WO2025235614A1

WO2025235614A1 - Liposome loading with polyanions

Info

Publication number: WO2025235614A1
Application number: PCT/US2025/028153
Authority: WO
Inventors: Timothy D. Heath; Lisa A. Krugner-Higby
Original assignee: Plumb Pharmaceuticals Inc
Current assignee: Plumb Pharmaceuticals Inc
Priority date: 2024-05-08
Filing date: 2025-05-07
Publication date: 2025-11-13
Anticipated expiration: 2026-11-08

Abstract

Provided herein is technology relating to incorporation of drugs into liposomes and particularly, but not exclusively, to methods for incorporating drugs into liposomes using a weak base and related compositions.

Description

LIPOSOME LOADING WITH POLYANIONS

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional Application No. 63/644,244, filed May 8, 2024, which is incorporated herein by reference in its entirety.

FIELD

Provided herein is technology relating to incorporation of drugs into liposomes and particularly, but not exclusively, to methods for incorporating drugs into liposomes using polyanions as a counter ion, preferably in conjunction with a weak base.

BACKGROUND

Controlled-release drug formulations can be produced by incorporating drugs into liposomes. These formulations have many advantages including, e.g., extending the duration of a drug’s effect following administration. Important considerations related to these technologies include the efficiency with which the drug is incorporated into liposomes and the release profile of the drug from the liposomes.

Some existing methods for incorporating drugs into liposomes employ passive aqueous capture. At best, this method incorporates only 50% of the drug into the liposomes and release rates after administration are very rapid. For example, previous data indicated that oxymorphone incorporation into dehydration-rehydration vesicles comprising egg phosphatidylcholine and cholesterol was 50% efficient and subsequent animal studies using these vesicles indicated that the release time was approximately 24 hours. In additional studies, incorporation of the drug into dehydration-rehydration vesicles comprising dipalmitoylphosphatidylcholine and cholesterol was only 7% efficient, although release times were more favorable at approximately 72 hours. In both cases, release rates were most rapid at early time points, resulting in high initial plasma concentrations of the drug.

Accordingly, technologies that combine efficient incorporation of drugs into liposomes with favorable release kinetics are needed.

SUMMARY

Gradient loading technologies provide an alternative to passive aqueous capture. In general, gradient loading methods improve the efficiency with which drugs are incorporated into liposomes. One illustrative example of this approach is the use of ammonium sulfate as a liposome loading agent. In particular, liposomes are prepared in an ammonium sulfate solution, which results in liposomes having ammonium sulfate in the intraliposomal space. Ammonium sulfate is also present in the extraliposomal space (e.g., in the bulk phase). The unincorporated ammonium sulfate in the bulk phase is eliminated by washing or other techniques. Then, the drug is added to the washed liposomes comprising ammonium sulfate in the intraliposomal space. As the system equilibrates, the drug is loaded into the liposomes by the process of base exchange. This approach is 99% efficient for a drug such as doxorubicin, which precipitates as an insoluble sulfate after transport into the liposomes. However, loading is less efficient for most drugs because most drug sulfate salts are highly soluble in aqueous solutions. For example, previous studies have indicated that liposomes are loaded with opioid drugs (e.g., oxymorphone, hydromorphone, and buprenorphine) using the ammonium sulfate gradient technique at loading efficiencies of approximately 35%.

Despite such moderate loading, a benefit of ammonium sulfate loading is the improved release profile of opioids loaded into liposomes. In particular, therapeutic concentrations of opioids in the blood are maintained for 2 to 3 weeks after a single subcutaneous injection. However, as for passively loaded opioids, the early release rate is rapid, which results in undesirably high plasma concentrations for the first few days following injection.

The present invention addresses this problem by utilizing polyanions, preferably polyanions comprising 3 or more negative charges, as the liposome loading agent (e.g., counter ion) in the drug loading process. The data presented herein shows that the use of such polyanionic liposome loading agents provides the unexpected technical effect of liposomes which can provide for the extended release of drug agents at therapeutic levels in vivo of greater, than for example, 6 weeks, 7 weeks or 8 weeks and/or up to 10 weeks.

Accordingly, provided herein is technology that provides highly efficient liposome loading with drugs and an extended release rate.

In some aspects, the present disclosure provides a composition comprising liposomes loaded with a bioactive agent, the liposomes further comprising a weak loading base and a polyanionic counter ion having three or more negative charges.

In some embodiments, the weak loading base is selected from the group consisting of pyridine, a pyridine derivative, an adenine, an adenine derivative, an aniline, and an aniline derivative. In some preferred embodiments, the pyridine derivative is pyridoxine, nicotinamide, or a nicotinamide derivative. In some embodiments, the polyanionic counter ion having three or more negative charges forms a salt with the weak base.

In some embodiments, the polyanionic counter ion having three or more negative charges is selected from the group consisting of sucrose octasulfate and polyvinylsulfonate.

In some embodiments, the bioactive agent is selected from the group consisting of chloroquine, doxycycline, hydromorphone, naltrexone, and buprenorphine. In some embodiments, the bioactive agent is an opioid. In some embodiments, the bioactive agent is selected from the group consisting of an antitumor agent, an anaesthetic, an analgesic, an antimicrobial agent, an antimalarial, a hormone, an antiasthmatic agent, a cardiac glycoside, an antihypertensive, a vaccine, an antiarrhythmic, an immunomodulator, a steroid, a monoclonal antibody, a neurotransmitter, a radionuclide, a radio contrast agent, a nucleic acid, a protein, a herbicide, a pesticide, and suitable combinations thereof.

In another aspect, the present disclosure provides a composition comprising liposomes loaded with a bioactive agent selected from the group consisting of chloroquine, doxycycline, hydromorphone, naltrexone, and buprenorphine, the liposomes further comprising a weak loading base and a polyanionic counter ion with three or more negative charges.

In some embodiments, the loading base is selected from the group consisting of pyridine, a pyridine derivative, an adenine, an adenine derivative, an aniline, and an aniline derivative. In some embodiments, the pyridine derivative is pyridoxine, nicotinamide, or a nicotinamide derivative. In some embodiments, the polyanionic counter ion with three or more negative charges forms a salt with the weak base. In some embodiments, polyanionic counter ion with three or more negative charges is selected from the group consisting of sucrose octasulfate and polyvinylsulfonate.

In further aspects, the compositions described above further comprise a loading medium in the extraliposomal space, the loading medium having a pH that is greater than the pKa of the loading base.

In some embodiments, the liposomes comprise phosphatidylcholine.

In some embodiments, the liposomes comprise: a) a phosphatidylcholine selected from the group consisting of distearoylphosphatidylcholine, hydrogenated soy phosphatidylcholine, hydrogenated egg phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dielaidoylphosphatidylcholine; b) a sphingomyelin; c)a neutral lipid; or d)an acidic phospholipid. In some embodiments, the liposomes comprise dipalmitoylphosphatidylcholine and optionally cholesterol. In some embodiments, the compositions further comprise an excipient and/or a pharmaceutically acceptable carrier.

In some embodiments, the amount of bioactive agent in the intraliposomal space is greater than approximately 80%, greater than approximately 85%, greater than approximately 90%, or greater than approximately 95% of the total amount of bioactive agent in the composition.

In some embodiments, the liposomes described above release therapeutic levels of the bioactive agent over an extended period in vivo. In some embodiments, the extended period is three or more weeks. In some embodiments, the extended period is four or more weeks. In some embodiments, the extended period is five or more weeks. In some embodiments, the extended period is six or more weeks. In some embodiments, the extended period is seven or more weeks. In some embodiments, the extended period is eight or more weeks. In some embodiments, the extended period is ten or more weeks. In some embodiments, the extended period is five to eight weeks. In some embodiments, the extended period is five to ten weeks. In some embodiments, the extended period is up to eight weeks. In some embodiments, the extended period is up to ten weeks.

In further aspects, the present invention provides methods of producing liposomes, the method comprising: a) dissolving lipids in a solvent to produce a lipid solution; and b) adding an aqueous solution comprising a weak loading base and a polyanionic counter ion, or a salt thereof, with three or more negative charges to the lipid solution to produce liposomes. In some embodiments, the weak loading base is selected from the group consisting of pyridine, a pyridine derivative, an adenine, an adenine derivative, an aniline, and an aniline derivative. In some embodiments, the pyridine derivative is pyridoxine, nicotinamide, or a nicotinamide derivative. In some embodiments, the polyanionic counter ion having three or more negative charges forms a salt with the weak base. In some embodiments, the polyanionic counter ion having three or more negative charges is selected from the group consisting of sucrose octasulfate and polyvinylsulfonate.

In some embodiments, the aqueous solution comprises an active agent. In some embodiments, the bioactive agent is selected from the group consisting of chloroquine, doxycycline, hydromorphone, naltrexone, and buprenorphine. In some embodiments, the bioactive agent is an opioid. In some embodiments, the bioactive agent is selected from the group consisting of an antitumor agent, an anaesthetic, an analgesic, an antimicrobial agent, an antimalarial, a hormone, an antiasthmatic agent, a cardiac glycoside, an antihypertensive, a vaccine, an antiarrhythmic, an immunomodulator, a steroid, a monoclonal antibody, a neurotransmitter, a radionuclide, a radio contrast agent, a nucleic acid, a protein, a herbicide, a pesticide, and suitable combinations thereof.

In some embodiments, the solvent is miscible with aqueous solutions or is a solvent that has significant aqueous solubility. In some embodiments, the solvent is an alcohol. In some embodiments, the solvent is selected from the group consisting of methanol, ethanol, 1- propanol, 2-propanol, 1-butanol, 2-butanol, and t-butanol. In some embodiments, the solvent is selected from the group consisting of a C3 and a C4 alcohol. In some embodiments, the C3 alcohol is 1 -propanol. In some embodiments, the C4 alcohol is 2-butanol.

In some embodiments, the lipids comprise phospholipids. In some embodiments, the phospholipids and the solvent are combined at a concentration of about 400 mg to 800 mg phospholipids per 1 ml of solvent. In some embodiments, the phospholipids and the solvent are combined at a concentration of about 500 mg to 700 mg phospholipids per 1 ml of solvent. In some embodiments, the phospholipids and the solvent are combined at a concentration of about 550 mg to 650 mg phospholipids per 1 ml of solvent.

In some embodiments, the lipids further comprise cholesterol. In some embodiments, the cholesterol and the solvent are combined at a concentration of about 50 mg to 250 mg cholesterol per 1 ml of solvent. In some embodiments, the cholesterol and the solvent are combined at a concentration of about 100 mg to 200 mg cholesterol per 1 ml of solvent. In some embodiments, the cholesterol and the solvent are combined at a concentration of about 125 mg to 175 mg cholesterol per 1 ml of solvent.

In some embodiments, the dissolving comprises warming the lipids and solvent to 40 to 85°C. In some embodiments, the methods further comprise warming the aqueous solution of an encapsulant to 40 to 85 °C prior to addition to the lipid solution.

In some embodiments, the lipids and solvent are dissolved in a vessel to provide a dissolved lipid composition and the aqueous solution is injected into the vessel containing the dissolved lipid composition.

In some embodiments, the methods further comprise cooling the liposomes to a temperature that is below the phase transition temperature of the lipids. In some embodiments, the methods further comprise the liposomes in an aqueous solution. In some embodiments, the methods further comprise washing the liposomes to remove liposome loading agent or encapsulant from the extraliposomal space.

In still further aspects, the present invention provides methods for preparing liposomes encapsulating a bioactive agent, the method comprising: contacting the composition of liposomes prepared by a method as described above with a solution comprising a bioactive agent under conditions such that the bioactive agent is transported to the intraliposomal space of the liposomes.

In still further aspects, the present invention provides methods for preparing liposomes encapsulating a bioactive agent, the method comprising: contacting a composition comprising liposomes comprising a weak loading base and a polyanionic counter ion having three or more negative charges in the intraliposomal space with a solution comprising a bioactive agent under conditions such that the bioactive agent is transported to the intraliposomal space of the liposomes.

In some embodiments, the weak loading base is selected from the group consisting of pyridine, a pyridine derivative, an adenine, an adenine derivative, an aniline, and an aniline derivative. In some embodiments, the pyridine derivative is pyridoxine, nicotinamide, or a nicotinamide derivative. In some embodiments, the polyanionic counter ion having three or more negative charges forms a salt with the weak base. In some embodiments, the polyanionic counter ion having three or more negative charges is selected from the group consisting of sucrose octasulfate and polyvinylsulfonate. In some embodiments, the active agent is selected from the group consisting of chloroquine, doxycycline, hydromorphone, naltrexone, and buprenorphine.

In some embodiments, the present invention provides a liposome composition made by the processes described above.

In some embodiments, the liposome compositions are provided for use in treatment of disease or condition in an animal.

In some embodiments, the liposome compositions are provided for use in administration to a human or animal subject.

In some embodiments, the liposome compositions are provided for use to treat a disease or condition in a subject. In some embodiments, the disease or condition is selected from the group consisting of pain and addiction.

In some embodiments, the composition is administered at a dosage of 1 to 100 mg/kg of active agent per dose. In some embodiments, the composition is administered at a dosage of 5 to 50 mg/kg of active agent per dose. In some embodiments, the composition is administered at a dosage of 10 to 300 mg/kg of active agent per dose.

In still further aspects, the present invention provides methods of treating a disease or condition, comprising: administering a liposome composition as described above to a subject in need thereof. In some embodiments, the disease or condition is selected from the group consisting of pain and addiction. In some embodiments, the composition is administered at a dosage of 1 to 100 mg/kg of active agent per dose. In some embodiments, the liposome composition is administered at a dosage of 5 to 50 mg/kg of active agent per dose. In some embodiments, the liposome composition is administered at a dosage of 10 to 300 mg/kg of active agent per dose.

In still further aspects, the present invention provides methods of treating a subject in need of pain reduction, the method comprising: a) administering to the subject a composition as described above; and b) assessing the subject’s pain.

Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present technology will become better understood with regard to the following drawings:

FIG. 1. Serum naltrexone concentrations with respect to time in Sprague Dawley rats following a 4 mg/Kg naltrexone dose, loaded liposomes using nicotinamide eprodisate.

FIG. 2. Serum naltrexone concentrations with respect to time in Sprague Dawley rats following 5, 10 or 20 mg/Kg naltrexone doses, loaded in liposomes using nicotinamide sucrose octasulfate.

FIG. 3. Serum naltrexone concentrations with respect to time in Sprague Dawley rats following 5, 10 or 20 mg/Kg naltrexone doses, loaded in liposomes using nicotinamide polyvinyl sulfonate.

It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.

DETAILED DESCRIPTION

Provided herein is a technology related to using poly anions, preferably poly anions comprising 3 or more negative charges, as the counter ion in systems for loading drugs into liposomes. The data presented herein shows that the use of such poly anionic liposome loading agents provides the unexpected technical effect of liposomes which can provide for the extended release of drug agents at therapeutic levels in vivo of greater, than for example, 6 weeks, 7 weeks or 8 weeks and/or up to 10 weeks.

In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that the sequences can be varied and still remain within the spirit and scope of the various embodiments disclosed herein. The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belongs. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control.

Definitions

To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the technology may be readily combined, without departing from the scope or spirit of the technology.

In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and/or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, and “the” include plural references. The meaning of “in” includes “in” and “on.” The term “lipid” refers to any suitable material resulting in a bilayer such that the hydrophobic portion of the lipid material orients toward the bilayer interior while the hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, and other like groups. Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).

Amphipathic lipids often find use as the primary lipid vesicle structural element. Examples of amphipathic compounds are phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid. Additionally, the amphipathic lipids described above may be mixed with other lipids including triacyglycerols and sterols.

“Phospholipid” refers to any one phospholipid or combination of phospholipids capable of forming liposomes. Phosphatidylcholines (PC), including those obtained from egg, soybeans, or other plant sources or those that are partially or wholly synthetic, or of variable lipid chain length and unsaturation find use in embodiments of the present technology. Synthetic, semisynthetic, and natural product phosphatidylcholines including, but not limited to, distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (soy PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC), dipalmitoylphosphatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC) are suitable phosphatidylcholines for use in this technology. All of these phospholipids are commercially available.

Further, phosphatidylglycerols (PG) and phosphatic acid (PA) are also suitable phospholipids for use in the present technology and include, but are not limited to, dimyristoylphosphatidylglycerol (DMPG), dilaurylphosphatidylglycerol (DLPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG) dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dilaurylphosphatidic acid (DLPA), and dipalmitoylphosphatidic acid (DPPA). Other suitable phospholipids include phosphatidylethanolamines, phosphatidylinositols, and phosphatidic acids containing lauric, myristic, stearoyl, and palmitic acid chains. Further, incorporation of polyethylene glycol (PEG) containing phospholipids is also contemplated by the present technology. It is contemplated by this technology to include cholesterol optionally in the liposomal formulation. Cholesterol is known to improve liposome stability and prevent loss of phospholipid to lipoproteins in vivo.

“Unilamellar liposomes”, also referred to as “single lamellar vesicles,” are spherical vesicles that include one lipid bilayer membrane that defines a single closed aqueous compartment. The bilayer membrane includes two layers (or “leaflets”) of lipids; an inner layer and an outer layer. The outer layer of the lipid molecules is oriented with the hydrophilic head portions toward the external aqueous environment and the hydrophobic tails pointed downward toward the interior of the liposome. The inner layer of the lipid lay directly beneath the outer layer with the lipids oriented with the heads facing the aqueous interior of the liposome and the tails oriented toward the tails of the outer layer of lipid.

“Multilamellar liposomes” also referred to as “multilamellar vesicles” or “multiple lamellar vesicles,” include more than one lipid bilayer membrane, which membranes define more than one closed aqueous compartment. The membranes are concentrically arranged so that the different membranes are separated by aqueous compartments, much like an onion.

The terms “bioactive agent” and “pharmaceutical agent” (e.g., a “drug”) are used interchangeably and include but are not limited to, an antibiotic, an analgesic, an anesthetic, an antiacne agent, an antibiotic, an antibacterial, an anticancer agent, an anticholinergic, an anticoagulant, an antidyskinetic, an antiemetic, an antifibrotic, an antifungal, an antiglaucoma agent, an anti-inflammatory, an antineoplastic, an antios teoporotic, an antipagetic, an antiParkinson’s agent, an antisporatic, an antipyretic, an antiseptic, an antithrombotic, an antiviral, an antimalarial, an antiparasitic, a calcium regulator, a keratolytic, and/or a sclerosing agent.

The terms “encapsulation” and “entrapped,” as used herein, refer to the incorporation or association of a biologically active (e.g., a pharmaceutical agent) in or with a liposome. The pharmaceutical agent may be associated with the lipid bilayer or present in the aqueous interior (“intraliposomal space”) of the liposome, or both. In one embodiment, a portion of the encapsulated pharmaceutical agent takes the form of a precipitated salt in the interior of the liposome. The pharmaceutical agent may also self-precipitate in the interior of the liposome. As used herein, a “liposome loading agent” refers to a substance (e.g., chemical, molecule, etc.) that promotes the movement of another substance (e.g., a drug) into the intraliposomal space (e.g., the lumen) of a liposome. The liposome loading agent may preferably be a counterion or counterion excipient that can initiate or facilitate drug loading and may also initiate or facilitate precipitation of the pharmaceutical agent in the aqueous interior of the liposome. Examples of liposome loading agents include, but are not limited to, weak bases and salts thereof as well as the acid, sodium or ammonium forms of monovalent anions such as chloride, acetate, lactobionate and formate; divalent anions such as aspartate, succinate and sulfate; and bivalent ions such as citrate and phosphate.

As used herein, the term “log P” refers to the logarithm of the partition coefficient (P) describing the ratio of concentrations of a compound in a mixture of two immiscible phases at equilibrium. In particular, the log P as used herein refers to the distribution between two reference phases, e.g., water and a non-aqueous-miscible liquid, e.g., an organic solvent, e.g., n-octanol. In some embodiments, logP values are theoretically calculated, e.g., using a group additivity approach or, in some embodiments, a method including factors such as dipole moment, molecular size, molecular shape, etc. See, e.g., Viswanadhan, et al (1989, J. Chem. Inf. Comput. Sci. 29(3): 163; Suzuki and Kudo (1990), J. Comput. Aided Mol. Des. 4(2): 155- 98, each incorporated herein by reference. Thus, the partition coefficient is a measure of how hydrophilic or hydrophobic a chemical substance is. Accordingly, partition coefficients are useful in estimating the distribution of drugs within the body.

As used herein, the “loading efficiency” refers to the amount of a substance that is incorporated into a liposome (e.g., in the intraliposomal space) by a liposome loading process relative to the total amount of the substance added. Generally, a liposome preparation is prepared for loading with a substance such as a drug; then, a known initial amount of the substance (e.g., drug) is added to the preparation. The substance (e.g., drug) is initially in the extraliposomal space and some amount of the substance (e.g., drug) moves into and becomes entrapped in the intraliposomal space. The ratio (e.g., expressed as a percentage, fraction, ratio, etc.) of entrapped substance (e.g., drug) relative to the known initial amount of the substance (e.g., drug) is a measure of the loading efficiency.

As used herein, “treat” or “treating” refers to: (i) preventing a pathologic condition (e.g., breast cancer; sepsis) from occurring (e.g. prophylaxis) or preventing symptoms related to the same; (ii) inhibiting the pathologic condition or arresting its development or inhibiting or arresting symptoms related to the same; or (iii) relieving the pathologic condition or relieving symptoms related to the same. As used herein, the terms “subject” and “patient” refer to any animal, such as a mammal like a dog, cat, bird, livestock, and preferably a human.

As used herein, the term “effective amount” refers to the amount of a composition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.

As used herein, the term “administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject. Exemplary routes of administration to the human body can be through the eyes (ophthalmic), mouth (oral), skin (transdermal, topical), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.), and the like.

As used herein, the term “pharmaceutical composition” refers to the combination of a biological agent with a carrier, inert or active, making the composition especially suitable for therapeutic use.

The terms “pharmaceutically acceptable” or “pharmacologically acceptable”, as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

As used herein, the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a disease or disorder through introducing in any way a therapeutic composition of the present technology into or onto the body of a subject. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

As used herein, “therapeutically effective dose” refers to an amount of a therapeutic agent sufficient to bring about a beneficial or desired clinical effect. The dose can be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired (e.g., aggressive versus conventional treatment). Description

The technology provided herein relates to the use of polyanions as the counterion to load drugs efficiently into liposomes. In some aspects, the poly anion counterions are used in conjunction with weak bases.

Liposomes

Liposomes, or lipid vesicles, are used for drug delivery to improve the therapeutic activity and increase the safety of a number of different pharmaceutical agents. Liposomal carrier systems (e.g., vesicles) are microscopic spheres of one or more lipid bilayers arranged around an aqueous core. The vesicles have been shown to be suitable as carriers for both hydrophilic and hydrophobic therapeutic agents owing to their unique combination of lipophilic and hydrophilic portions.

Liposomes are completely closed lipid bilayer membranes containing an entrapped volume. The bilayer membrane separates this surrounded volume (the “intraliposomal space” or “lumen”) from the bulk phase (the “extraliposomal space”). Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilameller vesicles (onion- like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). Liposomes may take other forms as well, e.g., multivesicular liposomes (MVL), which are lipid vesicles with multiple internal aqueous chambers formed by non- concentric layers and having internal membranes distributed as a network throughout the MVL.

In these various forms, the bilayer is composed of two lipid monolayers having a hydrophobic “tail” region and a hydrophilic “head” region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) “tails” of the lipid monolayers orient toward the center of the bilayer while the hydrophilic “heads” orient towards the aqueous phase.

Liposome formation

In a conventional liposome preparation such as that of Bangham et al. (J. Mol. Biol., 1965, 13: 238-252), phospholipids were suspended in an organic solvent that was then evaporated to dryness to leave a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase was added, the mixture was allowed to “swell”, and the resulting MLVs were dispersed by mechanical means to produce multilamellar vesicles. This preparation provided the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys, Acta., 1967, 135: 624-638) and multilamellar vesicles.

Subsequently, techniques for producing large unilamellar vesicles (LUVs) such as reverse phase evaporation, infusion procedures, and detergent dilution were used to produce liposomes. A review of these and other methods for producing liposomes may be found in the text Liposomes, Marc Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1. See also Szoka Jr. et al., (1980, Ann. Rev. Biophys. Bioeng., 9:467). One particular method for forming LUVs is described in Cullis et al., PCT Publication No. 87/00238, Jan. 16, 1986, entitled “Extrusion Technique for Producing Unilamellar Vesicles”.

In some embodiments, liposomes that are used in the present technology are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size and stability of the liposomes in the bloodstream.

Various types of lipids are used to produce liposomes. For example, amphipathic lipids that find use are zwitterionic, acidic, or cationic lipids. Examples of zwitterionic amphipathic lipids are phosphatidylcholines, phosphatidyl-ethanolamines, sphingomyelins, etc. Examples of acidic amphipathic lipids are phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, phosphatidic acids, etc. Examples of cationic amphipathic lipids are diacyl trimethylammonium propanes, diacyl dimethylammonium propanes, stearylamine, etc. Examples of neutral lipids include diglycerides, such as diolein, dipalmitolein, and mixed caprylin-caprin; triglycerides, such as triolein, tripalmitolein, trilinolein, tricaprylin, and trilaurin; and combinations thereof. Additionally, cholesterol or plant sterols are used in some embodiments, e.g., to make multivesicular liposomes.

In some embodiments, the major lipid component in the liposomes is phosphatidylcholine. Phosphatidylcholines having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well- known techniques. In general, less saturated phosphatidylcholines are more easily sized, particularly when the liposomes must be sized below approximately 0.3 microns, e.g., for purposes of filter sterilization. In some embodiments, phosphatidylcholines containing saturated fatty acids with carbon chain lengths in the range of approximately Cu to C22 are preferred. Phosphatidylcholines with monounsaturated or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids are used in some embodiments. Other suitable lipids include phosphonolipids in which the fatty acids are linked to glycerol via ether linkages rather than ester linkages (e.g., as found in some members of the Archaea). Liposomes useful in the present technology may also be composed of sphingomyelin or phospholipids with head groups other than choline, such as ethanolamine, serine, glycerol, and inositol. In some embodiments, liposomes include a sterol, preferably cholesterol, at molar ratios of from 0. 1 to 1 .0 of the cholesterol to the phospholipid). In some embodiments, the liposome compositions are distearoylphosphatidylcholine/ cholesterol, dipalmitoylphosphatidylcholine/ cholesterol, or sphingomyelin/ cholesterol. Methods used in sizing and filter-sterilizing liposomes are provided below.

A variety of methods are available for preparing liposomes as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871; 4,501,728; and 4,837,028; the text Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1, and Hope, et al., Chem. Phys. Lip. 40:89 (1986), each of which is incorporated herein by reference. One exemplary method produces multilamellar vesicles of heterogeneous sizes. In this method, the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film. Alternatively, the lipids may be dissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture that is in a more easily hydrated, microporous, powder-like form. This film or powder is covered with an aqueous solution (e.g., in some embodiments, an aqueous buffered solution) and allowed to hydrate, typically over a 15-60 minute period with agitation. The size distribution of the resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate.

Many different types of organic solvents such as ethers, hydrocarbons, halogenated hydrocarbons, and/or freons are used in some embodiments as the solvent in the lipid component. For example, diethyl ether, isopropyl ether, and other ethers; chloroform; tetrahydrofuran; halogenated ethers; esters, and combinations thereof find use in the present technology.

Several techniques are available for sizing liposomes to a desired size. One sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles less than approximately 0.05 microns in size. Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between approximately 0.1 and 0.5 microns, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination.

In some embodiments, extrusion of liposomes through a small-pore polycarbonate membrane or an asymmetric ceramic membrane provides an effective method for reducing liposome sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes to achieve a gradual reduction in liposome size. In some embodiments comprising use of extrusion methods, liposomes find use that have a size of from approximately 0.05 microns to approximately 0.15 microns. In some embodiments, liposomes are not extruded. For example, in some embodiments the liposomes are approximately 1 micron to 10 microns in diameter. While many technologies and sizes for liposomes are discussed herein, the technology is not dependent on the size of the liposomes; accordingly, there is no size preference for the liposome loading technology per se.

In some embodiments, liposomes are prepared, for example, by weighing out a quantity of a phosphatidylcholine (optionally cholesterol and/or optionally a phosphatidylglycerol) and dissolving them in an organic solvent, e.g., chloroform and methanol in a 1 :1 mixture (v/v) or alternatively in neat chloroform. The solution is evaporated to form a solid lipid phase such as a film or a powder, for example, with a rotary evaporator, spray dryer, or other method. The film or powder is then hydrated with an aqueous solution optionally containing an excipient and having a pH range from approximately 2.0 to approximately 7.4 to form a liposome dispersion. The lipid film or powder dispersed in the aqueous solution is heated to a temperature from approximately 25°C to approximately 70°C depending on the phospholipids used.

Multilamellar liposomes are formed, e.g., by agitation of the dispersion, preferably through the use of a thin-film evaporator apparatus such as is described in U.S. Pat. No. 4,935,171 or through shaking or vortex mixing. Unilamellar vesicles are formed by the application of a shearing force to an aqueous dispersion of the lipid solid phase, e.g., by sonication or the use of a microfluidizing apparatus such as a homogenizer or a French press. Shearing force can also be applied using injection, freezing and thawing, dialyzing away a detergent solution from lipids, or other known methods used to prepare liposomes. The size of the liposomes can be controlled using a variety of known techniques including controlling the duration of shearing force. In some embodiments, a homogenizing apparatus is employed to produce unilamellar vesicles having diameters of less than 200 nanometers at a pressure of 3,000 to 14,000 psi (e.g., 10,000 to 14,000 psi) and a temperature that is approximately at the aggregate transition temperature of the lipids.

In some exemplary embodiments, liposomes are prepared as described below in the Methods section of the included Examples.

According to some embodiments, liposomes are produced by combining lipids in chloroform, removing solvent to create a component mixture, suspending the lipid in a suitable liquid (e.g., an alcohol such as, e.g., t-butanol), and lyophilizing the suspension. Then, according to some embodiments for loading liposomes with a bioactive agent, the microporous lipid mass is subsequently hydrated using a weak base salt (e.g., a salt of a polyanion and a weak base).

In addition, provided herein are embodiments of the technology that eliminate the first steps (e.g., dissolving lipids in chloroform and removing the solvent) commonly used for the preparation of liposomes. For example, some embodiments comprise a step of dissolving lipids in a suitable liquid (e.g., an alcohol such as, e.g., t-butanol) directly without a preceding step of mixing the lipid components in chloroform. In some embodiments, dissolving lipids in a suitable liquid (e.g., an alcohol such as, e.g., t-butanol) is associated with heating the liquid to facilitate dissolving the lipid in the liquid. In some embodiments, the heating comprises providing an amount of heat to the liquid (e.g., a liquid comprising the lipid) that raises the temperature of the liquid sufficiently to dissolve the lipids therein (e.g. raising the temperature by 1 to 60 degrees (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 degrees Celsius).

Additional embodiments of liposome preparation methods provided herein eliminate the lyophilizing step. For example, some embodiments prepare liposomes directly from lipids provided as dry powders. In particular, some embodiments of the methods described herein comprise providing lipids (e.g., phospholipid, cholesterol, etc.) as powders, dissolving the lipids in a suitable liquid (e.g., an alcohol such as, e.g., 1- or 2-propanol), and adding a base salt (e.g., a salt of the polyanion and weak base) to the lipid solution to produce liposomes. In some embodiments, dissolving lipids in a suitable liquid (e.g., an alcohol such as, e.g., 1- or 2-propanol) is associated with heating the liquid to facilitate dissolving the lipid in the liquid. In some embodiments, the heating comprises providing an amount of heat to the liquid (e.g., a liquid comprising the lipid) that raises the temperature of the liquid sufficiently to dissolve the lipids therein (e.g. raising the temperature by 1 to 20 degrees (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 degrees Celsius).

Accordingly, in some embodiments, the present invention provides of producing liposomes, the method comprising: a) dissolving lipids in a solvent to produce a lipid solution; and b) adding an aqueous solution, preferably comprising the polyanion and weak base or a salt thereof to the lipid solution to produce liposomes. In some embodiments, the solvent is miscible with aqueous solutions or is a solvent that has significant aqueous solubility. In some embodiments, the solvent is an alcohol. In some embodiments, the solvent is selected from the group consisting of methanol, ethanol, 1 -propanol, 2-propanol, 2- butanol, 1 -butanol and t-butanol. In some embodiments, the solvent is selected from the group consisting of a C3 and a C4 alcohol. In some embodiments, the C3 alcohol is 1- propanol. In some embodiments, the C4 alcohol is 2-butanol.

In some embodiments, the dissolving step comprises warming the lipids and solvent to 40 to 85°C or 40 to 70°C. In some embodiments, the methods further comprise warming the aqueous solution to 40 to 85 °C or 40 to 70°C prior to addition to the lipid solution. In some embodiments, the lipids and solvent are dissolved in a vessel to provide a dissolved lipid composition and the aqueous solution is injected into the vessel containing the dissolved lipid composition. In some embodiments, the methods further comprise cooling the liposomes to a temperature that is below the phase transition temperature of the lipids.

In some embodiments, the aqueous solution comprises a liposome loading agent as described elsewhere herein. In some embodiments, the aqueous solution is selected from the group consisting of an acidic solution and a basic solution. In some embodiments, the aqueous solution comprises liposome loading agent selected from the group consisting of counter ions and salts thereof. In some embodiments, the aqueous solution comprises a weak base salt selected from the group consisting of a sulfate, an eprodisate, and an edisylate. In some embodiments, adding the weak base salt to the lipid solution comprises adding a first volume of the weak base salt to the lipid solution followed by adding a second volume of the weak base salt to the lipid solution. In some embodiments, the ratio of the first volume to the second volume is 5: 1 to 1:5.

In some embodiments, the aqueous solution comprises an encapsulant, e.g., a compound or molecule that is designated to be encapsulated into the intraliposomal space. Examples of encapsulants, and particularly bioactive agents, are described in detail herein. In some embodiments, the encapsulant is selected from the group consisting of a chemical bioactive agent and a biologic bioactive agent. In some embodiments, the chemical bioactive agent is an analgesic.

In some embodiments, the methods further comprise diluting the liposomes in an aqueous solution. In some embodiments, the methods further comprise washing the liposomes to remove liposome loading agents or encapsulants, if utilized, from the extraliposomal space.

In some embodiments, the present invention provides methods for preparing liposomes encapsulating a bioactive agent, the method comprising providing a composition of liposomes prepared with a liposome loading agent as described above and adding a bioactive agent to the composition of liposomes under conditions such that the bioactive agent is transported to the intraliposomal space of the liposomes.

In some embodiments, the present invention provides a liposome composition made by the methods described above. In some embodiments, the liposome compositions are used for treatment of disease or condition in an animal.

Liposome loading

Liposomes find use in pharmaceutical preparations, e.g., to improve the characteristics (e.g., bioavailability, pharmacokinetics, toxicity, etc.) of a drug or other bioactive agent (“pharmaceutical agent”) when administered to a patient. In particular, therapies employing bioactive agents can in many cases be improved by encapsulating the agent in liposomes rather than administering the free agent directly into the body. For example, incorporation of such agents in liposomes can change their activities, clearance rates, tissue distributions, and toxicities compared to direct administration. Liposomes themselves have been reported to have no significant toxicities in previous human clinical trials where they have been given intravenously. See. e.g., Richardson et al., (1979), Br. J. Cancer 40:35; Ryman et al., (1983) in “Targeting of Drugs” G. Gregoriadis, et al., eds. pp 235-248, Plenum, N.Y.; Gregoriadis G., (1981), Lancet 2:241, and Lopez-Berestein et al., (1985) J. Infect. Dis., 151 :704. Liposomes are reported to concentrate predominantly in the reticuloendothelial organs lined by sinusoidal capillaries, e.g., liver, spleen, and bone marrow, and phagocytosed by the phagocytic cells present in these organs.

When liposomes are used in a liposome drug delivery system, a bioactive agent such as a drug is entrapped in the liposome and then administered to the patient to be treated. For example, see Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Paphadjopoulos et al., U.S. Pat. No. 4.235,871; Schneider, U.S. Pat. No. 4,224.179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578. Alternatively, if the bioactive agent is lipophilic, it may associate with the lipid bilayer. Typically, the term “entrapment” includes both the drug in the aqueous volume of the liposome as well as drug associated with the lipid bilayer.

Liposome formulations for pharmaceutical applications can be made either by combining drug and lipid before formation of the vesicles or by “loading” lipid vesicles with drug after the liposomes have been formed. Upon administration to a patient, liposomes biodistribute and interact with cells in the body according to route of administration, vesicular composition, and vesicular size. Charge, chemistry, and bilayer components (e.g., the inclusion on the vesicle surface of protective polymers or targeting moieties) all change the way liposomes behave in the patient.

In some embodiments, the pharmaceutical agent is loaded into pre-formed liposomes using a loading procedure, for example, by using a pH gradient. In some embodiments, the pharmaceutical agent may precipitate in the interior of the liposome. This precipitation protects the pharmaceutical agent and the lipids from degradation (e.g., hydrolysis). In some embodiments, an excipient such as citrate or sulfate precipitates the pharmaceutical agent and can be utilized in the interior of the liposomes together with a gradient to promote drug loading.

In some embodiments, liposomal entrapment of bioactive agents is effected by employing transmembrane ion gradients (see, e.g., Int’l Pat. Appl. PCT/US1985/001501). Aside from inducing uptake, such transmembrane gradients also act to increase drug retention in the liposomes. For example, transmembrane pH gradients (ApH) influence the drug loading of certain weak acids and weak bases. See, for example, Jacobs, Quant. Biol. 8:30-39 (1940), Chapper, et al. in Regulation of Metabolic Processes in Mitochondria, Tager, et al. eds. Elsevier, Amsterdam, pp. 293-316 (1966), Crofts, J. Biol. Chem. 242:3352-3359 (1967), Crofts, Regulatory Functions of Biological Membranes, Jamefelt, ed., Elsevier Publishing Co., Amsterdam, pp. 247-263 (1968), Rottenberg, Bioenergetics 7:61-74 (1975), and Rottenberg, Methods in Enzmol. 55:547-569 (1979).

Liposome loading agents

The technology is not limited in the counter ion used for loading liposomes. In some preferred embodiments, the counter ion is a polyanion comprising three or more (for example, 3, 4, 5, 6, 7, 8 or more) negative charges. Exemplary polyanions include, but are not limited to, octasulfates (e.g., sucrose octasulfate) and polyvinylsulfonate.

The technology is not limited in the weak base used for loading liposomes. As used herein, a weak base is a chemical base that does not completely ionize (e.g., in an aqueous solution), e.g., a chemical base that is partially protonated (e.g., in an aqueous solution). Embodiments relate to the use of a weak base salt, e.g., a salt produced by the ionization of a weak base, e.g., by the acid form of the poly anion. Exemplary weak bases include pyridine, 2-methoxypyridine, pyridazine, adenine, aniline, pyridoxine, and nicotinamide and derivatives thereof. Furthermore, the technology is not limited in the phase of the weak base used for loading liposomes. For example, both adenine (solid at room temperature) and aniline (liquid at room temperature) find use in embodiments of the technology.

Pyridine is a heterocyclic organic base having a pKa of 5.3. Pyridine is a liquid that is miscible with water at all ratios. Pyridine is hydrophobic in the neutral form, with an estimated log P of approximately 1.3. When added to an acid solution, pyridine is protonated to form a pyridinium salt; the pH varies over the range of 3 to 7 depending on the proportion of pyridine to acid used.

2-methoxypyridine is a pyridine derivative having a pKa of 3.28. 2-methoxypyridine is a liquid with limited solubility in water. 2-methoxypyridine is hydrophobic in the neutral form, with an estimated log P of approximately 1.3 (similar to pyridine). When added to an acid solution, 2-methoxypyridine is protonated to form a highly soluble 2- methoxypyridinium salt; the pH varies over the range of 1 to 4 depending on the proportion of 2-methoxypyridine to acid used.

Pyridazine is a heterocyclic organic base having a pKa of 2.33. Pyridazine is a liquid that is miscible with water at all ratios. In contrast to pyridine, pyridazine is hydrophilic in the neutral form, with an estimated log P of approximately -0.7. When added to a an acid solution, pyridazine is protonated to form a pyridazinium salt; the pH varies over the range of 1 to 3 depending on the proportion of pyridazine to acid used.

Adenine (6-aminopurine) is a purine derivative having a pKa of 4.15. Adenine is a solid that forms soluble salts with acids.

Aniline (phenylamine, aminobenzene) is an organic compound with a pKa of 4.19. Aniline is a hydrophobic liquid that reacts with strong acids to form anilinium (phenylammonium) ions and that forms soluble salts with acids.

In preferred embodiments, a weak base salt of the polyanion and weak base is formed. In these embodiments, an acid form of the polyanion is generated, for example, by passage of the sodium salt of the poly anion over an appropriate column followed by regeneration with an acid such as HC1. Examples of weak base salts include, but are not limited to, nicotinamide sucrose octasulfate, nicotinamide polyvinyl sulfonate, pyridinium sucrose octasulfate, pyridinium polyvinyl sulfonate, 2-methoxypyridinium sucrose octasulfate, 2- methoxypyridinium polyvinyl sulfonate, pyridazinium sucrose octasulfate, and pyridazinium polyvinyl sulfonate.

In particular embodiments, a composition comprising liposomes is made using a weak base salt solution having a pH of at least 2, e.g., to allow for swelling of phospholipid (e.g., dipalmitoylphosphatidylcholine) during incubation (e.g., at 35 to 55°C, e.g., approximately 40°C, e.g., 42°C). It is further preferred that the pH of the weak base salt solution is below the pKa of the weak base to limit the amount of free base in the solution, thus stabilizing the liposome membranes. In some embodiments, liposomes are prepared in the weak base salt solution and excess weak base is eliminated by standard washing techniques (e.g., sedimentation in a centrifuge, dialysis, gel chromatography, etc.). Then, a drug, bioactive agent, pharmaceutical agent, etc. is added to the composition comprising liposomes. Incubating the composition comprising the drug and the liposomes for a period of at least 30 minutes to 72 hours (e.g., at least 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3.0 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 12 hours, 24 hours, 48 hours, 72 hours or from 6 hours to 24 hours, 12 hours to 24 hours, 24 to 48 hours, 6 hours to 72 hours, 12 hours to 72 hours or 24 to 72 hours) produces a composition comprising liposomes loaded with the drug (e.g., the drug moves into the liposomes (e.g., into the intraliposomal space)). Manipulation and control of the extraliposomal (e.g., bulk) phase pH provides for control of drug loading (e.g., to maximize loading (e.g., to maximize efficiency of drug loading)). For example, in some embodiments a buffer is provided in the extraliposomal phase to control the extraliposomal pH. For example, in some embodiments a buffer is added to particular compositions to maintain the extraliposomal pH at a value at which the drug to be loaded is predominantly in the protonated (e.g., charged) form. For example, for drugs that are weak bases, the protonated form is a charged form.

In some embodiments, the preferable external loading pH for pyridine salts is 6 to 8; further, in some embodiments, the external loading pH is lower than 6 for compositions comprising 2-methoxypyridine or pyridazine (but greater than the pKa of the 2- methoxypyridine or pyridazine).

Bioactive agents

In some embodiments, biological substances and/or therapeutic agents (e.g., “drugs”) are incorporated by encapsulation within liposomes (e.g., in the intraliposomal space). Examples of bioactive agents include but are not limited to antianginas, antiarrhythmics, antiasthmatic agents, antibiotics, antimalarials, antidiabetics, antifungals, antihistamines, antihypertensives, antiparasitics, antineoplastics, antivirals, cardiac glycosides, herbicides, hormones, immunomodulators, antibodies (e.g., monoclonal, human, humanized, chimeric, etc., antibodies), neurotransmitters, nucleic acids, pesticides, proteins, radio contrast agents, radionuclides, sedatives, analgesics, steroids, tranquilizers, vaccines, vasopressors, anesthetics, and/or peptides.

The drugs that can be incorporated into the dispersion system as therapeutic agents include chemicals as well as biologies. The term “chemicals” encompasses compounds that are classically referred to as drugs, such as antitumor agents, anesthetics, analgesics, antimicrobial agents, opiates, hormones, etc.

Of particular interest for inclusion in the liposome compositions of the present technology are analgesics, e.g., opiates and/or opioids (e.g., hydromorphone and buprenorphine), opioid antagonists (e.g., naltrexone), and quinoline drugs (e.g., a 4- aminoquinoline such as chloroquine).

The term “biologies” encompasses nucleic acids (e.g., DNA and RNA), proteins and peptides, and includes compounds such as cytokines, hormones (e.g., pituitary and hypophyseal hormones), growth factors, vaccines, etc.

Suitable antibiotics for inclusion in the liposome compositions of the present technology include, but are not limited to, loracarbef, cephalexin, cefadroxil, cefixime, ceftibuten, cefprozil, cefpodoxime, cephradine, cefuroxime, cefaclor, neomycin/polymyxin/bacitracin, dicloxacillin, nitrofurantoin, nitrofurantoin macrocrystal, nitrofurantoin/nitrofuran mac, dirithromycin, gemifloxacin, ampicillin, gatifloxacin, penicillin V potassium, ciprofloxacin, enoxacin, amoxicillin, amoxicillin/clavulanate potassium, clarithromycin, levofloxacin, moxifloxacin, azithromycin, sparfloxacin, cefdinir, ofloxacin, trovafloxacin, lomefloxacin, methenamine, erythromycin, norfloxacin, clindamycin/benzoyl peroxide, quinupristin/dalfopristin, doxycycline, amikacin sulfate, vancomycin, kanamycin, netilmicin, streptomycin, tobramycin sulfate, gentamicin sulfate, tetracyclines, framycetin, minocycline, nalidixic acid, demeclocy cline, trimethoprim, miconazole, colistimethate, piperacillin sodium/tazobactam sodium, paromomycin, colistin/neomycin/hydrocortisone, amebicides, sulfisoxazole, pentamidine, sulfadiazine, clindamycin phosphate, metronidazole, oxacillin sodium, nafcillin sodium, vancomycin hydrochloride, clindamycin, cefotaxime sodium, co-trimoxazole, ticarcillin disodium, piperacillin sodium, ticarcillin disodium/clavulanate potassium, neomycin, daptomycin, cefazolin sodium, cefoxitin sodium, ceftizoxime sodium, penicillin G potassium and sodium, ceftriaxone sodium, ceftazidime, imipenem/cilastatin sodium, aztreonam, cinoxacin, erythromycin/sulfisoxazole, cefotetan disodium, ampicillin sodium/sulbactam sodium, cefoperazone sodium, cefamandole nafate, gentamicin, sulfisoxazole/phenazopyridine, tobramycin, lincomycin, neomycin/polymyxin B/gramicidin, clindamycin hydrochloride, lansoprazole/clarithromycin/amoxicillin, alatrofloxacin, linezolid, bismuth subsalicylate/metronidazole/tetracycline, erythromycin/benzoyl peroxide, mupirocin, fosfomycin, pentamidine isethionate, imipenem/cilastatin, troleandomycin, gatifloxacin, chloramphenicol, cycloserine, neomycin/polymyxin B/hydrocortisone, ertapenem, meropenem, cephalosporins, fluconazole, cefepime, sulfamethoxazole, sulfamethoxazole/trimethoprim, neomycin/polymyxin B, penicillins, rifampin/isoniazid, erythromycin estolate, erythromycin ethylsuccinate, erythromycin stearate, ampicillin trihydrate, ampicillin/probenecid, sulfasalazine, sulfanilamide, sodium sulfacetamide, dapsone, doxycycline hyclate, trimenthoprim/sulfa, methenamine mandelate, plasmodicides, pyrimethamine, hydroxychloroquine, chloroquine phosphate, chloroquine diphosphate, trichomonocides, anthelmintics, atovaquone.

Pharmaceutical preparations

In some embodiments, liposome compositions prepared by the methods described herein are administered alone or in a mixture with a physiologically-acceptable carrier (such as physiological saline or phosphate buffer) selected in accordance with the route of administration and standard pharmaceutical practice. Generally, normal saline is employed as the pharmaceutically acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. In compositions comprising saline or other saltcontaining carriers, the carrier is preferably added following liposome formation. Thus, after the liposome is formed and loaded with a suitable drug, the liposome can be diluted into pharmaceutically acceptable carriers such as normal saline. These compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

Additionally, the composition may include lipid-protective agents that protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as alpha- tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.

The concentration of liposomes in the pharmaceutical formulations can vary widely, e.g., from less than approximately 0.05%, usually at least approximately 2 to 5% to as much as 10 to 30% by weight and are selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, liposomes composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration. The amount of liposomes administered will depend upon the particular drug used, the disease state being treated and the judgment of the clinician but will generally be between approximately 0.01 and approximately 50 mg per kilogram of body weight, preferably between approximately 0.1 and approximately 5 mg per kg of body weight.

In some embodiments, it is desirable to include polyethylene glycol (PEG)-modified phospholipids, PEG-ceramide, or ganglioside GMI -modified lipids to the liposomes. Addition of such components prevents liposome aggregation and provides for increasing circulation lifetime and increasing the delivery of the loaded liposomes to the target tissues. Typically, the concentration of the PEG-modified phospholipids, PEG-ceramide, or GMI -modified lipids in the liposome will be approximately 1 to 15%. In some embodiments, overall liposome charge is an important determinant in liposome clearance from the blood. Charged liposomes are typically taken up more rapidly by the reticuloendothelial system (Juliano, Biochem. Biophys. Res. Commun. 63: 651 (1975)) and thus have shorter half-lives in the bloodstream. Liposomes with prolonged circulation half-lives are typically desirable for therapeutic and certain diagnostic uses. For instance, liposomes that are maintained from 8, 12, or up to 24 hours in the bloodstream are particularly preferred.

In another example of their use, drug-loaded liposomes can be incorporated into a broad range of topical dosage forms including but not limited to gels, oils, emulsions, and the like. For instance, in some embodiments the suspension containing the drug-loaded liposomes is formulated and administered as a topical cream, paste, ointment, gel, lotion, and the like.

The present technology also provides liposome compositions in kit form. The kit will typically comprise a container that is compartmentalized for holding the various elements of the kit. The kit contains the compositions of the present inventions, preferably in dehydrated form, with instructions for their rehydration and administration.

In still other embodiments, the drug-loaded liposomes have a targeting moiety attached to the surface of the liposome. Methods of attaching targeting moieties (e.g., antibodies, proteins) to lipids (such as those used in the present particles) are known to those of skill in the art.

Dosage for the drug-loaded liposome formulations depends on the ratio of drug to lipid and the administrating physician’s and/or veterinarian’s opinion based on age, weight, and condition of the patient.

In some embodiments, compositions comprising liposomes encapsulating a bioactive agent are formulated with a buffering agent. The buffering agent may be any pharmaceutically acceptable buffering agent. Buffer systems include citrate buffers, acetate buffers, borate buffers, and phosphate buffers. Examples of buffers include citric acid, sodium citrate, sodium acetate, acetic acid, sodium phosphate and phosphoric acid, sodium ascorbate, tartaric acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic acid, imidazole, sodium bicarbonate and carbonic acid, sodium succinate and succinic acid, histidine, and sodium benzoate and benzoic acid.

In some embodiments, compositions comprising liposomes encapsulating a bioactive agent are formulated with a chelating agent. The chelating agent may be any pharmaceutically acceptable chelating agent. Chelating agents include ethylenediaminetetraacetic acid (also synonymous with EDTA, edetic acid, versene acid, and Sequestrene), and EDTA derivatives, such as dipotassium edetate, disodium edetate, edetate calcium disodium, sodium edetate, trisodium edetate, and potassium edetate. Other chelating agents include citric acid and derivatives thereof. Citric acid also is known as citric acid monohydrate. Derivatives of citric acid include anhydrous citric acid and trisodiumcitrate-dihydrate. Still other chelating agents include niacinamide and derivatives thereof and sodium deoxycholate and derivatives thereof.

In some embodiments, compositions comprising liposomes encapsulating a bioactive agent are formulated with an antioxidant. The antioxidant may be any pharmaceutically acceptable antioxidant. Antioxidants are well known to those of ordinary skill in the art and include materials such as ascorbic acid, ascorbic acid derivatives (e.g., ascorbylpalmitate, ascorbylstearate, sodium ascorbate, calcium ascorbate, etc.), butylated hydroxy anisole, buylated hydroxy toluene, alkylgallate, sodium meta-bisulfate, sodium bisulfate, sodium dithionite, sodium thioglycollic acid, sodium formaldehyde sulfoxylate, tocopherol and derivatives thereof, (d-alpha tocopherol, d-alpha tocopherol acetate, dl-alpha tocopherol acetate, d-alpha tocopherol succinate, beta tocopherol, delta tocopherol, gamma tocopherol, and d-alpha tocopherol polyoxyethylene glycol 1000 succinate) monothioglycerol, and sodium sulfite. Such materials are typically added in ranges from 0.01 to 2.0%.

In some embodiments, compositions comprising liposomes encapsulating a bioactive agent are formulated with a cryoprotectant. The cryoprotecting agent may be any pharmaceutically acceptable cryoprotecting agent. Common cryoprotecting agents include histidine, polyethylene glycol, polyvinyl pyrrolidine, lactose, sucrose, mannitol, and polyols.

In some embodiments, compositions comprising liposomes encapsulating a bioactive agent are formulated with an isotonicity agent. The isotonicity agent can be any pharmaceutically acceptable isotonicity agent. This term is used in the art interchangeably with iso-osmotic agent, and is known as a compound that is added to the pharmaceutical preparation to increase the osmotic pressure, e.g., in some embodiments to that of 0.9% sodium chloride solution, which is iso-osmotic with human extracellular fluids, such as plasma. Preferred isotonicity agents are sodium chloride, mannitol, sorbitol, lactose, dextrose and glycerol.

Compositions of the liposomes encapsulating a bioactive agent may optionally comprise a preservative. Common preservatives include those selected from the group consisting of chlorobutanol, parabens, thimerosol, benzyl alcohol, and phenol. Suitable preservatives include but are not limited to: chlorobutanol (0.3-0.9% w/v), parabens (0.01- 5.0%), thimerosal (0.004-0.2%), benzyl alcohol (0.5-5%), phenol (0.1-1.0%), and the like.

In some embodiments, compositions comprising liposomes encapsulating a bioactive agent are formulated with a humectant to provide a pleasant mouth-feel in oral applications. Humectants known in the art include cholesterol, fatty acids, glycerin, lauric acid, magnesium stearate, pentaerythritol, and propylene glycol.

In some embodiments, an emulsifying agent is included in the formulations, for example, to ensure complete dissolution of all excipients, especially hydrophobic components such as benzyl alcohol. Many emulsifiers are known in the art, e.g., polysorbate 60.

For some embodiments related to oral administration, it may be desirable to add a pharmaceutically acceptable flavoring agent and/or sweetener. Compounds such as saccharin, glycerin, simple syrup, and sorbitol are useful as sweeteners.

Administration and Therapy

Once the therapeutic agent has been loaded into the liposomes, the combination can be administered to a patient by a variety of techniques.

Preferably, the pharmaceutical compositions are administered parenterally, e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection. For example, see Raham et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578. Particular formulations that are suitable for this use are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Typically, the formulations comprise a solution of the liposomes suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers are used in embodiments of the technology, e.g., water, buffered water, 0.9% isotonic saline, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

Dosage for the liposome formulations will depend on the ratio of drug to lipid and the administrating physician’s opinion based on age, weight, and condition of the patient.

The methods of the present invention may be practiced in a variety of hosts. Preferred hosts include mammalian species, such as humans, non-human primates, dogs, cats, cattle, horses, sheep, and the like.

In other methods, the pharmaceutical preparations may be contacted with the target tissue by direct application of the preparation to the tissue. The application may be made by topical, “open”, or “closed” procedures. By “topical”, it is meant the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like. “Open” procedures are those procedures include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical preparations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue. “Closed” procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin. For example, the preparations may be administered to the peritoneum by needle lavage. Likewise, the pharmaceutical preparations may be administered to the meninges or spinal cord by infusion during a lumbar puncture followed by appropriate positioning of the patient as commonly practiced for spinal anesthesia or metrizamide imaging of the spinal cord. Alternatively, the preparations may be administered through endoscopic devices.

The compositions of the present invention that further comprise a targeting antibody on the surface of the liposome are particularly useful for the treatment of certain diseases.

The therapeutic use of liposomes can include the delivery of drugs that are normally toxic in the free form. In the liposomal form, the toxic drug may be directed away from the sensitive tissue where toxicity can result and targeted to selected areas where they can exert their therapeutic effects. Liposomes can also be used therapeutically to release drugs slowly, over a prolonged period of time, thereby reducing the frequency of drug administration through an enhanced pharmacokinetic profile. In addition, liposomes can provide a method for forming an aqueous dispersion of hydrophobic drugs for intravenous delivery.

The route of delivery of liposomes can also affect their distribution in the body. Passive delivery of liposomes involves the use of various routes of administration e.g., parenterally, although other effective administration forms, such as intraarticular injection, inhalant mists, orally active formulations, transdermal iotophoresis, or suppositories are also envisioned. Each route produces differences in localization of the liposomes.

Because dosage regimens for pharmaceutical agents are well known to medical practitioners, the amount of the liposomal pharmaceutical agent formulations that is effective or therapeutic for the treatment of a disease or condition in mammals and particularly in humans will be apparent to those skilled in the art. The optimal quantity and spacing of individual dosages of the formulations herein will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and such optima can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, e.g., the number of doses given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

The liposomes containing therapeutic agents and the pharmaceutical formulations thereof of the present technology and those produced by the processes thereof can be used therapeutically in animals (including man) in the treatment of infections or conditions which require: (1) repeated administrations, (2) the sustained delivery of the drug in its bioactive form, or (3) the decreased toxicity with suitable efficacy compared with the free drug in question.

The mode of administration of the liposomes containing the pharmaceutical agents and the pharmaceutical formulations thereof determine the sites and cells in the organism to which the compound will be delivered. The liposomes of the present technology can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. The preparations may be injected parenterally, for example, intravenously. For parenteral administration, they can be used, for example, in the form of a sterile aqueous solution that may contain other solutes, for example, enough salts or glucose to make the solution isotonic.

For the oral mode of administration, the liposomal therapeutic drug formulations of this technology can be used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. In the case of tablets, carriers that can be used include lactose, sodium citrate, and salts of phosphoric acid. Various disintegrants, such as starch, and lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.

For the topical mode of administration, the liposome-drug formulations of the present technology may be incorporated into dosage forms such as gels, oils, emulsions, and the like. Such preparations may be administered by direct application as a cream, paste, ointment, gel, lotion or the like.

For administration to humans in the curative, remissive, retardive, or prophylactic treatment of diseases the prescribing physician will ultimately determine the appropriate dosage of the drug for a given human subject, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature and severity of the patient’s disease. The dosage of the drug in liposomal form will generally be approximately that employed for the free drug. In some cases, however, it may be necessary to administer dosages outside these limits and, in some embodiments, the technology comprises administering dosages in excess of these limits due to the extended-release characteristics of the formulations.

The term “therapeutically effective” as it pertains to the compositions of the invention means that a biologically active substance present in the aqueous component within the vesicles is released in a manner sufficient to achieve a particular medical effect for which the therapeutic agent is intended. Examples, without limitation, of desirable medical effects that can be attained are chemotherapy, antibiotic therapy, and regulation of metabolism. Exact dosages will vary depending upon such factors as the particular therapeutic agent and desirable medical effect, as well as patient factors such as age, sex, general condition, and the like. Those of skill in the art can readily take these factors into account and use them to establish effective therapeutic concentrations without resort to undue experimentation.

Generally, however, the dosage range appropriate for human use includes the range of 0.1 to 6000 mg/m² of body surface area. For some applications, such as intravenous administration, the dose required may be quite small, but for other applications, such as subcutaneous and/or intraperitoneal administration, the dose desired to be used may be very large. While doses outside the foregoing dose range may be given, this range encompasses the breadth of use for practically all the biologically active substances.

The liposomes may be administered for therapeutic applications by any desired route, for example, intramuscular, intraarticular, epidural, intrathecal, intraperitoneal, subcutaneous, intravenous, intralymphatic, oral and submucosal, and by implantation under many different kinds of epithelia, including the bronchialar epithelia, the gastrointestinal epithelia, the urogenital epithelia, and various mucous membranes of the body.

In addition, the liposomes of the invention can be used to encapsulate compounds useful in agricultural applications, such as fertilizers, pesticides, and the like. For use in agriculture, the liposomes can be sprayed or spread onto an area of soil where plants will grow and the agriculturally effective compound contained in the vesicles will be released at a controlled rate by contact with rain and irrigation waters. Alternatively the slow-releasing vesicles can be mixed into irrigation waters to be applied to plants and crops. One skilled in the art will be able to select an effective amount of the compound useful in agricultural applications to accomplish the particular goal desired, such as the killing of pests, the nurture of plants, etc.

During the development of embodiments of the technology provided, experiments were conducted to collect data relevant to the in vivo use of the liposome preparations and delivery of pharmaceuticals to a subject. See, e.g., Example 1. In particular, during the experiments a pharmaceutical (e.g., naltrexone) was loaded into liposomes according to embodiments of the technology provided herein, e.g., using a salt weak base and polyanion counter ion. Furthermore, the data indicated that the technology provides extended release of the naltrexone when tested in in vivo. This extended release profile could not have been predicted based on the in vitro release results.

Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.

Examples

During the development of embodiments of the technologies described herein, experiments were conducted to evaluate the loading of drugs into liposomes using salts of polyanion and weak loading bases as described herein as well as their release profiles in vitro and in vivo.

Example 1:

Loading base salt solutions. The nicotinamide, pyridinium, or pyridoxinium salt of eprodisic acid was prepared by dissolving nicotinamide, pyridine, or pyridoxine in an eprodisic acid solution in a 2: 1 molar ratio (1 :1 charge ratio). The eprodisate salt solutions were diluted to 0.5 M with respect to eprodisate, filter sterilized and used for liposome preparation as described below. For sucrose octasulfate and polyvinyl sulfonate, the acid form was generated from the respective sodium salts by passage through an Amberlite IR 120 column, regenerated in the acid form with HC1 followed by extensive washing with water. Nicotinamide sucrose octasulfate was prepared by dissolving nicotinamide in a solution of sucrose octasulfate in the regenerated free acid form at an 8:1 molar ratio. Nicotinamide polyvinyl sulfonate was prepared by dissolving nicotinamide in a solution of polyvinyl sulfonate in the regenerated free acid form at a 1 : 1 molar ratio to the component vinylsulfonic acid monomer.

Liposome preparation. 80 pmol phospholipon 90H and 40 pmol cholesterol (chol) were combined in a 13 x 120 mm tube together with 100 microliter n-propanol. The mixture was warmed to 80°C to dissolve the lipids. A 0.7 mL aliquot of loading base salt solution, also at 80°C was added to the mixture, which was held at 80°C for 5 minutes. The mixture was then cooled on ice, and 2 mL of ice cold saline was added with vortexing. To eliminate the excess loading base, the suspension was further diluted with isotonic NaCl, and sedimented at 300 x g for 10 minutes. After aspiration of the supernatant, the pellet was resuspended two times in isotonic NaCl followed by sedimentation at 300 x g for 10 minutes. The final liposome pellet was resuspended in 1 mL of an appropriate loading medium. Various buffer choices were used for the loading medium as indicated below for the specific drug to be loaded in each experiment. Then, a sample was solubilized with 1:3:1 v/v/v chloroform:methanol:water and analyzed spectrophotometrically to measure loading base content. Drug for loading was added to the suspension and incubated at 22°C for 20 hours. Unencapsulated drug and released loading base were removed by diluting the solution 3 times with 8 mL of isotonic NaCl followed by sedimentation at 300 x g for 10 minutes. After the third wash, the liposome pellet was suspended in isotonic NaCl solution. An aliquot of the suspension was removed from each preparation and solubilized in 1 :3: 1 v/v/v chloroform:methanol:water, and the amounts of drug and loading base in the liposomes were quantified spectrophotometrically.

In vitro liposome drug release. Liposome samples were diluted to 3-5 mL and left on an orbital shaker at 22°C. At specific time points, the liposomes were sedimented in a centrifuge at 300 x g for 5 minutes and an aliquot of the clear supernatant was analyzed spectrophotometrically for drug and loading base content. After measurement, the solution was returned to the liposome suspension, which was vortexed and returned to the orbital shaker.

Pharmacokinetic experiments, rats. Liposomes loaded with naltrexone were injected subcutaneously into rats. The dose used was 4 mg/Kg in the experiment where the formulation was made with nicotinamide eprodisate. A range of doses, 5, 10, and 20 mg/Kg were used in the experiments where the formulations were made with either nicotinamide sucrose octasulfate, or nicotinamide polyvinyl sulfonate. Following injection, blood samples were taken from the rats, and serum was isolated by sedimenting the blood cells. Samples were taken after 4, 24, or 48 hours, and after 1,2,4,6,8,10, and 12 weeks. In some cases, samples were also taken from a second group of 4 rats after 72 and 96 hrs, and after 3, 5, 7, and 9 weeks post injection. Samples were stored at -80°C, and analyzed by an outside contract lab using LC/MS to determine naltrexone serum concentrations. The sensitivity of the assay permitted detection of naltrexone at concentrations between 0.02 and 25 ng/mL serum.

Table 1 - Loading of naltrexone with different loading base salts and subsequent release.

Loading. Loading with ultraweak base salts is very efficient regardless as to the choice of anion. Efficiency is always greater than 70%, and varies somewhat with the choice of loading base. (Table 1 , above)

In vitro release. The in vitro release rate is in all cases extremely low for all choices of loading base and anion. The lowest rates of release are seen when the anion is eprodisate though release rate is only somewhat greater when the anion is polyvinylsulfonate or sucrose octasulfate (Table 1, above). Pharmacokinetic results. The in vitro release of naltrexone occurs at quite similar rates regardless of the choice of nicotinamide salt used for loading. Therefore, one would expect the profile of serum naltrexone concentrations over time in vivo to be very similar regardless of which of the three anions were used. However, this is not the case. In the case of liposomes loaded using nicotinamide eprodisate (Fig 1), serum concentrations after a 4 mg/Kg dose are greater than 10 ng/mL 24 hours and 1 week after injection, and are at or above 1 ng/mL for only 2-4 weeks after injection. Furthermore, the serum naltrexone concentration falls rapidly below the detectable range (0.02 ng/mL) thereafter. In the log plot shown in figure 1, undetectable levels have been assigned the value of 0.01 ng/mL to avoid plotting log zero, which is minus infinity.

In contrast to the result with nicotinamide eprodisate, liposomes loaded using nicotinamide sucrose octasulfate (Fig 2) produce serum naltrexone concentrations after a 5 mg/Kg dose are no more than 3 ng/mL between 4 hours and 2 weeks after injection. Although levels fall below 1 ng/mL thereafter, they are close to Ing/mL at 4-6 weeks after injection, and are still detectable even 12 weeks after injection. Hence the effect of using sucrose octasulfate in place of eprodisate is to reduce the rapid in vivo release observed in the first week following injection, and to prolong the period during which serum naltrexone is detectable. As a result, it is possible to increase the dose given of the nicotinamide sucrose octasulfate formulation, which is not possible with the nicotinamide eprodisate formulation owing to the already high serum levels observed at 0- 1 week. Dose increase results in overall higher serum levels and, in the case of a 20 mg/Kg dose, a period of 8 weeks during which the serum concentration is above 1 ng/mL. 1 ng/mL is the generally accepted minimum therapeutic concentration for naltrexone.

In the case of the naltrexone formulation using nicotinamide polyvinyl sulfonate, the result is similar to that seen with sucrose octasulfate. Serum naltrexone levels are below 10 ng/mL at all but one time point, and naltrexone is detectable in the serum even after 12 weeks.

Conclusion. The use of the polyanions sucrose octasulfate and polyvinyl sulfonate as compared to divalent anions such as eprodisate has no substantial effect on loading efficiency or on the in vitro rate of release. However, despite the expected relationship between in vitro release and the in vivo release profile and lifetime, which would suggest a similar in vivo behavior for all of these anion choices, there is a marked improvement in the in vivo release profile when a polyanion such as sucrose octasulfate or polyvinyl sulfonate is used in place of a divalent anion such as eprodisate. All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

CLAIMS WE CLAIM:

1. A composition comprising liposomes loaded with a bioactive agent, the liposomes further comprising a weak loading base and a polyanionic counter ion having three or more negative charges.

2. The composition of claim 1 , wherein the weak loading base is selected from the group consisting of a pyridine, a pyridine derivative, an adenine, an adenine derivative, an aniline, and an aniline derivative.

3. The composition of claim 2, wherein the pyridine derivative is pyridoxine, nicotinamide, or a nicotinamide derivative.

4. The composition of any of the preceding claims, wherein the acid form of the polyanionic counter ion having three or more negative charges forms a salt with the weak base.

5. The composition of claim 1 , wherein the polyanionic counter ion having three or more negative charges is selected from the group consisting of sucrose octasulfate and polyvinylsulfonate.

6. The composition of any one of claims 1 to 5, wherein the bioactive agent is selected from the group consisting of chloroquine, doxycycline, hydromorphone, naltrexone, and buprenorphine.

7. The composition of any of claims 1 to 5, wherein the bioactive agent is an opioid.

8. The composition of any of claims 1 to 5, wherein the bioactive agent is selected from the group consisting of an antitumor agent, an anaesthetic, an analgesic, an antimicrobial agent, an antimalarial, a hormone, an antiasthmatic agent, a cardiac glycoside, an antihypertensive, a vaccine, an antiarrhythmic, an immunomodulator, a steroid, a monoclonal antibody, a neurotransmitter, a radionuclide, a radio contrast agent, a nucleic acid, a protein, a herbicide, a pesticide, and suitable combinations thereof.

9. A composition comprising liposomes loaded with a bioactive agent selected from the group consisting of chloroquine, doxycycline, hydromorphone, naltrexone, and buprenorphine, the liposomes further comprising a weak loading base and a polyanionic counter ion with three or more negative charges.

10. The composition of claim 9, wherein the loading base is selected from the group consisting of pyridine, a pyridine derivative, an adenine, an adenine derivative, an aniline, and an aniline derivative.

11. The composition of claim 10, wherein the pyridine derivative is pyridoxine, nicotinamide, or a nicotinamide derivative.

12. The composition of any one of claims 9 to 11, wherein the polyanionic counter ion with three or more negative charges forms a salt with the weak base.

13. The composition of any one of claims 9 to 12, wherein the polyanionic counter ion with three or more negative charges is selected from the group consisting of sucrose octasulfate and polyvinylsulfonate.

14. The composition of any one of claims 1 to 13, further comprising a loading medium in the extraliposomal space, the loading medium having a pH that is greater than the pKa of the loading base.

15. The composition of any one of claims 1 to 14, wherein the liposomes comprise phosphatidylcholine.

16. The composition of any of the preceding claims, wherein the liposomes comprise: a) a phosphatidylcholine selected from the group consisting of distearoylphosphatidylcholine, hydrogenated soy phosphatidylcholine, hydrogenated egg phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dielaidoylphosphatidylcholine; b) a sphingomyelin; c) a neutral lipid; or d) an acidic phospholipid.

17. The composition of any one of claims 1 to 16, wherein the liposomes comprise dipalmitoylphosphatidylcholine and optionally cholesterol.

18. The composition of any one of claims 1 to 17, further comprising an excipient and/or a pharmaceutically acceptable carrier.

19. The composition of any one of claims 1 to 18, wherein the amount of bioactive agent in the intraliposomal space is greater than approximately 80%, greater than approximately 85%, greater than approximately 90%, or greater than approximately 95% of the total amount of bioactive agent in the composition.

20. The composition of any one of claims 1 to 19, wherein the liposomes release therapeutic levels of the bioactive agent over an extended period in vivo.

21. The composition of claim 20, wherein the extended period is three or more weeks.

22. The composition of claim 20, wherein the extended period is four or more weeks.

23. The composition of claim 20, wherein the extended period is five or more weeks.

24. The composition of claim 20, wherein the extended period is six or more weeks.

25. The composition of claim 20, wherein the extended period is seven or more weeks.

26. The composition of claim 20, wherein the extended period is eight or more weeks.

27. The composition of claim 20, wherein the extended period is ten or more weeks.

28. The composition of claim 20, wherein the extended period is five to eight weeks.

29. The composition of claim 20, wherein the extended period is five to ten weeks.

30. The composition of claim 20, wherein the extended period is up to eight weeks.

31. The composition of claim 20, wherein the extended period is up to ten weeks.

32. A method of producing liposomes, the method comprising: a) dissolving lipids in a solvent to produce a lipid solution; b) adding an aqueous solution comprising a weak loading base and a polyanionic counter ion with three or more negative charges, or a salt thereof, to the lipid solution to produce liposomes.

33. The method of claim 32, further comprising contacting the liposomes produced in step b) with an aqueous solution comprising an active agent.

34. The method of any one of claims 32 to 33, wherein the active agent is selected from the group consisting of chloroquine, doxycycline, hydromorphone, naltrexone, and buprenorphine.

35. The composition of any of claims 32 to 33, wherein the bioactive agent is an opioid.

36. The composition of any of claims 32 to 33, wherein the bioactive agent is selected from the group consisting of an antitumor agent, an anaesthetic, an analgesic, an antimicrobial agent, an antimalarial, a hormone, an antiasthmatic agent, a cardiac glycoside, an antihypertensive, a vaccine, an antiarrhythmic, an immunomodulator, a steroid, a monoclonal antibody, a neurotransmitter, a radionuclide, a radio contrast agent, a nucleic acid, a protein, a herbicide, a pesticide, and suitable combinations thereof.

37. The method of any one of claims 32 to 36, wherein the solvent is miscible with aqueous solutions or is a solvent that has significant aqueous solubility.

38. The method of any one of claims 32 to 37 wherein the solvent is an alcohol.

39. The method of claim 38 wherein the solvent is selected from the group consisting of methanol, ethanol, 1 -propanol, 2-propanol, 1 -butanol, 2-butanol, and t-butanol.

40. The method of claim 38, wherein the solvent is selected from the group consisting of a C3 and a C4 alcohol.

41. The method of claim 40, wherein the C3 alcohol is 1 -propanol.

42. The method of claim 40, wherein the C4 alcohol is 2-butanol.

43. The method of any of claims 32 to 42, wherein the lipids comprise phospholipids.

44. The method of claim 43, wherein the phospholipids and the solvent are combined at a concentration of about 400 mg to 800 mg phospholipids per 1 ml of solvent.

45. The method of claim 44, wherein the phospholipids and the solvent are combined at a concentration of about 500 mg to 700 mg phospholipids per 1 ml of solvent.

46. The method of claim 45, wherein the phospholipids and the solvent are combined at a concentration of about 550 mg to 650 mg phospholipids per 1 ml of solvent.

47. The method of any of claims 32 to 46, wherein the lipids further comprise cholesterol.

48. The method of claim 37, wherein the cholesterol and the solvent are combined at a concentration of about 50 mg to 250 mg cholesterol per 1 ml of solvent.

49. The method of any of claims 47 to 48, wherein the cholesterol and the solvent are combined at a concentration of about 100 mg to 200 mg cholesterol per 1 ml of solvent.

50. The method of any of claims 47 to 49, wherein the cholesterol and the solvent are combined at a concentration of about 125 mg to 175 mg cholesterol per 1 ml of solvent.

51. The method of any of claims 32 to 50, wherein the dissolving comprises warming the lipids and solvent to 40 to 85°C.

52. The method of claim 51, further comprising warming the aqueous solution of an encapsulant to 40 to 85°C prior to addition to the lipid solution.

53. The method of any of claims 32 to 52, wherein the lipids and solvent are dissolved in a vessel to provide a dissolved lipid composition and the aqueous solution is injected into the vessel containing the dissolved lipid composition.

54. The method of any of claims 32 to 53, further comprising cooling the liposomes to a temperature that is below the phase transition temperature of the lipids.

55. The method of any of claims 32 to 54, further comprising diluting the liposomes in an aqueous solution.

56. The method of any of claims 32 to 55, further comprising washing the liposomes to remove liposome loading agent or encapsulant from the extraliposomal space.

57. A method for preparing liposomes encapsulating a bioactive agent, the method comprising: contacting the composition of liposomes prepared by a method according to any of claims 32 and 37 to 54 with a solution comprising a bioactive agent under conditions such that the bioactive agent is transported to the intraliposomal space of the liposomes.

58. A method for preparing liposomes encapsulating a bioactive agent, the method comprising: contacting a composition comprising liposomes comprising a weak loading base and a polyanionic counter ion having three or more negative charges in the intraliposomal space with a solution comprising a bioactive agent under conditions such that the bioactive agent is transported to the intraliposomal space of the liposomes

59. The method of claim 58, wherein the weak loading base is selected from the group consisting of pyridine, a pyridine derivative, an adenine, an adenine derivative, an aniline, and an aniline derivative.

60. The method of claim 59, wherein the pyridine derivative is pyridoxine, nicotinamide, or a nicotinamide derivative.

61 . The method of any one of claims 58 to 60, wherein the polyanionic counter ion having three or more negative charges forms a salt with the weak base.

62. The method of claim 61 , wherein the polyanionic counter ion having three or more negative charges is selected from the group consisting of sucrose octasulfate and polyvinylsulfonate.

63. The method of any one of claims 58 to 62, wherein the active agent is selected from the group consisting of chloroquine, doxycycline, hydromorphone, naltrexone, and buprenorphine.

64. The method of any one of claims 56 to 60, wherein the bioactive agent is an opioid.

65. The method of any one of claims 56 to 60, wherein the bioactive agent is selected from the group consisting of an antitumor agent, an anaesthetic, an analgesic, an antimicrobial agent, an antimalarial, a hormone, an antiasthmatic agent, a cardiac glycoside, an antihypertensive, a vaccine, an antiarrhythmic, an immunomodulator, a steroid, a monoclonal antibody, a neurotransmitter, a radionuclide, a radio contrast agent, a nucleic acid, a protein, a herbicide, a pesticide, and suitable combinations thereof.

66. A liposome composition made by the method of any of claims 32 to 65.

67. Liposome composition of claim 66 for use in treatment of disease or condition in an animal.

68. Liposome composition of any one of claims 1 to 31 for use in administration to a human or animal subject.

69. Liposome composition of any one of claims 1 to 31 for use to treat a disease or condition in a subject.

70. Use of claim 69, wherein the disease or condition is selected from the group consisting of pain relief and treatment of addiction.

71. Use of any one of claims 67 to 70, wherein the composition is administered at a dosage of 1 to 100 mg/kg of active agent per dose.

72. Use of claim 71, wherein the composition is administered at a dosage of 5 to 50 mg/kg of active agent per dose.

73. Use of claim 71, wherein the composition is administered at a dosage of 10 to 300 mg/kg of active agent per dose.

74. A method of treating a disease or condition, comprising: administering the liposome composition of any one of claims 1 to 31 and 66 to a subject in need thereof.

75. The method of claim 74, wherein the disease or condition is selected from the group consisting of pain relief and treatment of addiction.

76. The method of claim 74 or 75, wherein the composition is administered at a dosage of 1 to 100 mg/kg of active agent per dose.

77. The method of claim 76, wherein the composition is administered at a dosage of 5 to 50 mg/kg of active agent per dose.

78. The method of claim 76, wherein the composition is administered at a dosage of 10 to 300 mg/kg of active agent per dose.

79. A method of treating a subject in need of pain reduction, the method comprising: a) administering to the subject a composition according to any one of claims 1 to 31; and b) assessing the subject’s pain.