WO2013043830A1

WO2013043830A1 - Nanoparticle formulations of poorly soluble compounds

Info

Publication number: WO2013043830A1
Application number: PCT/US2012/056271
Authority: WO
Inventors: Gary Fujii; Su-Ming Chiang; Runjhun SRIVASTAVA; Alison FUJII; David Needham
Original assignee: Molecular Express, Inc.
Priority date: 2011-09-20
Filing date: 2012-09-20
Publication date: 2013-03-28

Abstract

The present invention provides liposomal nanoparticle formulations which can deliver high concentrations of poorly soluble compounds in aqueous solutions.

Description

NANOPARTICLE FORMULATIONS OF POORLY SOLUBLE COMPOUNDS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims priority to United States provisional application no. 61/536,966, filed September 20, 2011, which is hereby incorporated in its entirety, including all tables, figures and claims.

BACKGROUND OF THE INVENTION

[0002] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

[0003] It is estimated that -40% of biologically active compounds identified through combinatorial screening programs are difficult to formulate as a result of their lack of significant solubility in water. The general approach to deal with issues regarding solubility is to generate various salts of a poorly water-soluble molecule so as to improve solubility while retaining biological activity; to identify analogs or prodrugs with enhanced solubility; or to formulate the compound in the presence of a non-aqueous solvent system. Frequently, these approaches are not successful, and the molecule is abandoned early on in its development process; or the product is launched with suboptimal properties including poor bioavailability and/or the presence of extra excipients that pose usage limitations or undesirable physical characteristics.

[0004] When these types of situations arise, a nanoparticle formulation approach has proven to be very useful and invaluable in all stages of the drug development and has opened opportunities for revitalizing marketed products with suboptimal delivery. The term "nanoparticle" as used herein refers to particulate structures having a mean diameter of less than 1 micron and which are dispersible in aqueous media. The pharmaceutical industry has developed and marketed several nanoparticlate pharmaceuticals with major emphasis on intravenous products— for example, intravenous nutritional fat emulsion (Intralipid^®) and liposomal products (Doxil^®, AmBisome^®).

[0005] Liposomes are vesicles formed from one ("unilamellar") or more

("multilamellar") layers of phospholipid. Because of the amphipathic character of the phospholipid building blocks, liposomes typically comprise a hydrophilic layer presenting a hydrophilic external face and enclosing a hydrophilic core. The versatility of liposomes in the incorporation of hydrophilic/hydrophobic components, their non-toxic nature, biodegradability, biocompatibility, adjuvanticity, induction of cellular immunity, property of sustained release and prompt uptake by macrophages, makes them attractive candidates for the delivery of antigens. The inability to achieve high drug loading, the cost of ingredients and processing, and the restricted number of suitable excipients have hitherto limited the broader use of these formulation approaches.

[0006] There remains in the art a need for methods and compositions which can provide formulations that deliver high concentrations of poorly soluble compounds for a variety of applications.

BRIEF SUMMARY OF THE INVENTION

[0007] It is an object of the invention to provide nanoparticle compositions, methods for the manufacture thereof, and methods for the use thereof. Such compositions may be used to provide a stable, high concentration reservoir of water insoluble compounds such as triclosan.

[0008] In a first aspect, the invention relates to an aqueous composition comprising: particles comprising lipid and a water insoluble (hydrophobic) molecule, the particles having an average size of less than about 200 nm; and an aqueous carrier, wherein the aqueous composition has a water insoluble (hydrophobic) molecule concentration of at least about 1 mg/mL and is at approximately neutral pH.

[0009] The term "approximately neutral pH" as used herein refers to between about pH 5 and about pH 9. The term "water insoluble" or "hydrophobic" as used herein refers to a compound which has a solubility in water at approximately neutral pH of less than about 10^"4 g/mL, more preferably less than 10^"5 g/mL. The term "about" as used herein refers to +/- 10% of a given value.

[0010] In certain embodiments, the water insoluble molecule has a melting point greater than about 20°C, and preferably greater than about 37°C. Most preferably, the melting point is greater than about 20°C, and preferably greater than about 37°C, but less than the boiling point of the aqueous carrier. In certain embodiments, the water insoluble molecule is a diphenylether, and most preferably triclosan. [0011] In certain embodiments, the particles of the aqueous composition have an average size of less than about 50 nm; and/or the concentration of the water insoluble molecule in the aqueous composition at about 20°C, and preferably about 37°C, is at least about 5 mg/mL. Particle size can be selected, for example, by extrusion of an aqueous vehicle comprising the particles of the present invention through membranes having a preselected pore size and collecting the material flowing through the membrane. As described herein, such particles can form a composition having predominantly nanoparticles of less than about 200 nm, and preferably of less than about 50 nm. The term "predominantly" as used herein in this context means that at least 75%, more preferably 80%, and most preferably at least 90% of the particles are within the designated size range.

[0012] In certain embodiments, the particles comprise a pegylated lipid, and preferably the lipid component of the particles comprises at least 90% pegylated lipid. In a preferred embodiment, the particles comprise pegylated l,2-diacyl-sn-glycero-3- phosphoethanolamine, wherein the polyethylene glycol moiety on the pegylated 1 ,2- diacyl-sn-glycero-3-phosphoethanolamine has an average molecular weight of at least about 1000. Most preferably, the particles comprise pegylated 1 ,2-distearoyl-sn-glycero- 3-phosphoethanolamine, wherein the polyethylene glycol moiety on the pegylated 1,2- distearoyl-sn-glycero-3-phosphoethanolamine has an average molecular weight of at least about 1000. As described herein, such particles can form a composition having predominantly nanoparticles of less than about 200 nm, and preferably of less than about 50 nm, which comprise a lipid shell encapsulating a solid core of the insoluble compound.

[0013] In certain embodiments, the particles are predominantly unilamellar liposomes. Such unilamellar liposomes may comprise lipids having a transition temperature > 37°C, such as one or more lipids selected from the group consisting of DPPC, DPPG, DPPE, DSPC, DSPG, DSPE, and HSPC; and/or may comprise lipids having a transition temperature < 37°C, such as one or more lipids selected from the group consisting of DMPC, DMPG, DMPE, POPC, POPG, POPE, DOPC, DOPE, DOPG, soyPC, and eggPC. The unilamellar liposomes also preferably can comprise cholesterol.

[0014] In various embodiments, the aqueous compositions of the present invention are toxic or static to the growth of Gram negative and Gram positive bacteria and pathogenic fungi. In certain embodiments described hereinafter, the aqueous composition has a minimum inhibitory concentration for Staphylococcus aureus of less than about 0.5 μg lipid-triclosan particles/mL of culture medium in which the bacteria are grown, and a minimum inhibitory concentration for Acinetobacter baumannii of less than about 5 μg lipid-triclosan particles/mL of culture medium in which the bacteria are grown.

[0015] The aqueous compositions of the present invention may be formulated as a variety of products in which bactericidal or bacteriostatic are sought. Such products include, but are not limited to, disinfectants, paints or other surface coatings, bactericidal or bacteriostatic agents for topical or internal use such as skin washes and oral treatments, medical devices, pharmaceutical compositions, fabrics, etc.

[0016] In related aspects, the present invention relates to methods of treating a bacterial or fungal infection in a subject comprising administering a pharmaceutical composition according the present invention.

[0017] In another aspect, the present invention relates to methods for preparing a particle composition comprising lipid and a water insoluble (hydrophobic) molecule, comprising:

a. combining at a temperature greater than the melting point of the water insoluble molecule but less than the boiling point of the water insoluble molecule (i) an aqueous solution comprising one or more lipids and having an approximately neutral pH, and (ii) a solution of the water insoluble molecule in a solvent that is miscible in the aqueous solvent, to provide a mixture; homogenizing the mixture at a temperature greater than the melting point of the water insoluble molecule but less than the boiling point of the water insoluble molecule; cooling the mixture; and collecting particles having an average size of less than about 200 nm from the cooled mixture; or b. adding an aqueous solvent having an approximately neutral pH to a dried mixture which comprises one or more lipids and the water insoluble molecule; homogenizing the mixture formed thereby at a temperature greater than the melting point of the water insoluble molecule but less than the boiling point of the water insoluble molecule ; cooling the mixture; and collecting particles having an average size of less than about 200 nm from the cooled mixture.

[0018] In a related aspect, the present invention relates to methods for preparing a lipid-triclosan particle composition, comprising:

a. combining at a temperature greater than the melting point of triclosan but less than the boiling point of triclosan (i) an aqueous solution comprising one or more lipids and having an approximately neutral pH, and (ii) a solution of triclosan in a solvent that is miscible in the aqueous solvent, to provide a lipid-triclosan mixture; homogenizing the lipid-triclosan mixture at a temperature greater than the melting point of triclosan but less than the boiling point of triclosan; cooling the lipid-triclosan mixture; and collecting lipid-triclosan particles having an average size of less than about 200 nm from the cooled lipid-triclosan mixture; or b. adding an aqueous solvent having an approximately neutral pH to a dried mixture which comprises one or more lipids and triclosan; homogenizing the mixture formed thereby at a temperature greater than the melting point of triclosan but less than the boiling point of triclosan; cooling the lipid-triclosan mixture; and collecting lipid-triclosan particles having an average size of less than about 200 nm from the cooled lipid-triclosan mixture.

[0019] As used herein, "homogenization" refers to any of a number of high shear dispersion methods known in the art, including sonication, microfluidization, and extrusion. [0020] As discussed above, the water insoluble molecule has a melting point greater than about 20°C, and preferably greater than about 37°C. Most preferably, the melting point is greater than about 20°C, and preferably greater than about 37°C, but less than the boiling point of the aqueous carrier. In certain embodiments, the water insoluble molecule is a diphenylether, and most preferably triclosan.

[0021] In certain embodiments, the methods comprise selecting particles having an average size of less than about 50 nm; and/or the concentration of the water insoluble molecule in the aqueous composition at about 20°C, and preferably about 37°C, is at least about 5 mg/mL. Particle size can be selected, for example, by extrusion of an aqueous vehicle comprising the particles of the present invention through membranes having a preselected pore size and collecting the material flowing through the membrane. As described herein, such particles can form a composition having predominantly nanoparticles of less than about 200 nm, and preferably of less than about 50 nm. The term "predominantly" as used herein in this context means that at least 75%, more preferably 80%, and most preferably at least 90% of the particles are within the designated size range.

[0022] In certain embodiments, the particles comprise a pegylated lipid, and preferably the lipid component of the particles comprises at least 90% pegylated lipid. Most preferably, the particles comprise pegylated l,2-distearoyl-sn-glycero-3- phosphoethanolamine, wherein the polyethylene glycol moiety on the pegylated 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine has an average molecular weight of at least about 1000. As described herein, such particles can form a composition having predominantly nanoparticles of less than about 200 nm, and preferably of less than about 50 nm, which comprise a lipid shell encapsulating a solid core of the insoluble compound.

[0023] In certain embodiments, the particles are predominantly unilamellar liposomes. Such unilamellar liposomes may comprise lipids having a transition temperature > 37°C, such as one or more lipids selected from the group consisting of DPPC, DPPG, DPPE, DSPC, DSPG, DSPE, and HSPC; and/or may comprise lipids having a transition temperature < 37°C, such as one or more lipids selected from the group consisting of DMPC, DMPG, DMPE, POPC, POPG, POPE, DOPC, DOPE, DOPG, soyPC, and eggPC. The unilamellar liposomes also preferably can comprise cholesterol. [0024] In various embodiments, the aqueous compositions of the present invention are toxic or static to the growth of Gram negative and Gram positive bacteria and pathogenic fungi. In certain embodiments described hereinafter, the aqueous composition has a minimum inhibitory concentration for Staphylococcus aureus of less than about 0.5 μg lipid-triclosan particles/mL of culture medium in which the bacteria are grown, and a minimum inhibitory concentration for Acinetobacter baumannii of less than about 5 μg lipid-triclosan particles/mL of culture medium in which the bacteria are grown.

[0025] In certain embodiments, the method providing the insoluble compound (e.g., triclosan) as a solution in a polar aprotic solvent that is miscible in the aqueous solvent. For example, triclosan may be provided at least 100 mg/mL in dimethyl sulfoxide. Other polar aprotic solvents, such as dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, and acetonitrile may also find use in the present invention. It is noted that the polar aprotic solvent does not form all or part of a solvent for the final nanoparticle preparation, but is used to aid in the dispersion of the insoluble compound during formation of the nanoparticles.

[0026] It is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

[0027] As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

[0028] Fig. 1 depicts in schematic form free triclosan (A), liposomal triclosan nanoparticles (B), and solid core triclosan nanoparticles having a lipid shell. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is described using triclosan as an example of a poorly soluble compound for formulation into nanoparticles. Triclosan has a melting point of about 55-57 °C and a boiling point 120 °C and, as such, provides an ideal model compound for demonstration of the present invention. The skilled artisan will understand, however, that the methods described herein are generally applicable to formulation of poorly soluble compounds having the necessary physical characteristics with regard to melting point.

[0030] The incidence of infection caused by antibiotic-resistant gram-positive and gram-negative bacteria continues to increase globally both in the hospital and in the community. For instance, population-based estimates of the incidence of invasive S. aureus infections have ranged from a low of approximately 30 per 100,000 based on data at a single Canadian hospital [11], up to 600 per 100,000. In addition, it has been reported that more people now die of methicillin-resistant S. aureus (MRSA) infection in US hospitals than of HIV/ AIDS and tuberculosis combined. Although the use of vancomycin and of newer antimicrobial agents such as linezolid and daptomycin has controlled some recalcitrant MRSA infections, the rapid development of resistance to these agents is a major concern. Also, among current gram-negative bacterial infections, multi-drug resistant (MDR) Acinetobacter species represent an enormous clinical challenge, with a crude mortality of 26-68%, as well as increased length of stay in the intensive care unit. However, our therapeutic options for these two pathogens are extremely limited because of the toxicities of current agents and emerging resistance.

[0031] Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is an antibacterial and antifungal agent. Triclosan has been used since 1972, and it is present in soaps (0.10- 1.00%), deodorants, toothpastes, shaving creams, mouth washes, and cleaning supplies, and is infused in an increasing number of consumer products, such as kitchen utensils, toys, bedding, socks, and trash bags. At in-use concentrations, triclosan acts as a biocide, with multiple cytoplasmic and membrane targets. Triclosan has been shown to be effective in reducing and controlling bacterial contamination on the hands and on treated products. More recently, showering or bathing with 2% triclosan has become a recommended regimen for the decolonization of patients whose skin is carrying methicillin-resistant Staphylococcus aureus (MRSA) following the successful control of MRS A outbreaks in several clinical settings.

[0032] The present invention provides several types of nanoscale formulations of triclosan. With a solubility of 30 μΜ at 20°C and an estimated solubility of 100 micromolar at 37 °C, triclosan can readily achieve these levels in solution, if enough material is present to provide a stable source for controlled release. The present invention can provide such a stable reservoir of triclosan, which will continuously "bathe" the treated material with at least 25 micromolar triclosan.

[0033] Two main classes of nanoparticle formulations are described herein. First, a nanoparticle comprising a liposomal formulation that encapsulate the hydrophobic triclosan molecule (referred to herein as "L-triclosan"). In these formulations, triclosan is expected to be encapsulated at -100 micromolar in the liposome interior and embedded within the hydrophobic bilayer of the liposome, thereby maximizing its clinical application as an i.v. drug. For formulations that are composed of low transition temperature lipids (<37°C), drug release is expected to occur by simple desorption from the bilayer. For lipid compositions with higher transition temperatures, drug release could occur if extravasation of the liposomes to the sites of infection facilitates the exposure to bacterially- and tissue/immune-derived lipases that degrade liposomal lipid components.

[0034] The second type of nanoparticles provide a monolayer of PEG-lipid coating around a solid core of the drug (referred to herein as "SM-triclosan"). These formulations are designed to yield a range of drug dissolution rates dictated purely by the surface areata- volume ratio of the drug mass in a more or less finely divided state. For the smallest nanoparticles (10s of nm), we follow the examples of Lukyanov et al., but modify those techniques to provide much higher quantities of drug in the aqueous phase. While Torchilin et al. have disclosed the use of, DSPE-PEG micelles as delivery vehicles for poorly water soluble drugs, such as paclitaxel, and camptothecin, these methods provide samples containing 15 μg to 200 μg of compound per mL. The present methods can provide 100-1,000 times these amounts— i.e., 25mM triclosan in a 5mM solution of micellar DSPE-PEG2000.

[0035] Also, in Torchilin's work, drug-loaded micelles were prepared by dispersing a dry film of the mixture of micelle-forming material (PEG-PE) and drug in an aqueous buffer solution. The main reason for this is the fact that the melting point of paclitaxel is 213-216°C. As described herein, our method takes advantage of the lower melting point of triclosan, ~ 55-57°C. Given triclosan's solubility (-120 μΜ at an elevated

temperature), the pre-existing micelles are rapidly swollen by triclosan. At room and body temperature, these swollen triclosan micelles (25 to 50nm) are expected to have a solid core, but with maximum surface area (-100 m² per gm). In order to provide reduced surface areas per mass of material, with resultant slower dissolution and release profiles for triclosan, one may increase the triclosan: lipid ratio and emulsify to form larger nanoparticles that range from 100-200nm (referred to herein as "E-triclosan").

[0036] These formulation strategies can provide different drug-carrying capacities, release (dissolution) rates, pharmacokinetics and biodistribution properties.

[0037] Definitions

[0038] "Administration" as it applies to a human, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, experimental subject, cell, tissue, organ, or biological fluid, refers without limitation to contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition to the subject, cell, tissue, organ, or biological fluid, and the like. "Administration" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" also encompasses in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell.

[0039] An "agonist," as it relates to a ligand and receptor, comprises a molecule, combination of molecules, a complex, or a combination of reagents, that stimulates the receptor. For example, an agonist of granulocyte-macrophage colony stimulating factor (GM-CSF) can encompass GM-CSF, a mutein or derivative of GM-CSF, a peptide mimetic of GM-CSF, a small molecule that mimics the biological function of GM-CSF, or an antibody that stimulates GM-CSF receptor.

[0040] An "antagonist," as it relates to a ligand and receptor, comprises a molecule, combination of molecules, or a complex, that inhibits, counteracts, downregulates, and/or desensitizes the receptor. "Antagonist" encompasses any reagent that inhibits a constitutive activity of the receptor. A constitutive activity is one that is manifest in the absence of a ligand/receptor interaction. "Antagonist" also encompasses any reagent that inhibits or prevents a stimulated (or regulated) activity of a receptor. By way of example, an antagonist of GM-CSF receptor includes, without implying any limitation, an antibody that binds to the ligand (GM-CSF) and prevents it from binding to the receptor, or an antibody that binds to the receptor and prevents the ligand from binding to the receptor, or where the antibody locks the receptor in an inactive conformation.

[0041] "Effective amount" encompasses, without limitation, an amount that can ameliorate, reverse, mitigate, prevent, or diagnose a symptom or sign of a medical condition or disorder. Unless dictated otherwise, explicitly or by context, an "effective amount" is not limited to a minimal amount sufficient to ameliorate a condition.

[0042] The term "subject" as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. In certain embodiments, subjects are "patients," i.e., living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology. Preferred are subjects who have an existing Plasmodium infection.

[0043] "Therapeutically effective amount" is defined as an amount of a reagent or pharmaceutical composition that is sufficient to show a patient benefit, i.e., to cause a decrease, prevention, or amelioration of the symptoms of the condition being treated. When the agent or pharmaceutical composition comprises a diagnostic agent, a

"diagnostically effective amount" is defined as an amount that is sufficient to produce a signal, image, or other diagnostic parameter. Effective amounts of the pharmaceutical formulation will vary according to factors such as the degree of susceptibility of the individual, the age, gender, and weight of the individual, and idiosyncratic responses of the individual (see, e.g., U.S. Pat. No. 5,888,530 issued to Netti, et al).

[0044] "Treatment" or "treating" (with respect to a condition or a disease) is an approach for obtaining beneficial or desired results including and preferably clinical results. For purposes of this invention, beneficial or desired results with respect to a disease include, but are not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality of life of one suffering from a disease, and/or prolonging survival. Likewise, for purposes of this invention, beneficial or desired results with respect to a condition include, but are not limited to, one or more of the following:

improving a condition, curing a condition, lessening severity of a condition, delaying progression of a condition, alleviating one or more symptoms associated with a condition, increasing the quality of life of one suffering from a condition, and/or prolonging survival.

[0045] Liposomal preparation:

[0046] The preparation of liposomes is well known in the prior art. In general, liposomes have been made by a number of different techniques including ethanol injection (Batzri et al., Biochem. Biophys. Acta. 298: 1015, 1973); ether infusion (Deamer et al., Biochem. Biophys. Acta. 443:629, 1976; Schieren et al., Biochem. Biophys. Acta. 542: 137, 1978); detergent removal (Razin, Biochem. Biophys. Acta. 265:24 1972);

solvent evaporation (Matsumato et al., J. Colloid Interface Sci. 62: 149, 1977);

evaporation of organic solvents from chloroform in water emulsions (REV's) (Szoka Jr. et al., Proc. Natl. Acad. Sci. USA, 75:4194, 1978); extrusions of MLVs or EUV's through a nucleopore polycarbonate membrane (Olson et al., Biochem. Biophys. Acta. 557:9, 1979); freezing and thawing of phosopholipid mixtures (Pick, Arch. Biochem. Biophys. , 212: 186, 1981), as well as sonication and homogenization. By convention, liposomes are categorized by size, and a 3-letter acronym is used to designate the type of liposome being discussed. Multilamellar vesicles are generally designated "MLV." Small unilamellar vesicles are designated "SUV," and large unilamellar vesicles are designated "LUV." These designations are sometimes followed by the chemical composition of the liposome. For a discussion of nomenclature and a summary of known types of liposomes, see Storm et al., PSIT, 1 : 19-3, 1998.

[0047] The liposomal compositions of the invention may further comprise, either as part of the liposome itself or as part of the vehicle in which the liposomes are suspended, various excipients, carriers, auxiliary substances, modulating agents, and the like.

[0048] A carrier, which is optionally present, is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLGA. See, e.g., Jeffery et al., Pharm. Res. 10:362, 1993; McGee et al., J. Microencapsul. 14: 197, 1997; O'Hagan et al, Vaccine 11 : 149, 1993. Such carriers are well known to those of ordinary skill in the art.

[0049] Pharmaceutical compositions

[0050] The nanoparticles of the present invention may be formulated as

pharmaceutical compositions for parenteral or enteral delivery. A typical pharmaceutical composition for administration to an animal comprises a pharmaceutically acceptable vehicle such as aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. See, e.g., Remington's Pharmaceutical Sciences, 15th Ed., Easton ed. , Mack Publishing Co., pp 1405-1412 and 1461- 1487 (1975); The National Formulary XIV, 14th Ed., American Pharmaceutical Association, Washington, DC (1975) . Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art.

[0051 ] Oral compositions

[0052] Oral care products containing the nanoparticles of the present invention include: rinses, spray, gels, creams, other toothpastes, tooth powders, denture cleaning tablets, dental floss, interproximal simulators, mints, chewing gums, pet treats and pet main meals..

[0053] Textile compositions

[0054] The nanoparticles of the present invention may be formulated for providing antimicrobial properties to a fabric. By way of example, the textile substrate itself may be made from woven, non-woven, or knit fabric and made from any natural or man-made fiber. Examples of fibers include, but are not limited to, paper, glass fiber matting, cotton, polyester, polyamide, ramie, acetate, polyolefin, acrylic, and lycra, or any blends thereof. Of these, polyester, polyamide, particularly nylon (-6 or -6,6), and lycra, and especially, blends of nylon and lycra are preferred. Also, the particularly preferred textiles are those which are knit. The durable, long-lasting, antimicrobial characteristics are most evident on these preferred textile substrates. The inclusion of any standard dye, dyestuff, or colorant utilized within a textile dyeing process is also contemplated. The amount of dye or colorant may need to be adjusted from usual levels to compensate for the added triclosan treatment. The ratio of wt % between the weight of fabric and the weight of triclosan nanoparticles within the dye bath should be from about 100:0.01 to about 100: 1. Preferably, this range is from about 100:0.03 to about 100:0.6, and most preferably from about 100:0.1 to about 100:0.25. Any other standard textile additives, such as dyes, sizing compounds, ultra violet absorbers, and softening agents may also be incorporated within or introduced onto the surface of the treated fabric substrate. The treated fabric may be incorporated into a garment, a table linen, a bathroom linen, a napery linen, a bar towel, or any other type of fabric of which antimicrobial properties are desirous.

[0055] Medical devices

[0056] Medical articles may be either fabricated from or coated or treated with the nanoparticles of the present invention include, but are not limited to, catheters including urinary catheters and vascular catheters (e.g. peripheral and central vascular catheters), wound drainage tubes, arterial grafts, soft tissue patches, gloves, shunts, stents, tracheal catheters, wound dressings, sutures, guide wires and prosthetic devices (e.g. heart valves and LVADs). Vascular catheters which may be prepared according to the present invention include, but are not limited to, single and multiple lumen central venous catheters, peripherally inserted central venous catheters, emergency infusion catheters, percutaneous sheath introducer systems and thermodilution catheters, including the hubs and ports of such vascular catheters.

[0057] Skin wash compositions:

[0058] Skin wash formulations using the triclosan nanoparticles of the present invention as a biocidal agent may be formulated without the use a surfactant or nonaqueous solvent which tend to cause skin irritation. This is particularly troublesome to health care workers, who are employing skin washes several times a day.

Examples

[0059] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.

[0060] Example 1. Lipid film preparation [0061] Stock solution of Hydrogenated Soy Phosphatidylcholine (HSPC, Lipoid), Cholesterol (Choi, NOF), Distearoylphophatidyl glycerol (DSPG, Lipoid), Triclosan ( Sigma) were prepared in chloroform : Methanol 1 : 1 solvent system at proper

concentration as desired. Lipid formula for the liposomal sample is HSPC:CHOL:DSPG 2:1:0.1 (mole ratio) for liposomal triclosan. To prepare a lipid film, proper amount of each lipid component was measured and was mixed in tube and desired amount of triclosan ( based on lipid to triclosan w/w ratio ) was added to the tube, the lipid to triclosan weight ratio is 50 : 1. An identical sample of lipid film were prepared without triclosan as a control. The dried lipid films were placed under vacuum for at least 48 hours to remove residual organic solvents.

[0062] Example 2: Liposome preparation :

[0063] To the dried lipid films, an appropriate amount of buffer, lOmM Sodium Succinate in 9% Sucrose pH 5.5, was added to obtain a l.Omg/ml triclosan concentration. Probe sonication (1/8" Stapped Microtip, Sonic&materials ) was applied to the lipid drug mixture at around 10% power with a 65°C water bath on a Branson Sonifier for 15-20 minutes, until a transparent liposome solution reached. The liposome was centrifuged at 3600 rpm for 10 minutes to remove any precipitation. The liposome was then sterile filtered through a 0.2uM polyethersulfone syringe filter into sterile, pyrogenic free tubes. Liposome size distribution was determined by Microtrac (Microtrac-UPA150,

Honeywell). Triclosan and lipids concentration of the liposomal preparation was confirmed by HPLC assay.

[0064] Example 3: Triclosan Nanoparticles

[0065] Materials: DSPE-PEG-2000 (Ammonium Salt) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Irgasan

(Triclosan) and Dimethyl Sulfoxide (Anhydrous DMSO) were purchased from Sigma- Aldrich (St. Louis, MO). Deionized water was used in all experiments. Syringe driven 0.22um PES membrane filters were bought from Millipore (Billerica, MA).

[0066] Micelle Preparation: To prepare solid core triclosan micelles, 5mM DSPE- PEG-2000 in water and 500 mg/ml Triclosan stock in DMSO were prepared. Then, appropriate amounts of the triclosan stock was added slowly to the DSPE-PEG-2000 solution preheated to 65-70°C, to achieve a final mole ratio of 5:1 (Triclosan to DSPE- PEG-2000). The solution was subjected to sonication in a Branson 1510 bath sonicator at 60-63C until all the white precipitation was in solution. The sample was allowed to cool to room temperature before sterile filtration through a 0.22um PES membrane.

[0067] Example 4: Analysis

[0068] Micelle Size Measurement: The micelle size (hydrodynamic diameter) was measured by dynamic light scattering (DLS) using a Microtrac® UPA 150 (Honeywell TAC, Fort Washington, PA) ultrafine particle analyzer. Dilutions for size measurement were made in a 5mM DSPE-PEG-2000 aqueous solution.

[0069] Triclosan Concentration Measurement: HPLC analysis was performed to measure triclosan concentration in the final sample.

[0070] Example 5: Antibacterial properties of nanoparticles

[0071] Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays of SM-triclosan and L-triclosan (HSPC:Chol:DSPG) formulations were performed in cation-supplemented Mueller-Hinton Broth (MHB) by a microdilution technique according to Clinical and Laboratory Standards Institute guidelines (CLSI; Ref. National Committee for Clinical Laboratory Standards. 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th ed. Approved standard. NCCLS document M7-A6. National Committee for Clinical Laboratory Standards, Wayne, PA). MIC was determined as the lowest drug

concentration preventing visible turbidity after 18 h of incubation at 37°C. The MBC was determined from the MIC test plates by subculturing to MHB agar plates, and reported as the lowest concentration of antibiotic producing a 99.9% reduction in bacterial count relative to the initial inoculum.

[0072] Data from MIC and MBC assays of MRSA (LAC USA300) and A. baumannii are presented in the following table. For MRSA, the MIC values for L-triclosan and SM- triclosan were substantially lower than for free triclosan and the vancomycin control. Similar comparative MIC data were seen for A. baumannii. MBC data followed a similar trend. DSPE-PEG-2000 alone and liposomal controls (without triclosan) showed low to no inhibition and bactericidal activities.

[0073] Example 6: In vitro cytotoxicity assay

[0074] The toxicity of the triclosan nanoparticle preparations were screened against Vero cells (ATCC CCL-81) and MDA-MB-231 cells (ATCC HTB-26). The Vera cells were cultured in Minimum Essential Medium with Earle's Balanced Salts (MEM/EBSS, HyClone, Logan, UT) and the MDA-MB-231 cells in Roswell Park Memorial Institute medium-1640 (RPMI-1640, HyClone, Logan, UT); both media supplemented with 10% heat- inactivated fetal bovine serum (Biocell, Rancho Dominguez, CA), 100 units/mL penicillin, 100 μg/mL streptomycin and 292 μg/mL L-glutaimine (antibiotics from Mediatech, Manassas, VA), in 75-cm2 flasks (BD Falcon, Franklin Lakes, NJ) at 37°C in a 7.5% C02 atmosphere. The cultures were grown to >80% confluency before harvested for seeding. The cells were seeded at 6320 cells per well in poly-D-lysine coated, 96-well plates (BD Falcon, Franklin Lakes, NJ). The seeded cells were incubated for 2 days, until confluent. The media was discarded and 100 μL· of supplemented media with triclosan liposomes, triclosan nanoparticles, free triclosan in DMSO or the appropriate negative controls (liposome only, DSPE-PEG-2000 only or succinate buffer only) was added to each well; the maximum final triclosan concentration tested in each form was 200 μg/mL, a 2-times serial dilution with a total of ten dilutions per test sample was assessed. The cells were incubated with the test samples for 16 hours at 37°C in a 7.5% C02 atmosphere. The media containing test materials was then replaced with 100 μL· of fresh supplemented media and 10 of alamarBlue (Life Technologies, Carlsbad, CA). The plates were returned to 37°C in a 7.5% C02 atmosphere for an additional 3 hours of incubation. The plates were then analyzed spectrofluorometrically (544 excitation, 590 emission) by a Fluorskan Ascent Microplate Fluorometer (Thermo Sci., Hudson, NH).

[0075] The results suggested that triclosan in nanoparticle or free form is toxic to half the population of MDA-MB-231 cells, a breast cancer cell line, at -25 μg/mL final triclosan concentration. And the triclosan in nanoparticle or free form is toxic to half the population Vero cells, a normal African green monkey kidney cell line, at >25 and <50 μg/mL final triclosan concentration. The toxicity of the triclosan is almost independent to the preparation method of triclosan tested.

[0076] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. [0077] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

Table 1. MIC and MBC comparison of free triclosan to the novel triclosan

nanoparticles vs our prototype MRSA and Acinetobacter baumannii test strains.

_Λ c . Acinetobacter

MRSA ,

baumannii

MIC MBC MIC MBC

(Mg/mL) (Mg/mL) (Mg/mL) (Mg/mL)

SM-triclosan 0.125 2 1 4

L- Triclosan 0.125 4 2 4

Free triclosan 4 8 32 64

Vancomycin 1 8 N/A N/A

Amikacin 2 8 1 4

N/A - no inhibition at all concentrations tested

[0078] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0079] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0080] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms

"comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0081] Other embodiments are set forth within the following claims.

Claims

We claim:

1. An aqueous composition comprising: particles comprising lipid and a water insoluble (hydrophobic) molecule, the particles having an average size of less than about 200 nm; and an aqueous carrier, wherein the aqueous composition has a water insoluble (hydrophobic) molecule concentration of at least about 1 mg/mL and is at approximately neutral pH.

2. An aqueous composition according to claim 1 , wherein the water insoluble molecule has a melting point greater than about 20°C.

3. An aqueous composition according to claim 1, wherein the water insoluble molecule has a melting point greater than about 37°C.

4. An aqueous composition according to claim 2, wherein the water insoluble molecule is a diphenylether.

5. An aqueous composition according to claim 4, wherein the diphenyl ether compound is triclosan, whereby the particles are lipid-triclosan particles.

6. An aqueous composition according to claim 5, wherein the lipid-triclosan particles have an average size of less than about 50 nm and a triclosan concentration of at least about 5 mg/mL.

7. An aqueous composition according to claim 6, wherein the lipid-triclosan particles comprise a pegylated lipid.

8. An aqueous composition according to claim 7, wherein the lipid component of the lipid-triclosan particles comprises at least 90% pegylated lipid.

9. An aqueous composition according to claim 5, wherein the lipid-triclosan particles comprise pegylated l,2-distearoyl-sn-glycero-3-phosphoethanolamine, wherein the polyethylene glycol moiety on the pegylated l,2-distearoyl-sn-glycero-3- phosphoethanolamine has an average molecular weight of at least about 1000.

10. An aqueous composition according to claim 5, wherein the lipid-triclosan particles are predominantly unilamellar liposomes.

11. An aqueous composition according to claim 10, wherein the unilamellar liposomes comprise lipids having a transition temperature > 37 °C.

12. An aqueous composition according to claim 11, wherein the unilamellar liposomes comprise one or more lipids selected from the group consisting of DPPC, DPPG, DPPE, DSPC, DSPG, DSPE, HSPC, and cholesterol.

13. An aqueous composition according to claim 10, wherein the unilamellar liposomes comprise lipids having a transition temperature < 37 °C.

14. An aqueous composition according to claim 13, wherein the unilamellar liposomes comprise one or more lipids selected from the group consisting of DMPC, DMPG, DMPE, POPC, POPG, POPE, DOPC, DOPE, DOPG, soyPC, eggPC, and cholesterol.

15. An aqueous composition according to claim 5, wherein the lipid- triclosan particles are predominantly nanoparticles comprising a lipid shell encapsulating a solid triclosan core.

16. An aqueous composition according to claim 5, wherein the aqueous composition has a minimum inhibitory concentration for Staphylococcus aureus of less than about 0.5 μg lipid-triclosan particles/mL of culture medium, and a minimum inhibitory

concentration for Acinetobacter baumannii of less than about 5 μg lipid-triclosan particles/mL of culture medium.

17. A method according to claim 1, wherein the collected particles have a minimum inhibitory concentration less than the free diphenylethers for Gram negative and Gram positive bacteria and pathogenic fungi.

18. An aqueous composition according to one of claims 1-17, wherein particles can are formulated as a disinfectant, or incorporated into a paint, surface coating, or a bactericidal or bacteriostatic agent.

19. A formulation for topical application to the skin or oral mucosa comprising an aqueous composition according to one of claims 1-17.

20. A medical device comprising an aqueous composition according to one of claims 1-17.

21. A pharmaceutical composition comprising an aqueous composition according to one of claims 1-17.

22. A fabric comprising an aqueous composition according to one of claims 1-17.

23. A method of treating a bacterial or fungal infection in a subject comprising administering a pharmaceutical composition according to claim 21 to the subject.

24. A method for preparing a particle composition comprising lipid and a water insoluble (hydrophobic) molecule, comprising: a. combining at a temperature greater than the melting point of the water insoluble molecule but less than the boiling point of the water insoluble molecule (i) an aqueous solution comprising one or more lipids and having an approximately neutral pH, and (ii) a solution of the water insoluble molecule in a solvent that is miscible in the aqueous solvent, to provide a mixture; homogenizing the mixture at a temperature greater than the melting point of the water insoluble molecule but less than the boiling point of the water insoluble molecule; cooling the mixture; and collecting particles having an average size of less than about 200 nm from the cooled mixture; or b. adding an aqueous solvent having an approximately neutral pH to a dried mixture which comprises one or more lipids and the water insoluble molecule; homogenizing the mixture formed thereby at a temperature greater than the melting point of the water insoluble molecule but less than the boiling point of the water insoluble molecule ; cooling the mixture; and collecting particles having an average size of less than about 200 nm from the cooled mixture.

25. A method for preparing a lipid-triclosan particle composition, comprising: a. combining at a temperature greater than the melting point of triclosan but less than the boiling point of triclosan (i) an aqueous solution comprising one or more lipids and having an approximately neutral pH, and (ii) a solution of triclosan in a solvent that is miscible in the aqueous solvent, to provide a lipid-triclosan mixture; homogenizing the lipid-triclosan mixture at a temperature greater than the melting point of triclosan but less than the boiling point of triclosan; cooling the lipid-triclosan mixture; and collecting lipid-triclosan particles having an average size of less than about 200 nm from the cooled lipid-triclosan mixture; or b. adding an aqueous solvent having an approximately neutral pH to a dried mixture which comprises one or more lipids and triclosan; homogenizing the mixture formed thereby at a temperature greater than the melting point of triclosan but less than the boiling point of triclosan; cooling the lipid-triclosan mixture; and collecting lipid-triclosan particles having an average size of less than about 200 nm from the cooled lipid-triclosan mixture.

26. A method according to claim 24 or 25, wherein the homogenizing step comprises sonication.

27. A method according to claim according to claim 25, wherein the collecting step comprises collecting particles having an average size of less than about 50 nm and a triclosan concentration of at least about 5 mg/mL.

28. A method according to claim 25, wherein the method comprises combining at a temperature greater than the melting point of triclosan but less than the boiling point of triclosan (i) an aqueous solution comprising one or more lipids and (ii) a solution of triclosan in a polar aprotic solvent that is miscible in the aqueous solvent to provide the lipid-triclosan mixture.

29. A method according to claim 28, wherein the solution of triclosan comprises at least 100 mg/mL in dimethyl sulfoxide.

30. A method according to claim 24 or 25, wherein the aqueous solution comprising one or more lipids comprises a pegylated lipid.

31. A method according to claim 30, wherein the aqueous solution comprising one or more lipids comprises at least 90% pegylated lipid.

32. A method according to claim 24 or 25, wherein the pegylated lipid is pegylated l,2-distearoyl-sn-glycero-3-phosphoethanolamine, wherein the polyethylene glycol moiety on the pegylated l ,2-distearoyl-sn-glycero-3-phosphoethanolamine has an average molecular weight of at least about 1000.

33. A method according to claim 24 or 25, wherein the collected particles are predominantly unilamellar liposomes.

34. A method according to claim 24 or 25, wherein the unilamellar liposomes comprise lipids having a transition temperature > 37°C.

35. A method according to claim 24 or 25, wherein the unilamellar liposomes comprise lipids having a transition temperature < 37°C.

36. A method according to claim 25, wherein the collected lipid- triclosan particles are predominantly nanoparticles comprising a lipid shell encapsulating a solid triclosan core.

37. A method according to claim 24 or 25, wherein the collected particles have a minimum inhibitory concentration for Staphylococcus aureus of less than about 0.5 μg particles/mL of culture medium, and a minimum inhibitory concentration for

Acinetobacter baumannii of less than about 5 μg particles/mL of culture medium.