WO2016013031A1

WO2016013031A1 - Liposome composition and method of preparing the liposome

Info

Publication number: WO2016013031A1
Application number: PCT/IN2015/050073
Authority: WO
Inventors: Subhas Bhowmick; Manish Kumar UMRETHIA; Kuntal MAITI; Jayesh Kumar HADIA; Nitesh Kumar PATEL
Original assignee: Sun Pharma Advanced Research Company Ltd.
Priority date: 2014-07-25
Filing date: 2015-07-23
Publication date: 2016-01-28

Abstract

The present invention relates to a method of loading an ionizable drug having a group selected from (Formule I or Formule II) or pharmaceutically acceptable salts thereof into a liposome and the liposomal composition of drug produced therewith.

Description

LIPOSOME COMPOSITION AND METHOD OF PREPARING THE LIPOSOME

FIELD OF THE INVENTION

The present invention relates to a method of loading an ionizable drug having a group selected fro

or pharmaceutically acceptable salts thereof into a liposome and the long circulating liposomal composition of drug produced therewith. BACKGROUND OF THE INVENTION

Liposomes have been widely studied and used as carriers for a variety of agents such as drugs, nutrients, diagnostic reagents, and genetic material. Liposomes are artificially prepared spherical vesicle composed of a lamellar phase lipid bilayer. Liposomes or lipid vesicles are often composed of phosphatidylcholine-enriched phospholipids and may also contain mixed lipid chains with surfactant properties such as egg phosphatidyl ethanolamine. Since liposomes consist of non-toxic lipids, they generally have low toxicity and therefore are useful in a variety of pharmaceutical applications. Liposomes are a recognized drug delivery system which can improve the therapeutic activity and increase the safety of a number of different pharmaceutical agents. Liposome-encapsulated drugs often have biodistributions and toxicities which differ greatly from those of free drug. The use of liposomes increases the therapeutic index of many drugs, and also offers drug targeting and controlled release. To be useful in medical treatments, liposome formulations should have an efficient drug to lipid ratio, i.e. a higher percentage of drug entrapment, a practical shelf-life and be capable of reproducible preparation. General techniques of preparing drug loaded liposomes has been extensively studied in the past. The liposome formulations for pharmaceutical applications can be made either by combining drug and lipid before formation of the vesicles (passive loading technique), or by "loading^" drug into lipid vesicles after they are formed (active loading technique). There are various methods by which liposomal vesicles can be prepared, such as mechanical dispersion methods including lipid film hydration, sizing using sonication, extrusion; solvent evaporation methods such as ethanol injection, ether injection, reverse phase evaporation; detergent removal methods. For drug encapsulation, there is a need to increase the entrapment efficiency such that the drug to lipid ratio is as high as possible, while maintaining the original chemical integrity of both drug and lipid. Once administration to a patient has occurred, drug release is a factor. Rapid release of pharmaceuticals from liposomes reduce the bio-distribution benefits sought in utilizing lipid vesicle carriers. Accordingly, efforts to optimize pharmaceutical loading and to reduce the rate of release of pharmaceuticals from lipid vesicles and make them long circulating in plasma have continued. Further, the liposome formulations should be stable such that they remain intact during shipping and upon storage for the shelf life. At present, there is no single universal encapsulation method that offers stable encapsulation of most drugs; each drug requires a different approach to manage all of its properties.

The prior art does not disclose methods of loading an ionizable drug having a group selected from

such as losartan, candesartan or pharmaceutically acceptable salt thereof into liposomes that have high percentage of drug loading and long blood circulating nature when administered parenterally. While attempting to make a liposome composition of these ionizable drugs such as losartan or candesartan or pharmaceutically acceptable salts thereof, the inventors faced the problem of poor drug loading and /or short circulating liposome. Particularly, the inventors found that the salts of the transition metal ions of strong acid provided liposomes that showed very poor loading. Surprisingly, and unexpectedly, however, when salts of the transition metal ions of weak acid were employed, the method provided liposomes with very good, efficient loading of the drug. Not only the drug loading was satisfactory, the so prepared liposomes were long circulating in the plasma. This was indeed surprising. SUMMARY OF THE INVENTION

The present invention provides a method of loading an ionizable drug having a group selected from

or pharmaceutically acceptable salts thereof into a liposome, said method comprising the steps of:

a) providing preformed liposomes containing an internal solution comprising a weak acid salt of a transition metal;

b) adding the ionizable drug to the external solution, and

c) incubating the external solution of step 'b' with the preformed liposomes of step

'a' for a sufficient time to load the drug into the liposomes.

The present invention further provides a liposomal composition prepared by the method described above.

BRIEF DESCRIPTION OF FIGURES

Figure 1: represents a graph showing the plasma concentration versus time data of losartan after intravenous administration of reference solution; liposomal composition of example 1 and liposomal composition of comparative example A according to study provided in example 5.

DETAILED DESCRIPTION OF THE INVENTION

The phrase 'percentage drug loading' and 'percentage drug entrapment' has been used interchangeably in the specification. It refers to the percentage by weight of the total drug which is loaded or entrapped inside the liposome vesicle.

The term 'long circulating' as used herein means that the liposomes escape the reticuloendothelial system (RES) which is responsible for removal of foreign particles from the bloodstream and thus circulates in bloodstream for prolonged period. Long circulating liposomes entrap the drug and increase the mean residence time of the drug in the body as compared to mean residence time from a non-liposomal solution composition. Mean residence time is an average time a drug molecule spends in the body. The liposome composition of the present invention increases the mean residence time to more than 6 hours, preferably more than 10 hours.

The phrase 'internal solution" and the "external solution" as used herein are related to the intermediate steps while preparing the drug loaded liposomes. The method of the present invention is based on active drug loading into the preformed liposomes. The preformed liposomes before loading the drug into it contain an internal solution which is aqueous solution of weak acid salt of a transition metal ion. The phrase "external solution' refers to a medium in which an ionizable drug such as losartan or its pharmaceutically acceptable salt is added, and this external solution is incubated with the preformed liposome for loading the ionizable drug. Suitably, the external solution may comprise one or more of a sucrose phosphate buffer, sucrose histidine buffer, HEPES buffer, sucrose phosphate EDTA buffer, acetate buffer, citrate buffer, sucrose HEPES buffer, HEPES EDTA buffer, HEPES saline, histidine and sucrose.

or pharmaceutically acceptable salts thereof into a liposome, said method comprising steps of:

b) adding the ionizable drug or its pharmaceutically acceptable salt to the external solution, and

c) incubating the external solution of step 'b' with the preformed liposomes of step 'a' for a sufficient time to load the drug into the liposomes.

The present invention further provides a long circulating liposomal composition produced by the method of the invention described above. The liposomal composition comprises the ionizable drug or its pharmaceutically acceptable salt; a weak acid salt of a transition metal cation and vesicle forming lipids selected from one or more phospholipids, one or more steroidal lipid, one or more polymer derivatised lipid or mixtures thereof.

Suitably, a drug suitable for the method of the present invention is an ionizable drug having a group selected from

or pharmaceutically acceptable salts thereof. Suitably, these ionizable drugs are weakly acidic in nature. The pharmaceutically acceptable salts include, any suitable salt that is pharmaceutically acceptable. Suitable pharmaceutically acceptable salts include, but are not limited to alkaline metal salts like sodium, potassium etc., alkaline earth metal salts like magnesium, calcium etc., medoxomil, cilexetil, mesylate, hydrochloride, hydrobromide, etc.

Preferably, the drugs having the group

include, but are not limited to, candesartan, losartan, irbesartan, valsartan, olmesartan, EXP-3174 or their active metabolites, esters or pharmaceutically acceptable salts thereof.

H ^N Preferably, the drugs having the group H include, but are not limited to azilsartan or their active metabolites, esters or pharmaceutically acceptable salts thereof. For example the drug is azilsartan medoxomil potassium.

In preferred embodiments, the method of the present invention comprises an ionizable drug selected from losartan, candesartan, EXP-3174, olmesartan or their esters or pharmaceutically acceptable salts thereof, such as candesartan cilexetil, losartan potassium, olmesartan medoxomil. The ionizable drug is present in the liposomal composition in therapeutically active amounts, suitable to achieve the desired therapeutic effect. In one specific embodiment, the ionizable drug or active agent is losartan or its pharmaceutically acceptable salts, having the following structure:

In one embodiment, the ionizable drug or active agent is candesartan or its pharmaceutically acceptable salts, having the following structure:

In one embodiment, the ionizable drug is EXP-3174, which is an active metabolite of losartan and have the following structure:

In another embodiment, the ionizable drug or active agent is irbesartan or its pharmaceutically acceptable salts, having the followin structure:

In another embodiment, the ionizable drug or active agent is valsartan or its pharmaceutically acceptable salts, having the following structure:

In another embodiment, the ionizable drug or active agent is olmesartan or its pharmaceutically acceptable salts, having the following structure:

In specific embodiment, the ionizable drug as above is an Angiotensin II receptor blocker. Angiotensin II receptor blocker and other RAAS inhibitors are anti-hypertensives and are also known to reduce the levels of collagen I and III and basement membrane collagen IV in various experimental models of fibrosis and reverse renal and cardiac fibrosis in hypertensive patients. Further, it has been shown to inhibit collagen I production in tumors. The liposomal composition prepared by the method of the present invention are suitable for use as a collagen modifying agent to improve the distribution and/or penetration of chemotherapeutics into solid tumours. In one aspect, the liposomal composition produced by the method of the present invention, are useful in treatment of cancers/tumours such as breast, pancreatic, colorectal, pancreatic, colon, lung, skin, ovarian, cervix, prostate, gastric, gastrointestinal, stomach, head and neck, kidney, liver cancer or metastatic lesions of these cancers. In one aspect, the liposomal composition produced by the method of the present invention, when administered in combination with other anticancer agents, provides an improved method of treating cancers or tumors, such as solid tumors. In one specific embodiment, the ionizable drug is losartan or its pharmaceutically acceptable salt, an angiotensin II receptor blocker. Suitable salts include losartan potassium or losartan sodium. Any other salt may be used. As used herein in some embodiments, the term ' losartan' is intended to include losartan or its pharmaceutically acceptable salts. A preferred salt is losartan potassium. Losartan or its pharmaceutically acceptable salt may be present in the liposomal composition prepared according to the method of the present invention, in an amount ranging from about 0.0001 %w/v to about 10.0 %w/v, preferably from about 0.1 %w/v to about 1.0 % w/v.

The weak acid salt of a transition metal according to the present invention is a salt of a transition metal cation with a weak acid anion. The transition metal cation suitable for use in the present invention may be selected from a monovalent, divalent, trivalent or a tetravalent cation, preferably a divalent or trivalent cation, more preferably a divalent cation. In preferred embodiments, the transition metal/cation is selected from but not limited to copper (monovalent Cu⁺ or divalent Cu⁺²), nickel (Ni⁺²), iron (Fe⁺² or Fe⁺³), cobalt (Co⁺² or Co⁺³), zinc (Zn⁺²), manganese (Mn⁺²), strontium (Sr⁺²), molybdenam (Mo) and the like. The weak acid anion forming a salt with transition metal according to the present invention include but is not limited to acetate, glutamate, gluconate, tartrate, formate, citrate or glycinate. Accordingly, non-limiting examples of the weak acid that can form a salt with transition metal includes acetic acid, gluconic acid, tartaric acid, glutamic acid, citric acid, formic acid, or glycinic acid.

According to preferred embodiments, the weak acid salt of a transition metal include, but are not limited to, copper acetate, copper gluconate, nickel acetate and nickel gluconate. The weak acid salt of transition metal used in preparing liposomes of the present invention is used in the internal solution at a concentration ranging from about 100 mM to about 600 mM, preferably at a concentration ranging from about 150 mM to about 300 mM. In specific embodiments, the liposome composition formed by the method of the present invention contains copper acetate in an amount ranging from about 0.0001 % to about 5.0 % weight by volume of the liposomal composition.

Suitably, the liposomal composition prepared according to the method of the present invention is composed of one or more vesicle forming lipid, selected from di-aliphatic chain lipid, such as phospholipids; diglycerides; di-aliphatic glycolipids; single lipids such as sphingomyelin or glycosphingolipid; steroidal lipids; hydrophilic polymer derivatised lipids, or mixtures thereof. Preferably, the vesicle forming lipid comprises one or more phospholipids, one or more steroidal lipids, and one or more hydrophilic polymer derivatised lipids.

The one or more phospholipids that may be used in the liposomal composition prepared according to method of the present invention comprises phospholipids that form bilayer vesicular structure. The phospholipids that may be used include, but are not limited to, phospholipid such as phosphatidyl choline (PC); phosphatidyl ethanolamine(PE); phosphatidyl serine(PS), phosphatidylglycerol(PG), phosphatidylionositol (PI), sphingomyelin, phosphatidic acid (PA), lecithin; phosphatidylcholine lipid derivatives such as dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (EPC), hydrogenated egg phosphatidylcholine (HEPC), partially hydrogenated egg phosphatidylcholine (PHEPC), distearylphosphatidyl choline (DSPC), dipalmitoyl phosphatidyl choline (DPPC), soy phosphatidyl choline (SPC), hydrogenated soy phosphatidyl choline (HSPC), diarachidoyl phosphatidyl choline, dimyristoyl phosphatidyl ethanolamine (DMPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), distearoyl phosphatidyl ethanolamine (DSPE), diarachidoyl phosphatidyl ethanolamine (DAPE) and dipalmitoyl phosphatidyl glycerol (DPPG) and the like. These phospholipids may be fully saturated or partially hydrogenated. They may be naturally occurring or synthetic. The preferred phospholipid is hydrogenated soy phosphatidyl choline (HSPC). The steroidal lipids that may be used in the liposomal composition prepared according to method of the present invention include, but not limited to cholesterol or its derivatives such as cholesteryl sulfate and its salts (CS), cholesteryl hemisuccinate and its salts, cholesterol phosphate and its salts, cholesteryl phosphocholine and other hydroxycholesterol or amino cholesterol derivatives, cholesteryl succinate, cholesteryl oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG); and coprostanol, cholestanol, cholestane, cholic acid, Cortisol, corticosterone, hydrocortisone, calciferol and the like. Preferably the steroidal lipid is cholesterol. In one embodiment, cholesterol is used in an amount such that the liposomes have greater than 20 mole percent cholesterol. Preferably, greater than 30 mole percent cholesterol is used, more preferably the cholesterol amount ranges from about 40 mole percent to 60 mole percent. According to one embodiment, the liposome prepared according to the method of the present invention is a polymerized liposome. Such liposomes can be prepared by incorporating one or more lipids that are derivatized by a polymer, along with one or more phospholipids, and optionally, one or more steroids. The examples of the polymers that are used to derivatize the lipids include, but are not limited to, polyethylene glycol (PEG); Poly acrylamide (PAA), which is formed from the acryl amide subunits i.e. readily formed from polymerized acryl amide; Poly (2-alkyl-2-oxozoline) such as Poly(2-methyl-2-oxozoline); Poly (amino) acids such as synthetic poly( amino acid)s for example, poly(glutamic acid) (PGA); Poly (glycerol) such as hyperbranched PG (HPG) with molar masses ranging from 150 Da to 540; poly (vinylpyrrohdone) and the like. In one preferred embodiment, the hydrophilic polymer e.g., polyethylene glycol (PEG) having a molecular weight between 1-10 Kdaltons, preferably 2-5 Kdaltons (PEG-derivatized phospholipids) may be used.

Preferably, the polymer derivatised lipid that may be used includes a hydrophilic polymer derivatised lipid. Non-limiting examples of hydrophilic polymer derivatised lipid, include, polyethylene glycol conjugated phosphatidyl ethanolamine, (PEG-PE), methoxy polyethylene glycol conjugated hydrogenated soy phosphatidylcholine (mPEG-HSPC), methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE). Other hydrophilic polymers which may be suitable includes polylactic acid, polyglycolic acid, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose. In preferred embodiments, the hydrophilic polymer derivatised lipid is methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE). In one specific embodiment, the polymerized lipid may be used in an amount ranging from 0.01 % to 5.0 % weight by volume, preferably, about 0.1 % to 1.0 % weight by volume.

The one or more vesicle forming lipid, that are used in the liposome prepared according to the method of the present invention, is present in an amount such that the molar ratio of ionizable drug to vesicle forming lipids is in the range of about 0.0001: 1 to about 0.5: 1, preferably from about 0.01 : 1 to about 0.5: 1. In one embodiment, the liposomal composition prepared according to the method of the present invention comprises one or more phospholipids, one or more steroidal lipids and one or more polymer derivatised lipids. The lipids are present in amounts such that the molar ratio of phospholipids: steroidal lipid: polymer derivatised lipid ranges from about 30-75 : 21-60 : 1- 15preferably from about 45-55 : 40-50 : 2-7. In specific embodiment, the liposomal composition prepared according to the method of the present invention comprises one or more phospholipids, one or more steroidal lipids and one or more polymer derivatised lipids, wherein the molar ratio of phospholipids: steroidal lipid: polymer derivatised lipid is about 50 : 45 : 5.

In some specific embodiments, the long circulating liposomal composition prepared according to the method of the present invention comprises an ionizable drug losartan or candesartan or EXP- 3174 or their pharmaceutically acceptable salts, a weak acid salt of a transition metal cation and vesicle forming lipids comprising one or more phospholipids, one or more steroidal lipid and one or more polymer derivatised lipid. Preferably, the lipids are present in amounts such that the molar ratio of phospholipids: steroidal lipid: polymer derivatised lipid ranges from about 30-75 : 21-60 : 1-15. In one embodiment, the liposomes prepared by the method of the present invention are unilamellar liposomes. The vesicles have a mean particle size ranging from about 50 nm to about 200 nm, preferably from about 60 nm to about 120 nm. In one specific embodiment, the liposomal composition prepared according to the method of the present invention are long circulating in blood and the drug has a mean residence time of greater than 8 hours

In one specific embodiment, the liposomal composition prepared according to the method of the present invention comprises losartan potassium, vesicle forming lipids including a mixture of hydrogenated soy phosphatidyl choline (HSPC); cholesterol and methoxy polyethylene glycol- distearoylphosphatidyl ethanolamine (mPEG-DSPE), and copper acetate which is used as a weak acid salt of the transition metal ion. Preferably, the lipids are present in amounts such that the molar ratio of HSPC : Cholesterol: mPEG-DSPE may vary from about 30-75 : 21-60 : 1-15. It was found by the inventors that in one embodiment wherein the vesicle forming lipid is a mixture of hydrogenated soy phosphatidyl choline (HSPC); cholesterol and methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-DSPE), the method of the present invention preferably requires use of higher mole percentage of cholesterol, preferably greater than 20 mole percentage to achieve higher drug loading. The inventors while attempting to encapsulate an ionizable drug such as those defined in the invention, into a liposomal vesicle, faced a problem of poor/non-efficient drug loading particularly when a strong acid salt of a transition metal such as sulphate, nitrate, phosphate salt of transition metal was used. For instance, when copper sulphate was used, the percentage drug loading was very less, for example, as low as 10 % or even lesser in some cases. In one specific embodiment, wherein copper sulphate was used as gradient to load losartan into liposomes, the percentage drug loading observed was only 7.36%. In another specific embodiment, wherein copper sulphate was used as gradient to load candesartan into liposomes, the percentage drug loading observed was only 54.31%. Surprisingly, however, when a weak acid salt of a transition metal, such as acetate, or gluconate salt of a transition metal, for instance copper acetate or copper gluconate or niclek acetate was used, inventors unexpectedly found that the drug loading could be achieved at levels greater than 80%, even greater than 90%. Further, it was surprisingly found that such liposomes were long circulating in nature. This observation of efficient drug loading and long circulating nature of liposome, in fact was indeed surprising and unexpected. This was all the more unexpected in view of the fact that inventor's prior attempts of preparing liposomes using alkaline earth metal ion salts such as calcium acetate, although resulted in acceptable drug loaded liposomes, these liposomes were not long circulating in nature. This was evidenced by low mean residence time of losartan in plasma (<2hours) upon parenteral injection of these liposomes (in-vivo pharmacokinetics study). In contrast, the liposomes prepared by using a weak acid salt of a transition metal cation, such as for example, weak acid salt of copper, viz. copper acetate, are long circulating in nature, which upon intravenous injection remain and circulates in plasma for a long period of time. This is evidenced by longer mean residence time of losartan in plasma which is more than 6 hours, preferably more than 10 hours, upon intravenous injection of the liposome prepared by the method according to the present invention.

In one preferred aspect, the pH of the internal solution is less than the pH of the external solution. In another preferred aspect, the pH of the internal solution is more than the pH of the external solution. The pH of the internal solution may range from about pH 3.0 to 9.0, preferably from about 5.0 to 9.0 while the pH of the external solution may range from about 5.0 to 9.0, preferably from about 6.0 to 7.0. In one aspect, the method of the present invention does not necessitate presence of a trans-membrane pH gradient or use of an ionophore for achieving drug loading.

According to one embodiment, the method of the present invention involves following steps of : preparing an aqueous solution of weak acid salt of transition metal ion, in water for injection. The weak acid salt of transition metal used in preparing liposomes of the present invention may be taken in amounts ranging from about 100 mM to about 600 mM, preferably in an amount ranging from about 150 mM to about 300 mM. Suitably, the weak acid salt of transition metal helps in active loading of the drug into the liposomes in higher amounts. The lipids are then dissolved in a suitable organic solvent such as ethanol or ether and injecting the lipidic solution into the aqueous solution of weak acid salt of transition metal, to form liposome suspension having multilamellar vesicles. The liposomal vesicles maybe prepared by alternative techniques such as lipid film formation and hydration. Subjecting the liposome suspension having multilamellar vesicles to particle size reduction by techniques known in the art (such as extrusion, homogenization etc,) so as to obtain preformed liposomes, which are mostly unilamellar having a mean particle size, preferably in the range of 50-150 nm. Exchanging the external solution, i.e. the extra-liposomal medium with a suitable solvent/buffer at optimum condition, by methods such as diafiltration or dialysis or ultrafiltration or tangential flow filtration, causing removal of transition metal cation present in the extra-liposomal environment. The resulting liposomal composition contained the weak acid salt of transition metal in internal solution/aqueous medium and solvent/buffer solution in external solution (extra-liposomal medium). Adding suitable buffer solution/pH stabilizer, so as to maintain desired pH in the external medium. Adding the ionizable drug or its pharmaceutically acceptable salt to the external solution or extra-liposomal medium and incubating the liposome containing internal solution with the external solution containing drug for sufficient period of time, usually ranging from about 5 minutes to 120 minutes or longer to effect loading of drug into the liposome. This is usually carried out at suitable temperature preferably about 25°C to 75°C. Sterilizing the liposomal composition by membrane filtration, using a 0.2 micron membrane filter.

In one embodiment, while preparing the pre-formed liposomes, the liposomes may be passively co-encapsulated with another active agent/drug along with the weak acid salt of the transition metal. Using this approach, two or more drugs may be incorporated into the liposome by combining passive and active methods of loading. The method of the present invention provides high percentage of drug loading or drug entrapment into the liposomes, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90%. In one preferred embodiment, the long circulating liposomal composition prepared by the method of the present invention encapsulates more than 80% by weight of the added drug into the liposomes. It is to be understood that various possible sub-steps and various possible processing conditions that gives the desired effect are within the scope of this invention.

The liposomal composition prepared by the method of the present invention is sterile and storage stable at 2-8°C for the shelf life and is thus suitable for parenteral administration.

Hereinafter, the invention will be more specifically described with reference to examples. The examples are not intended to limit the scope of the invention and are merely used as illustrations. EXAMPLE 1

The example represents one specific embodiment of the present invention that makes use of copper acetate as the weak acid salt of the transition metal.

The internal solution was prepared by dissolving copper acetate (200 mM) in water for injection, having a pH of 5.0-6.0. The lipids i.e. hydrogenated soy phosphatidyl choline (HSPC); cholesterol and Methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG- 2000-DSPE) at a molar ratio of 50:45:5, were dissolved in ethanol. The lipid solution was injected into the internal solution, to form preformed liposomes. The preformed liposomal suspension with multilamellar vesicles so formed was subjected to size reduction to form nano sized preformed liposomes. This was followed by diafiltration to remove the unentrapped copper ions from the external medium/solution by using Sucrose-EDTA-phosphate buffer, pH 7. Subsequently, the external solution was exchanged with sucrose-phosphate buffer, pH 7. To this external solution/medium, losartan potassium was added and incubated for about 10 minutes to 120 minutes to effect drug loading. The liposomal composition was sterilized by membrane filtration using a 0.2 micron membrane filter.

The percentage drug entrapment was determined and was found to be 98.02%. The particle size distribution of the liposomes was checked with Malvern Zetasizer and it was observed that D₁₀ is 63.5nm, D50 is 92.6nm (i.e. 50% of the particles have a mean particle size of 92.6 nanometer/nm/nms), and D90 is 136.0nm. The liposomal composition so prepared was subjected to storage stability study at 2-8°C and the composition was found to be physically and chemically stable for at least 4 months.

Following the same procedure as above, liposomes were prepared for other drugs viz. candesartan and EXP-3174, by use of appropriate buffering system. The examples provided acceptable drug loading. EXAMPLE 2

The example represents another specific embodiment of the present invention that makes use of copper gluconate as the weak acid salt of the transition metal.

The internal solution was prepared by dissolving copper gluconate (200 mM) in water for injection, having a pH of 7.0-9.0. The specified amounts of lipids i.e. hydrogenated soy phosphatidyl choline (HSPC); cholesterol and Methoxy polyethylene glycol - distearoylphosphatidyl ethanolamine (mPEG-2000-DSPE) in molar ratio (50:45:5) were dissolved in ethanol. The lipid solution was added into the copper gluconate solution, to form preformed liposomes. The liposomal suspension with multilamellar vesicles so formed was subjected to size reduction to form nano sized preformed liposomes. This was followed by diafiltration to remove the untrapped copper ions from the external medium using sucrose- EDTA-phosphate buffer (pH 7). Subsequently, the external solution was exchanged with sucrose/phosphate buffer pH 7. To this external solution/medium, ionizable drug losartan potassium was added and incubated for about 10 minutes to 120 minutes to effect drug loading. The liposomal composition was sterilized by membrane filtration using a 0.2 micron membrane filter.

The percentage entrapment of drug into the liposomes was found to be 90.02%. The particle size distribution of the liposomes was checked with Malvern Zetasizer and it was observed that Dio is 58.7 nm, D50 is 83.5 nm and D90 is 121nm. Following the same procedure as above, liposomes were prepared for other drugs viz. candesartan and EXP-3174, by use of appropriate buffering system. The examples provided acceptable drug loading.

EXAMPLE 3

The example represents another specific embodiment of the present invention that makes use of Nickel acetate as the weak acid salt of the transition metal.

The internal solution was prepared by dissolving nickel acetate (200 mM) in water for injection, having a pH of 5.0-7.0 The specified amounts of lipids i.e. hydrogenated soy phosphatidyl choline (HSPC); cholesterol and Methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine (mPEG-2000-DSPE) in molar ratio (50:45:5) were dissolved in ethanol. The lipid solution was added into the Nickel acetate solution, to form preformed liposomes. The liposomal suspension with multilamellar vesicles so formed was subjected to size reduction to form nano sized preformed liposomes. This was followed by diafiltration to remove the untrapped nickel ions from the external medium using sucrose-EDTA-phosphate buffer. Subsequently, external solution was exchanged with sucrose/phosphate buffer pH 6. To this external solution/medium, losartan potassium was added and incubated to effect drug loading. The liposomal composition was sterilized by membrane filtration using a 0.2 micron membrane filter. The percentage entrapment of losartan potassium into the liposomes was found to be 100 %. The particle size distribution of the liposomes was checked with Malvern Zetasizer and it was observed that Dio is 63.6 nm, D50 is 89.1 nm and D90 is 125nm.

Following the same procedure as above, liposomes were prepared for other drugs viz. candesartan and EXP-3174, by use of appropriate buffering system. The examples provided acceptable drug loading. EXAMPLE 4

The liposomal composition of the present invention prepared as per Example 1 and comparative example A were subjected to in vitro plasma stability study. The liposomal compositions were diluted to 1000 times in plasma and were incubated for 24 hours. The percentage of drug released from the liposomal compositions at different time points were analyzed and the observations are given below in Table 1 :

Table 1 :

In case of liposomal composition of Example 1 of the present invention, the analysis revealed that at any point of time within 7 hours, at least 74% of the drug remained entrapped inside the liposomes even at 1000 times dilution (2ppm) in plasma. In case of liposomal composition of comparative example A, the analysis revealed that after 7 hours, only 21 % of the drug remained 5 entrapped inside the liposomes at 1000 times dilution (2ppm) in plasma.

EXAMPLE 5

The composition of Example 1 and comparative example A were subjected to in-vivo plasma pharmacokinetic study in Sprague Dawley rats following single intravenous (IV) injection and pharmacokinetic parameters were evaluated. The pharmacokinetic parameters were studied and 10 were compared with conventional (non-liposomal) losartan injection solution. The conventional losartan potassium injectable solution was a solution of losartan potassium in saline (called hereinafter as Reference).

The composition of example 1 ; comparative example A and the reference were injected intravenously at the dose of 20mg/kg to separate groups of Sprague Dawley rats. Approximately 15 500μΕ of blood was collected at 0.083, 0.5, 1, 4, 8, 24 and 48 hours post dosing. Plasma was separated and was stored at about -70°C until analysis for the drug content. The pharmacokinetic parameters were determined from the concentration obtained after analysis using win Nonlin software version 5.1 by non- compartmental model.

Figure 1 represents the plasma concentration time data of losartan after IV administration of 20 liposomal composition of Example 1 ; liposomal composition of comparative example A and conventional losartan injectable solution (Reference). The table 2 below provides the values of pharmacokinetic parameters observed after IV administration of composition of Example 1; comparative example A and Reference.

Table 2: Pharmacokinetic parameters:

It was found that the liposomal composition of the present invention (Example 1 , wherein a weak acid salt of a transition metal was used) retained the drug Losartan within themselves for a longer period of time, leading to an increase in Mean Residence Time (MRT) in blood. This indicates the long circulating nature of the liposomal composition of the present invention. The Mean Residence Time (MRT) of losartan was found to be about 11 hours in case of the liposomal composition of the present invention. However, in case of conventional, non-liposomal losartan injection as well as in case of composition of comparative example A wherein a weak acid salt of other than transition metal (calcium acetate) was used, the Mean Residence Time (MRT) of losartan in plasma was found to be less than 2.0 hours.

COMPARATIVE EXAMPLE A

This example provides method of preparing liposomes and liposomal composition so prepared using weak acid salt of a alkaline earth metal such as calcium acetate:

The internal solution was prepared by dissolving calcium acetate (200 mM) in water for injection, having a pH of 5.0-7.0. Calcium acetate aqueous solution (200mM) having a pH of 7-8 was prepared using water for injection. The specified amounts of lipids i.e. hydrogenated soy phosphatidyl choline (HSPC); cholesterol and methoxy polyethylene glycol- distearoylphosphatidyl ethanolamine (mPEG-DSPE) in a molar ratio of 55:40:5 were dissolved in ethanol. The lipid solution so prepared was injected into calcium acetate solution, to form nano sized preformed liposomes. The liposomal suspension so formed was subjected to size reduction to form preformed liposomes. This was followed by diafiltration using sucrose solution to exchange the calcium metal ions present in the extra-liposomal external medium/solution. Aqueous solution of losartan potassium, was added and incubated to the above external solution to effect loading. The percentage entrapment of drug into the liposomes was determined and was found to be 87.23%. The particle size distribution of the liposomes was checked with Malvern Zetasizer and it was observed that D₁₀ is 72.4 nm, D50 is 104.0 nm and D90 is 151.0 nm.

Claims

Claims:

1. A method of loading an g having a group selected from

or pharmaceutically acceptable salts thereof into a liposome, said method comprising steps of: a. providing preformed liposomes containing an internal solution comprising a weak acid salt of a transition metal;

b. adding the ionizable drug to the external solution, and

c. incubating the external solution of step 'b' with the preformed liposomes of step 'a' for a sufficient time to load the drug into the liposomes.

2. The method as claimed in claim 1, wherein the liposome comprises one or more phospholipids, one or more steroidal lipids, and one or more hydrophilic polymer derivatised lipids.

3. The method as claimed in claim 1, wherein the drug having a group selected from

/ \

I is any one of candesartan, irbesartan, losartan, valsartan, olmesartan, EXP- 3174 or their active metabolites, esters or pharmaceutically acceptable salts thereof.

4. The method as claimed in claim 1, wherein the drug having a group selected from

is any one of azilsartan or their active metabolites, esters or pharmaceutically acceptable salts thereof.

5. The method as claimed in claim 1, wherein the transition metal is selected from copper, nickel, iron, cobalt, manganese or zinc and wherein the weak acid is selected from acetic, gluconic, tartaric, glutamic, citric, formic, or glycinic acid.

6. The method as claimed in claim 1, wherein the weak acid salt of transition metal is selected from copper acetate, copper gluconate, nickel acetate or nickel gluconate.

7. The method as claimed in claim 1, wherein the weak acid salt of transition metal is used in the internal solution at a concentration ranging from about 100 mM to about 600 mM.

5 8. The method as claimed in claim 1, wherein the ionizable drug and the vesicle forming lipids are taken in amounts such that the molar ratio of ionizable drug to lipids ranges from about 0.01 : 1 to about 0.5: 1.

9. The method as claimed in claim 1, wherein the pH of the internal solution ranges from about 5.0 to 9.0 and the pH of the external solution ranges from about 6.0 to 7.0.

10 10. A liposomal composition produced by the process of claim 2.

11. The liposomal composition as in claim 10, wherein the molar ratio of phospholipids: steroidal lipid: polymer derivatised lipid ranges from about 30-75 : 21-60 : 1-15.

12. The liposomal composition as in claim 11, wherein the molar ratio of phospholipids: steroidal lipid: polymer derivatised lipid ranges from about 45-55 : 40-50 : 2-7.

15 13. The liposomal composition as in claim 12, wherein the molar ratio of phospholipids: steroidal lipid: polymer derivatised lipid is about 50 : 45 : 5.

14. The liposomal composition as in claim 11, wherein the liposomal composition is long circulating in blood and the drug has a mean residence time of greater than 8 hours.

15. The liposomal composition as in claim 11, wherein the liposomes have a particle size ranging 20 from about 50 nm to 200 nm.

16. The liposomal composition as in claim 15, wherein the liposomes are unilamellar liposomes.

17. The liposomal composition of claim 10 for use as a collagen modifying agent to improve the distribution and/or penetration of chemotherapeutics into solid tumours.

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