapsulated therein. By way of example, and without intending to imply any limitations, the encapsulated drugs may be one or more antitubercular drugs (AID). Background of Invention The need to administer multiple ATD daily for 6-9 months is responsible for patient non-compliance as well as drug related hepatotoxicity, which result in therapeutic failure. Another consequence of incomplete/irregular treatment is the emergence of drug resistance. Controlled release drug delivery systems employing biodegradable polymers have been extensively studied over the past decade. Of the several procedures available such as double-emulsion solvent evaporation, microemulsion, gelification of anionic polysaccharides etc., none is perfect in terms of particle size, drug encapsulation efficiency and drug release kinetics. Further, mutidrug encapsulation in single formulation is not yet reported. Encapsulation of ATD in nanoparticles in synthetic polymers is known in the art. Such an encapsulation has the advantages of a better delivery system, a better uptake of drug and a better drug encapsulation efficiency. Such a synthetic polymer normally comprises a polymer of polylactide-co-glycolide, which is expensive and contributes to a higher end cost of the drug. Objects of the invention An object of this invention is to propose nanoparticles of a natural polymer having one or more of drugs encapsulated therein and to a process for the preparation thereof. Another object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof and wherein the drugs are ATD drugs selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated and obviates the disadvantages associated with the known art. Still another object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) which provides a prolonged and sustained release of said drugs. Yet another object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said plurality of drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) and capable of being modulated to entrap maximum drug. A further object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) and capable of distributing the drug(s) evenly to different organs where tubercle bacteria reside. A still further object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated which is ready for oral use without requiring any further treatment. Yet a further object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated which does not exhibit hepatotoxicity. BRIEF DESCRIPTION OF THE INVENTION According to this invention, there is provided a process for the preparation of alginate nanoparticles having one or more drugs comprising singularly or combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated therein comprising: i) preparing an aqueous solution of said drugs or drugs along with natural polymer alginate in Distilled Water/Normal Saline/Phosphate Buffer Saline, (DW/NS/PBS), and 0.2:l-10v/v of an organic solvent such as methanol is added to enhance the solubility of hydrophobic drugs, characterized by natural olymer; ii) mixing calcium chloride with drug/alginate solution in 18:1-22:1 v/v ratio, with a molarity of calcium chloride maintained between 15-20M; iii) mixing chitosan solution with drug/alginate solution of step (ii) in 8:1-12:1 v/v ratio with an alginate/chitosan ratio maintained between 4:1-7:1 w/w; iv) allowing the nanoparticles to mature for 1-18 h recovering the nanoparticles by centrifugation at 19,000-37,000 rpm, at 4°C-30°C for 30 min-45 min. Further, according to this invention there is provided alginate nanoparticles having a plurality of drugs encapsulated threin. In accordance with this invention, an aqueous solution of drug or drugs and alginate is prepared in DW/NS/PBS with an alginate: drug ratio of 1:6-1:8 w/w and a final alginate concentration of 0.05-0.07% w/v. Methanol may be added (if one of the drug is hydrophobic) in a ratio of 0.2:1-10 v/v. The mixture can comprise an excess amount of drug solution or sodium alginate solution, but which would only constitute a waste. To the mixture is added an aqueous solution of calcium chloride (drug-alginate solution: calcium chloride solution-18:1-22:1 v/v) with a molarity of calcium chloride maintained between 15-20mM. To the mixture is further added an aqueous solution of chitosan (drug-alginate solution: chitosan solution=8:1-12:1 v/v) with an alginatexhitosan ratio maintained preferably, but not limited to a ratio between 4:1-7:1 w/w. The nanoparticles formed are left as such for l-18h and subsequently collected by centrifugation at 19,000-37,000 rpm, at 4°C-30°C for 30 min-45 min. A process for the preparation of alginate nanoparticles having ATD encapsulated is explained by the following example: 22.5mg INH, 22.5mg PZA, 22.5mg RIF and 22.5mg EMB were put in 20 ml DW (with 0.2 ml methanol) containing 12 mg alginate (i.e. alginate concentration = 0.06% w/v). 1 ml of 18mM calcium chloride is added to the above mixture followed by the addition of 2ml of chitosan solution (containing 2mg chitosan). The final mixture is kept at such for 12 h followed by centrifugation at 19,000 rpm, at 4°C for 30 min to collect the nanoparticles. Oral administration of the nanoparticles encapsulating 4 ATD was found to produce sustained drug levels in the plasma (RIF-9 days, EMB-7 days, INH/PZA-14 days) and in the tissues (lungs, liver and spleen) for 15 days, thereby suggesting a fortnightly therapeutic regimen. The nanopartciles were administered orally to tuberculosis-infected guinea pigs and the results are given hi Table 1. Table 1-Colony forming units (cfu) of M.tuberculosis in lungs of guinea pigs after drug treatment. (Table Removed) Determination of drug content in alginate nanoparticles- The drug encapsulation efficiency was as under: RIF - 80-90% INH/PZA - 70-90% EMB - 88-95% The alginate nanoparticles did not produce any hepatotoxicity as assessed by plasma bilirubin, alanine transaminase and alkaline phosphatase. WE CLAIM: 1. A process for the preparation of alginate nanoparticles having one or more drugs comprising singularly or combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated therein comprising- i) preparing an aqueous solution of said drugs or drugs along with natural polymer alginate in Distilled Water/ Normal Saline/Phosphate Buffer Saline, (DW/NS/PBS), and 0.2:l-10v/v of an organic solvent such as methanol is added to enhance the solubility of hydrophobic drugs, characterized by natural olymer; ii) mixing calcium chloride with drug/alginate solution in 18:1-22:1 v/v ratio, with a molarity of calcium chloride maintained between 15-20M; iii) 'mixing chitosan solution with drug/alginate solution of step (ii) in 8:1-12:1 v/v ratio with an alginate/chitosan ratio maintained between 4:1-7:1 w/w; iv) allowing the nanoparticles to mature for 1-18 h recovering the nanoparticles by centrifugation at 19,000-37,000 rpm, at 4°C-30°C for 30 min-45 min. 2. A process as claimed in claim 1, wherein aqueous solution of said alginate is 0.05-0.07% w/v and mixed with the drugs preferably at a ratio of 1:6-1:8 w/w (alginate:drug)

Full Text

Field of Invention
This invention relates to natural polymer nanoparticles having one or more tuberculosis
drugs encapsulated therein, which can be administered orally, and to a process for the
preparation thereof. In particular this invention relates to sodium alginate nanoparticle
having one or more of drugs encapsulated therein. By way of example, and without
intending to
imply any limitations, the encapsulated drugs may be one or more antitubercular drugs
(AID).
Background of Invention
The need to administer multiple ATD daily for 6-9 months is responsible for patient non-compliance as well as drug related hepatotoxicity, which result in therapeutic failure. Another consequence of incomplete/irregular treatment is the emergence of drug resistance.
Controlled release drug delivery systems employing biodegradable polymers have been extensively studied over the past decade. Of the several procedures available such as double-emulsion solvent evaporation, microemulsion, gelification of anionic polysaccharides etc., none is perfect in terms of particle size, drug encapsulation efficiency and drug release kinetics. Further, mutidrug encapsulation in single formulation is not yet reported.
Encapsulation of ATD in nanoparticles in synthetic polymers is known in the art. Such an encapsulation has the advantages of a better delivery system, a better uptake of drug and a better drug encapsulation efficiency. Such a synthetic polymer normally comprises a polymer of polylactide-co-glycolide, which is expensive and contributes to a higher end cost of the drug.
Objects of the invention
An object of this invention is to propose nanoparticles of a natural polymer having one or more of drugs encapsulated therein and to a process for the preparation thereof.
Another object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof and wherein the drugs are ATD drugs selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated and obviates the disadvantages associated with the known art.
Still another object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) which provides a prolonged and sustained release of said drugs.
Yet another object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said plurality of drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) and capable of being modulated to entrap maximum drug.
A further object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) and capable of distributing the drug(s) evenly to different organs where tubercle bacteria reside.
A still further object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated which is ready for oral use without requiring any further treatment.
Yet a further object of this invention is to propose alginate nanoparticles having one or more of drugs encapsulated therein and a process for the preparation thereof, said drugs being selected from rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated which does not exhibit hepatotoxicity.
BRIEF DESCRIPTION OF THE INVENTION
According to this invention, there is provided a process for the preparation of alginate nanoparticles having one or more drugs comprising singularly or combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) encapsulated therein comprising:
i) preparing an aqueous solution of said drugs or drugs along with natural polymer alginate in Distilled Water/Normal Saline/Phosphate Buffer Saline, (DW/NS/PBS), and 0.2:l-10v/v of an organic solvent such as methanol is added to enhance the solubility of hydrophobic drugs, characterized by natural olymer;
ii) mixing calcium chloride with drug/alginate solution in 18:1-22:1 v/v ratio, with a molarity of calcium chloride maintained between 15-20M;
iii) mixing chitosan solution with drug/alginate solution of step (ii) in 8:1-12:1 v/v ratio with an alginate/chitosan ratio maintained between 4:1-7:1
w/w;
iv) allowing the nanoparticles to mature for 1-18 h recovering the nanoparticles by centrifugation at 19,000-37,000 rpm, at 4°C-30°C for 30 min-45 min.
Further, according to this invention there is provided alginate nanoparticles having a plurality of drugs encapsulated threin.
In accordance with this invention, an aqueous solution of drug or drugs and alginate is prepared in DW/NS/PBS with an alginate: drug ratio of 1:6-1:8 w/w and a final alginate concentration of 0.05-0.07% w/v. Methanol may be added (if one of the drug is hydrophobic) in a ratio of 0.2:1-10 v/v. The mixture can comprise an excess amount of drug solution or sodium alginate solution, but which would only constitute a waste. To the mixture is added an aqueous solution of calcium chloride (drug-alginate solution: calcium chloride solution-18:1-22:1 v/v) with a molarity of calcium chloride maintained between 15-20mM. To the mixture is further added an aqueous solution of chitosan (drug-alginate solution: chitosan solution=8:1-12:1 v/v) with an alginatexhitosan ratio maintained preferably, but not limited to a ratio between 4:1-7:1 w/w. The nanoparticles formed are left as such for l-18h and subsequently collected by centrifugation at 19,000-37,000 rpm, at 4°C-30°C for 30 min-45 min.
A process for the preparation of alginate nanoparticles having ATD encapsulated is explained by the following example:
22.5mg INH, 22.5mg PZA, 22.5mg RIF and 22.5mg EMB were put in 20 ml DW (with 0.2 ml methanol) containing 12 mg alginate (i.e. alginate concentration = 0.06% w/v). 1 ml of 18mM calcium chloride is added to the above mixture followed by the addition of 2ml of chitosan solution (containing 2mg chitosan). The final mixture is kept at such for 12 h followed by centrifugation at 19,000 rpm, at 4°C for 30 min to collect the nanoparticles.
Oral administration of the nanoparticles encapsulating 4 ATD was found to produce sustained drug levels in the plasma (RIF-9 days, EMB-7 days, INH/PZA-14 days) and in the tissues (lungs, liver and spleen) for 15 days, thereby suggesting a fortnightly therapeutic regimen. The nanopartciles were administered orally to tuberculosis-infected guinea pigs and the results are given hi Table 1.
Table 1-Colony forming units (cfu) of M.tuberculosis in lungs of guinea pigs after drug treatment.
(Table Removed)
Determination of drug content in alginate nanoparticles-
The drug encapsulation efficiency was as under:
RIF - 80-90%
INH/PZA - 70-90%
EMB - 88-95%
The alginate nanoparticles did not produce any hepatotoxicity as assessed by plasma bilirubin, alanine transaminase and alkaline phosphatase.

WE CLAIM:
1. A process for the preparation of alginate nanoparticles having one or
more drugs comprising singularly or combination of rifampicin (RIF),
isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB)
encapsulated therein comprising-
i) preparing an aqueous solution of said drugs or drugs along with natural polymer alginate in Distilled Water/ Normal Saline/Phosphate Buffer Saline, (DW/NS/PBS), and 0.2:l-10v/v of an organic solvent such as methanol is added to enhance the solubility of hydrophobic drugs, characterized by natural olymer;
ii) mixing calcium chloride with drug/alginate solution in 18:1-22:1 v/v ratio, with a molarity of calcium chloride maintained between 15-20M;
iii) 'mixing chitosan solution with drug/alginate solution of step (ii) in 8:1-12:1 v/v ratio with an alginate/chitosan ratio maintained between 4:1-7:1 w/w;
iv) allowing the nanoparticles to mature for 1-18 h recovering the nanoparticles by centrifugation at 19,000-37,000 rpm, at 4°C-30°C for 30 min-45 min.
2. A process as claimed in claim 1, wherein aqueous solution of said alginate is 0.05-0.07% w/v and mixed with the drugs preferably at a ratio of 1:6-1:8 w/w (alginate:drug)

Documents:

2666-DEL-2005-Abstract-(13-01-2012).pdf

2666-DEL-2005-Abstract-(13-06-2012).pdf

2666-del-2005-Abstract-(18-10-2012).pdf

2666-del-2005-abstract.pdf

2666-DEL-2005-Claims-(13-01-2012).pdf

2666-DEL-2005-Claims-(13-06-2012).pdf

2666-del-2005-Claims-(18-10-2012).pdf

2666-del-2005-claims.pdf

2666-DEL-2005-Correspondence Others-(13-01-2012).pdf

2666-DEL-2005-Correspondence Others-(13-06-2012).pdf

2666-del-2005-Correspondence-Others-(18-10-2012).pdf

2666-del-2005-correspondence-others.pdf

2666-del-2005-correspondence-po.pdf

2666-DEL-2005-Description (Complete)-(13-06-2012).pdf

2666-del-2005-description (complete).pdf

2666-DEL-2005-Form-1-(13-01-2012).pdf

2666-del-2005-form-1.pdf

2666-del-2005-Form-13-(18-03-2009).pdf

2666-del-2005-form-18.pdf

2666-del-2005-form-2.pdf

2666-del-2005-form-26.pdf

2666-DEL-2005-GPA-(13-01-2012).pdf

2666-del-2005-Joint Affidavit.pdf

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Patent Number

256134

Indian Patent Application Number

2666/DEL/2005

PG Journal Number

19/2013

Publication Date

10-May-2013

Grant Date

07-May-2013

Date of Filing

05-Oct-2005

Name of Patentee

LIFECARE INNOVATIONS PVT. LTD.

Applicant Address

B-589, SUSHANT LOK, PHASE, 1, GURGAON-122 002, HARYANA, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	PROF. G.K. KHULLER	DEPARTMENT OF BIOTECHNOLOGY, POST GRADUATE INSTITUTE OF MEDICAL EDUCATION AND RESEARCH, CHANDIGARH-160 012, INDIA.
2	ZAHOOR AHMED	DEPARTMENT OF BIOTECHNOLOGY, POST GRADUATE INSTITUTE OF MEDICAL EDUCATION AND RESEARCH, CHANDIGARH-160 012, INDIA.
3	RAJESH PANDEY	DEPARTMENT OF BIOTECHNOLOGY, POST GRADUATE INSTITUTE OF MEDICAL EDUCATION AND RESEARCH, CHANDIGARH-160 012, INDIA.
4	SADHNA SHARMA	DEPARTMENT OF BIOTECHNOLOGY, POST GRADUATE INSTITUTE OF MEDICAL EDUCATION AND RESEARCH, CHANDIGARH-160 012, INDIA.

PCT International Classification Number

A61K 9/20

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA