RU2777601C1 - Liposome-containing composition - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine and pharmaceuticals. The first invention of the group is a liposome-containing composition for suppressing the binding of lipopolysaccharides to TLR4, wherein the liposomes contain 0.7 to 3.0% mol. of eritoran or a pharmaceutically acceptable salt thereof, 0.5 to 3.0% mol. of a pegylated phospholipid, and 0 to 10% mol. of sterol as calculated for the liposome, wherein the pegylated phospholipid constitutes 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. The second invention of the group is a method for producing said composition, including producing a solution containing 0.7 to 3.0% mol. of eritoran or a pharmaceutically acceptable salt thereof, 0.5 to 3.0% mol. of a pegylated phospholipid, and a solvent as calculated for all components of the liposomes subject to use; evaporating the solvent from the solution forming a thin film; dispersing the thin film in a buffer solution forming a liquid dispersion, and extruding the liquid dispersion through a filter forming a composition.

EFFECT: group of inventions provides compositions with increased activity in terms of suppression of IL-6 and/or TNF-ɑ production.

20 cl, 5 dwg, 11 tbl, 26 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention relates to a composition containing liposomes.

BACKGROUND OF THE INVENTION

[0002] Eritoran, also referred to as "E5564", is a lipopolysaccharide analog containing two sugar moieties and four long chain fatty acid moieties. Eritoran has a molecular weight of approximately 1401. Methods for preparing eritoran are described in US Pat. Nos. 5,530,113; 5 681 824; 5 750 664; 5 935 938 and 6 184 366 and also in WO 96/39411. These documents are incorporated herein by reference. Eritoran-based formulations with varying hydrodynamic micelle diameters have reportedly been prepared by controlling the pH value and the concentration of counterions in solution. This is reported in US Pat. No. 6,906,042, which is incorporated herein by reference.

[0003] Eritoran is a lipid A analog that acts as a Toll-like receptor 4 (TLR4) antagonist. Eritoran has the following structure:

.

.

[0004] The activity of lipid A analogues has been reported to be altered by interactions with lipoprotein cholesterol. These lipoproteins are typically present in human serum and include low density lipoproteins (LDL) and high density lipoproteins (HDL). The study postulated that changes in plasma lipoprotein profiles could alter both the efficacy and pharmacodynamic profiles of lipophilic drugs. In particular, the study noted that binding of eritoran to HDL resulted in a time-dependent loss of drug activity. See Daniel P. Rossignol, et al., “Safety, Pharmacokinetics, Pharmacodynamics, and Plasma Lipoprotein Distribution of Eritoran (E5564) during Continuous Intravenous Infusion into Healthy Volunteers” Antimicrobial Agents & Chemotherapy, Sep. 2004, p. 3233-3240, which is incorporated herein by reference.

Short description

[0005] One of the objectives of the present invention is to provide a composition containing erytoran, which is characterized by higher rates for forms of activity in vivo or in the presence of HDL (for example, human HDL), preferably forms of activity involving the suppression of the production of interleukin-6 (IL-6 ), or forms of activity involving the suppression of the production of TNF-α than eritoran itself or micellized erytoran. The present inventors have found that a liposome containing erytoran and a pegylated phospholipid performs better in in vivo activity forms or in the presence of HDL (e.g., human HDL), and that adjusting the amounts of erytoran and pegylated phospholipid enables liposomes to exhibit much higher performance. for activity forms.

[0006] The present invention provides the following embodiments.

[1] Composition containing liposomes, where the liposomes contain from 0.7 to 3.0 mol. % eritoran or its pharmaceutically acceptable salt and from 0.5 to 3.0 mol. % of pegylated phospholipid in terms of liposome.

[2] The composition according to [1], wherein the pharmaceutically acceptable salt is the tetrasodium salt.

[3] The composition according to p. [1] or p. [2], where the liposomes contain from 1.0 to 2.5 mol. % eritoran or its pharmaceutically acceptable salt and from 1.0 to 2.5 mol. % of pegylated phospholipid in terms of liposome.

[3-1] The composition according to any one of paragraphs [1]-[3], where the liposomes contain 1.0 mol. % eritoran or its pharmaceutically acceptable salt and 1.0 mol. % of pegylated phospholipid in terms of liposome.

[3-2] The composition according to any one of paragraphs [1]-[3], where the liposomes contain 1.0 mol. % eritoran or its pharmaceutically acceptable salt and 2.5 mol. % of pegylated phospholipid in terms of liposome.

[3-3] The composition according to any one of paragraphs [1]-[3], where the liposomes contain 2.5 mol. % eritoran or its pharmaceutically acceptable salt and 1.0 mol. % of pegylated phospholipid in terms of liposome.

[3-4] The composition according to any one of paragraphs [1]-[3], where the liposomes contain 2.5 mol. % eritoran or its pharmaceutically acceptable salt and 2.5 mol. % of pegylated phospholipid in terms of liposome.

[4] The composition according to any one of [1] to [3], wherein the pegylated phospholipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

[5] The composition according to p. [4], where the liposomes contain 1.0 mol. % eritoran or its pharmaceutically acceptable salt and 1.0 mol. % 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in terms of liposome.

[6] Composition according to p. [4], where the liposomes contain 1.0 mol. % eritoran or its pharmaceutically acceptable salt and 2.5 mol. % 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in terms of liposome.

[7] Composition according to p. [4], where the liposomes contain 2.5 mol. % eritoran or its pharmaceutically acceptable salt and 1.0 mol. % 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in terms of liposome.

[8] The composition according to p. [4], where the liposomes contain 2.5 mol. % eritoran or its pharmaceutically acceptable salt and 2.5 mol. % 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in terms of liposome.

[9] The composition according to any one of [1]-[8], wherein the liposomes further contain phosphatidylcholine.

[10] The composition according to [9], wherein the phosphatidylcholine is distearoylphosphatidylcholine.

[11] The composition according to any one of [1]-[10], wherein the liposomes further contain an antioxidant.

[12] The composition of [11], wherein the antioxidant is butylated hydroxyanisole.

[13] The composition according to any one of paragraphs [1]-[12], where the liposomes additionally contain from 0 to 10 mol. % sterol in terms of liposome.

[14] The composition of [13], wherein the sterol is cholesterol.

[15] The composition according to [13], wherein the liposomes do not contain a sterol.

[16] The composition according to any one of [1]-[15], wherein the liposomes have an average particle size of 100 to 120 nm as measured by dynamic light scattering (DLS) analysis.

[17] The composition according to any one of [1]-[16], wherein the liposomes have a polydispersity coefficient of 0.20 or less.

[18] The method of obtaining a composition containing liposomes, including obtaining a solution containing from 0.7 to 3.0 mol. % eritoran or its pharmaceutically acceptable salt, from 0.5 to 3.0 mol. % pegylated phospholipid and solvent, based on all liposome components to be used;

evaporating the solvent from the solution to form a thin film;

dispersion of a thin film in a buffer solution to form a liquid dispersion, and

extruding the liquid dispersion through a filter to form a composition.

[19] The method according to [18], wherein the solution additionally contains phosphatidylcholine and an antioxidant.

[20] The method according to [18] or [19], wherein the solvent is a combination of chloroform and methanol.

[21] The method according to any one of [18] to [20], wherein the dispersion step is carried out by sonication.

[22] The method according to any one of [18]-[21], wherein the method further comprises adjusting the pH of the composition formed in the extrusion step to 6.2-6.8.

[0007] The present invention further provides for the following embodiments.

[A] The composition according to any of the embodiments [1]-[22] for use in inhibiting lipopolysaccharide binding to TLR4, for use in inhibiting TLR4 dimerization, for use in inhibiting TLR4 signaling, for use in inhibiting IL-6 production, for use in the treatment or prevention of diseases that are mediated by TLR4 activation or IL-6 production, for use in the suppression of TNF-α production, or for use in the treatment or prevention of diseases that are mediated by TNF-α production.

[B] A method for suppressing lipopolysaccharide binding to TLR4, suppressing TLR4 dimerization, suppressing TLR4 signaling, suppressing IL-6 production, treating or preventing diseases that are mediated by TLR4 activation or IL-6 production, for use in suppressing TNF-α production or for use in the treatment or prevention of diseases that are mediated by the production of TNF-α, comprising the introduction of an effective amount of the composition according to any of the embodiments of [1]-[22] in need of this subject.

[C] Use of a composition according to any of the embodiments of [1]-[22] to inhibit the binding of lipopolysaccharides to TLR4, to inhibit the dimerization of TLR4, to inhibit TLR4 signaling, to inhibit the production of IL-6, to treat or prevent diseases that are mediated TLR4 activation or IL-6 production, for use in suppressing TNF-α production, or for use in the treatment or prevention of diseases that are mediated by TNF-α production.

[D] The use of the composition according to any one of the embodiments of [1]-[22] in the manufacture of a medicament for inhibiting the binding of lipopolysaccharides to TLR4, for inhibiting dimerization of TLR4, for inhibiting TLR4 signaling, for inhibiting the production of IL-6, for treating or preventing diseases that are mediated by TLR4 activation or IL-6 production, for use in suppressing TNF-α production, or for use in treating or preventing diseases that are mediated by TNF-α production.

[0008] With respect to embodiments [A]-[D], the features described in the <Composition> section below can be given.

[0009] According to the present invention, a composition containing erytoran can be provided that is characterized by higher rates for forms of activity in vivo or in the presence of HDL (for example, human HDL), preferably forms of activity involving the suppression of the production of IL-6, or forms of activity involving inhibition of TNF-α production than erytoran alone or micellized erytoran.

Brief description of graphic materials

[0010] FIG. 1 demonstrates in vivo IL-6 suppression activity for the formulations of Examples 1-4.

In FIG. 2-1 shows in vivo IL-6 inhibition activity for the dilute formulations of Examples 1, 11 and 13.

In FIG. 2-2 demonstrate in vivo IL-6 inhibition activity for the dilute formulations of Examples 1-4.

In FIG. 2-3 demonstrate in vivo IL-6 inhibition activity for the dilute formulations of Examples 5 and 6.

In FIG. 3 demonstrates TNF-α suppression activity for micellized eritoran and the formulations of Examples 1 to 4 in the presence of human HDL.

DETAILED DESCRIPTION

[0011] <Definition>

As used herein, the singular means "one or more" or "at least one" unless otherwise indicated.

[0012] <Composition>

One embodiment relates to a composition containing liposomes, where the liposomes contain from 0.7 to 3.0 mol. % eritoran or its pharmaceutically acceptable salt and from 0.5 to 3.0 mol. % of pegylated phospholipid in terms of liposome. Liposomes containing less eritoran or a pharmaceutically acceptable salt thereof have been found to perform better in vivo forms of activity or in the presence of HDL (eg, human HDL).

[0013] The composition may contain, in addition to liposomes, disks based on a lipid bilayer. The composition may contain, in addition to liposomes, lipid bilayers. The formulation may contain, in addition to liposomes, lipid bilayer discs and lipid bilayers. The lipid bilayer used herein does not form either a liposome or a lipid bilayer disc.

[0014] The liposomes contain eritoran or a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable salt" is well known in the art. Examples of a pharmaceutically acceptable salt include, without limitation, alkali metal salts such as lithium, sodium or potassium salts; and alkaline earth metal salts, for example calcium or magnesium salts. The pharmaceutically acceptable salt of erytoran is preferably a sodium salt, such as a tetrasodium salt.

[0015] The amount of eritoran or its pharmaceutically acceptable salt contained in liposomes is from 0.7 to 3.0 mol. %, preferably from 1.0 to 2.5 mol. % in terms of liposome. Adjustment of the amount of eritoran or its pharmaceutically acceptable salt within the above range or up to the above point provides a composition with much higher performance for forms of activity.

[0016] The liposomes contain a pegylated phospholipid. The PEGylated phospholipid contains a polyethylene glycol-exposed (“PEG”) moiety covalently linked to the phospholipid. The molecular weight of the PEG group is preferably from about 500 to about 5000, more preferably from about 1000 to about 3000, and even more preferably about 2000. The PEGylated phospholipid preferably contains acyl groups containing from 14 to 18 carbon atoms, and more preferably acyl groups, containing 18 carbon atoms. Examples of the PEGylated phospholipid include, without limitation, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000). These pegylated phospholipids may be used alone or in combination. The pegylated phospholipid is preferably DSPE-PEG2000 having the following structure:

.

.

[0017] The amount of pegylated phospholipid contained in the liposome is from 0.5 to 3.0 mol. %, preferably from 1.0 to 2.5 mol. % in terms of liposome. Adjusting the amount of pegylated phospholipid within the above range or up to the above point provides a formulation with much higher performance for activity forms.

[0018] Preferably, the amount of eritoran or its pharmaceutically acceptable salt and pegylated phospholipid in the liposome is 1.0 mol. % and 1.0 mol. %, 1.0 mol. % and 2.5 mol. %, 2.5 mol. % and 1.0 mol. % or 2.5 mol. % and 2.5 mol. %, respectively, in terms of liposome.

[0019] Liposomes may additionally contain phosphatidylcholine. Examples of phosphatidylcholine include, without limitation, soy phosphatidylcholine, egg phosphatidylcholine, dielaidoylphosphatidylcholine (DEPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (syn. 1,2-distearoyl-sn-glycero-3-phosphocholine) (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleophosphatidylcholine (POPC), dibegenoylphosphatidylcholine (DBPC) and dimyristoylphosphatidylcholine (DMPC). These varieties of phosphatidylcholine can be used alone or in combination. Phosphatidylcholine is preferably a DSPC having the following structure:

.

.

[0020] The term "soy phosphatidylcholine" refers to a composition based on phosphatidylcholine containing a number of different mono-, di-, tri-unsaturated and saturated fatty acids. Typically, soy phosphatidylcholine provides palmitic acid in an amount of from about 12% to about 33% by weight, stearic acid in an amount from about 3% to about 8% by weight, oleic acid in an amount from about 4% to about 22% by weight , linoleic acid in an amount from about 60% to about 66% by weight and linolenic acid in an amount from about 5% to about 8% by weight.

[0021] The term "egg phosphatidylcholine" refers to a composition based on phosphatidylcholine containing a number of different saturated and unsaturated fatty acids. Typically, egg phosphatidylcholine provides palmitic acid at about 34% by weight, stearic acid at about 10% by weight, oleic acid at about 31% by weight, and linoleic acid at about 18% by weight.

[0022] The amount of phosphatidylcholine contained in the liposome is preferably 86 to 98 mol. %, more preferably from 89 to 97 mol. %, and even more preferably from 92 to 96 mol. % in terms of liposome.

[0023] Liposomes may additionally contain an antioxidant. Examples of the antioxidant include, but are not limited to, butylated hydroxyanisole (BHA), butylated hydroxytoluene, ascorbic acid, sodium ascorbate, ascorbyl palmitate, sodium sulfite, sodium bisulfite, cysteinate hydrochloride, sodium dithionate, monosodium glutamate, glutathione, propyl gallate, alpha-tocopherol, alpha-tocopherol hydrosuccinate and salts of ethylenediaminetetraacetic acid. These antioxidants can be used alone or in combination. The antioxidant is preferably BHA.

[0024] The amount of antioxidant contained in the liposome is preferably 0.02 to 0.12 mol. %, more preferably from 0.04 to 0.10 mol. % and even more preferably from 0.06 to 0.08 mol. % in terms of liposome.

[0025] Liposomes may additionally contain a sterol. Examples of a sterol include, without limitation, cholesterol, campesterol, β-sitosterol, stigmasterol, and ergosterol. These sterols can be used alone or in combination. The sterol is preferably a cholesterol having the following structure:

.

.

[0026] The amount of sterol contained in the liposome is preferably from 0 to 30 mol. %, more preferably from 0 to 10 mol. %, even more preferably from 0 to 6 mol. % and most preferably 0% based on liposome. Reducing the amount of sterol provides a composition with much higher performance for forms of activity.

[0027] Preferably, the liposomes consist only of eritoran or a pharmaceutically acceptable salt thereof, a pegylated phospholipid, phosphatidylcholine, and an antioxidant. More preferably, the liposomes consist only of eritoran or a pharmaceutically acceptable salt thereof, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], distearoylphosphatidylcholine, and butylated hydroxyanisole. The use of these components provides a formulation with much higher rates for activity forms. One of skill in the art will appreciate that liposomes may contain materials such as water and buffer within their internal space.

[0028] The components of liposomes and their amounts as described above may be applicable to lipid bilayer discs and lipid bilayers.

[0029] The composition may further contain optional components. Examples of the optional component include, without limitation, pharmaceutically acceptable excipients such as lactose, dextrose, sucrose, sorbitol, mannitol, starch varieties, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose and polyacrylic acids; lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers; suspending means; preservatives such as methyl, ethyl and propylhydroxybenzoates; pH adjusting agents such as inorganic and organic acids and bases; sweeteners and flavors.

[0030] Although the average particle size of the liposomes can be suitably adjusted by a publicly known method, the liposomes have an average particle size (Z-ave) of preferably 80 to 140 nm and more preferably 100 to 120 nm measured by dynamic light scattering (DLS).

[0031] Liposomes have a polydispersity index (PdI) of preferably 0.20 or less, more preferably 0.10 or less, and even more preferably 0.05 or less. Although the lower limit of PdI is not particularly limited, it is, for example, 0.01.

[0032] The composition can be used to suppress the binding of lipopolysaccharides (LPS) to TLR4. The composition can be used to suppress the dimerization of TLR4. The formulation can be used to suppress TLR4 signaling. The composition can be used to suppress the production of IL-6.

[0033] The composition can be used to treat or prevent diseases that are mediated by TLR4 activation or IL-6 production. Examples of such a disease include, without limitation, sepsis; disease caused by the Ebola virus; disease caused by the Marburg virus; pain; septicemia, including without limitation endotoxemia; endotoxemia resulting from gram-negative bacteremia with accompanying symptoms of fever, generalized inflammation, disseminated intravascular coagulation, hypotension, acute renal failure, acute respiratory distress syndrome, adult respiratory distress syndrome (ARDS), hepatocellular destruction and/or heart failure, and various forms of septicemia shock, including without limitation endotoxic shock.

[0034] the Composition can be used to suppress the production of TNF-α. The composition can be used to treat or prevent diseases that are mediated by the production of TNF-α. Examples of such a disease include, without limitation, sepsis; disease caused by the Ebola virus; disease caused by the Marburg virus; pain; rheumatoid arthritis; psoriasis; diabetes; dyslipidemia, including, without limitation, hyperlipidemia, primary hyperlipidemia, hypercholesterolemia, familial combined hyperlipidemia, hyperlipoproteinemia, hypolipoproteinemia, and hypertriglyceridemia; and osteoporosis.

[0035] Appropriate dosages and administration of the composition can be determined by one of ordinary skill in the art depending on the age, weight and health of the subjects, the disease being treated or prevented, the route of administration, etc.

[0036] The composition is preferably administered parenterally and more preferably administered intravenously.

[0037] The dosage form of the composition may be injectable or infusion.

[0038] The amount of injectable formulation to be administered in terms of eritoran may be from 0.001 to 20 mg eritoran/kg body weight per dose, preferably from 0.01 to 10 mg eritoran/kg body weight per dose. The injection formulation may be administered 1 to 6 times per day, preferably 1 to 3 times per day. The injection formulation may be administered over a period of 1 to 10 days, preferably 1 to 5 days.

[0039] The amount of injectable formulation to be administered in terms of eritoran may be from 0.001 to 10 mg eritoran/kg body weight/hour, preferably from 0.003 to 5 mg eritoran/kg body weight/hour. The infusion formulation may be administered over a period of 0.5 to 6 hours/day, preferably 1 to 3 hours/day. The infusion formulation may be administered over a period of 1 to 10 days, preferably 1 to 5 days.

[0040] The term “subject” refers to an animal, preferably a mammal and more preferably a human, that is the object of treatment or prevention. Examples of mammals include, without limitation, mice, rats, hamsters, gerbils, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, giraffes, platypuses, primates such as humans, monkeys, chimpanzees, and great apes. The subject is preferably a human.

[0041] <Obtaining method>

One embodiment relates to a process for preparing a formulation containing liposomes as defined above. In particular, the method includes obtaining a solution containing from 0.7 to 3.0 mol. % eritoran or its pharmaceutically acceptable salt, from 0.5 to 3.0 mol. % pegylated phospholipid and solvent, based on all liposome components to be used; evaporating the solvent from the solution to form a thin film; dispersing a thin film in a buffer solution to form a liquid dispersion; and extruding the liquid dispersion through a filter to form a composition.

[0042] Regarding the components of the liposomes to be used in the production step and their amounts, you can refer to the information on the components of the liposomes and their amounts, which are described in the above section <Composition>.

[0043] Any solvent can be used in the preparation step, provided that it dissolves the components of the liposome. Examples of the solvent include, without limitation, halogenated hydrocarbons such as chloroform and dichloromethane, and alcohols such as methanol and ethanol. The solvent is preferably a combination of chloroform and methanol.

[0044] The dispersion step is preferably carried out by sonication. The buffer solution preferably contains a saccharide such as sucrose. The temperature of the buffer solution is preferably 50 to 90°C and more preferably 60 to 80°C.

[0045] The filter used in the extrusion step preferably comprises two or more filter elements that are superimposed on each other. The number of filter elements is preferably 2 to 6, more preferably 3 to 5, and even more preferably 4. The filter elements preferably have different pore sizes, respectively. The pore size of the filter elements is preferably 0.05 to 1.5 µm, more preferably 0.08 to 1.0 µm, and even more preferably 0.1 to 0.8 µm. When the number of filter elements is 4, the pore size of each filter element is preferably 0.1 µm, 0.2 µm, 0.4 µm and 0.8 µm, respectively. The filtration in the extrusion step is preferably performed two or more times, more preferably 3 to 7 times, and even more preferably 4 to 6 times.

[0046] The method may further include adjusting the pH of the composition that is formed in the extrusion step, preferably to a value in the range of 6.2 to 6.8, and more preferably 6.4 to 6.6. The pH can be adjusted with an alkali metal hydroxide such as NaOH and KOH or an inorganic acid such as HCl. The composition, the pH of which has been adjusted, can be sterilized through a sterilizing filter.

Examples

[0047] <Get composition>

(Materials)

The examples used the following materials from the indicated suppliers: eritoran (from Eisai Co., Ltd.); cholesterol (cholest., from Wako Pure Chemical (Wako)); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, from Nippon Pure Chemical); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000, from Genzyme); butylated hydroxyanisole (BHA, from Wako); chloroform (from Wako); methanol (from Wako); sucrose (from Wako); anhydrous NaH ₂ PO ₄ (from Wako); 1 n. HCl (from Wako); 1 n. NaOH (from KANTO Chemical) and water purified using Milli-Q Gradient A10 (Merck Millipore).

[0048] (Unit)

The examples used the following equipment from the indicated vendors: Scales: METTLER AT250 and PG503; pH meter: HORIBA pH/ION METER D-53; rotary evaporator: EYELA N-1000; water bath with digital control: EYELA SB-1000; vacuum pump: SATO VACUUM MACHINERY Oil Rotary Vacuum Pump SW-100.

[0049] Example 1

Obtaining a composition containing the components listed in table 1

Table 1

Example 1: 2.5 mol. % eritoran, 2.5 mol. % DSPE-PEG2000, 6 mol. % cholest. Material mm Mol. % in 5 ml lot Eritoran 1.00 2.50 7.01 mg* Cholesterol 2.40 6.00 4.64 mg DSPC 35.57 88.93 140.54 mg DSPE-PEG2000 1.00 2.50 14.03 mg BHA 0.03 0.07 0.03 mg Total 40.00 100.00 166.24 mg

*: mg eritoran as tetrasodium salt

[0050] Example 1 used the following reagents.

(1) 0.1 N NaOH

One ml 1 N. NaOH was added to a 10 ml volumetric flask and then made up to volume with purified water.

(2) 0.1 N HCl

One ml 1 N. HCl was added to a 10 ml volumetric flask and then made up to volume with purified water.

(3) Sucrose buffer solution

Twenty g of sucrose was weighed into a 200 ml glass beaker and approximately 180 ml of purified water was added. Then 24 mg of NaH ₂ PO ₄ was added and the pH was adjusted to 6.5 by adding 0.1N. NaOH and 0.1 N. HCl. The solution was added to a 200 ml volumetric flask and made up to volume with purified water.

(4) Chloroform/methanol mixture

Fifty ml of chloroform and 100 ml of methanol were mixed in a glass bottle.

(5) Eritoran solution

Forty mg of eritoran was weighed into a glass bottle and 10 ml of chloroform/methanol was added. Complete dissolution of eritoran was ensured by shaking.

(6) DSPC solution

Two point five tenths of a g of DSPC was weighed into a glass bottle and 60 ml of chloroform/methanol was added. Ensured complete dissolution of DSPC by shaking.

(7) Cholesterol solution

Ninety mg of cholesterol was weighed into a glass bottle and 30 ml of chloroform/methanol was added. Complete dissolution of cholesterol was ensured by shaking.

(8) DSPE-PEG2000 solution

Five hundred mg of DSPE-PEG2000 was weighed into a glass bottle and 20 ml of chloroform/methanol was added. Ensured complete dissolution of DSPE-PEG2000 by shaking.

(9) BHA solution

Four mg of BHA were weighed into a glass bottle and 10 ml of chloroform/methanol was added. Complete dissolution of BHA was ensured by shaking.

[0051] (1) Preparation of a thin film

To obtain the composition shown in Table 1, 1.75 ml of erytoran solution, 3.37 ml of DSPC solution, 1.55 ml of cholesterol solution, 0.56 ml of DSPE-PEG2000 solution and 60 μl of BHA solution were mixed in a 50 ml round bottom flask . After making sure that all components were completely dissolved, the solvent was removed using a rotary evaporator to obtain a thin film from the components. The round bottom flask was placed in a vacuum chamber overnight to remove residual solvent.

[0052] (2) Preparation of liquid dispersion

The sucrose buffer solution was heated to 70° C. in a water bath, and 5 ml of the heated sucrose buffer solution was added to a round bottom flask containing a thin film. The mixture was subjected to ultrasonic treatment in an ultrasonic bath until the thin film was completely dispersed.

[0053] (3) Preparation of composition

The extruder was mounted with PC filters having a pore size of 0.1 µm, 0.2 µm, 0.4 µm, 0.8 µm stacked on top of each other, and the temperature was set to 70° C. in a circulating water bath. The extruder was attached to a No. ₂ canister with a regulator and safety valve. The assembled extruder was allowed to warm up and then loaded with the liquid dispersion. The extruder was closed and the liquid dispersion was allowed to equilibrate to 70°C. The N ₂ bottle piping was opened and the N ₂ pressure was slowly increased until a steady stream of liquid dispersion exiting the outlet hose was observed. The filtration steps were repeated 4 more times. The filtrate after the 5th filtration was collected in a clean tube and the pH of the filtrate was adjusted to 6.5 using 0.1N HCl. NaOH and/or 0.1 N. HCl after cooling to room temperature. The filtrate was sterilized using a syringe filter (Whatman PES filter material with a polypropylene body and a pore size of 0.2 μm) and stored in a refrigerator without freezing until further use in experiments. It was confirmed by dynamic light scattering analysis that the composition thus obtained contained liposomes.

[0054] Example 2

Obtaining a composition containing the components listed in table 2

table 2

Example 2: 1.0 mol. % eritoran, 2.5 mol. % DSPE-PEG2000, 6 mol. % cholest. Material mm Mol. % in 5 ml lot Eritoran 1.00 1.00 7.01 mg* Cholesterol 6.00 6.00 11.60 mg DSPC 90.43 90.43 357.27 mg DSPE-PEG2000 2.50 2.50 35.07 mg BHA 0.07 0.07 0.06 mg Total 100.00 100.00 411.01 mg

*: mg eritoran as tetrasodium salt

[0055] In example 2, the following reagents were used.

(1) 0.1 N NaOH

One ml 1 N. NaOH was added to a 10 ml volumetric flask and then made up to volume with purified water.

(2) 0.1 N HCl

One ml 1 N. HCl was added to a 10 ml volumetric flask and then made up to volume with purified water.

(3) Sucrose buffer solution

Twenty g of sucrose was weighed into a 200 ml glass beaker and approximately 180 ml of purified water was added. Then 24 mg of NaH ₂ PO ₄ was added and the pH was adjusted to 6.5 by adding 0.1N. NaOH and 0.1 N. HCl. The solution was added to a 200 ml volumetric flask and made up to volume with purified water.

(4) Chloroform/methanol mixture

Fifty ml of chloroform and 100 ml of methanol were mixed in a glass bottle.

(5) BHA solution

Six mg of BHA was weighed into a glass bottle and 10 ml of chloroform/methanol was added. Complete dissolution of BHA was ensured by shaking.

[0056] (1) Preparation of a thin film

To obtain the composition shown in Table 2, 6.7 mg of erytoran, 368.3 mg of DSPC, 11.9 mg of cholesterol and 34.9 mg of DSPE-PEG2000 were weighed into a 50 ml round bottom flask. Eight ml of chloroform and 3 ml of methanol were added and the flask was shaken gently. After making sure that all components were completely dissolved, 40 μl of BHA solution was added. The solvent was removed using a rotary evaporator to obtain a thin film from the components. The round bottom flask was placed in a vacuum chamber overnight to remove residual solvent.

[0057] (2) Preparation of liquid dispersion

The sucrose buffer solution was heated to 70° C. in a water bath, and 5 ml of the heated sucrose buffer solution was added to a round bottom flask containing a thin film. The mixture was subjected to ultrasonic treatment in an ultrasonic bath until the thin film was completely dispersed.

[0058] (3) Preparation of composition

The extruder was mounted with PC filters having a pore size of 0.1 µm, 0.2 µm, 0.4 µm, 0.8 µm stacked on top of each other, and the temperature was set to 70° C. in a circulating water bath. The extruder was attached to a No. ₂ canister with a regulator and safety valve. The assembled extruder was allowed to warm up and then loaded with the liquid dispersion. The extruder was closed and the liquid dispersion was allowed to equilibrate to 70°C. The N ₂ bottle piping was opened and the N ₂ pressure was slowly increased until a steady stream of liquid dispersion exiting the outlet hose was observed. The filtration steps were repeated 4 more times. The filtrate after the 5th filtration was collected in a clean tube, and after cooling to room temperature, the pH of the filtrate was adjusted to 6.5 using 0.1N HCl. NaOH and/or 0.1 N. HCl. The filtrate was sterilized using a syringe filter (Whatman PES filter material with a polypropylene body and a pore size of 0.2 μm) and stored in a refrigerator without freezing until further use in experiments. It was confirmed by dynamic light scattering analysis that the composition thus obtained contained liposomes.

[0059] Example 3

Obtaining a composition containing the components listed in table 3

Table 3

Example 3: 1 mol. % eritoran, 1 mol. % DSPE-PEG2000, 6 mol. % cholest. Material mm Mol. % in 5 ml lot Eritoran 1.00 1.00 7.01 mg* Cholesterol 6.00 6.00 11.60 mg DSPC 91.93 91.93 363.19 mg DSPE-PEG2000 1.00 1.00 14.03 mg BHA 0.07 0.07 0.16 mg Total 100.00 100.00 395.99 mg

*: mg eritoran as tetrasodium salt

[0060] Example 3 used the following reagents.

(1) 0.1 N NaOH

One ml 1 N. NaOH was added to a 10 ml volumetric flask and then made up to volume with purified water.

(2) 0.1 N HCl

One ml 1 N. HCl was added to a 10 ml volumetric flask and then made up to volume with purified water.

(3) Sucrose buffer solution

Twenty g of sucrose was weighed into a 200 ml glass beaker and approximately 180 ml of purified water was added. Then 24 mg of NaH ₂ PO ₄ was added and the pH was adjusted to 6.5 by adding 0.1N. NaOH and 0.1 N. HCl. The solution was added to a 200 ml volumetric flask and made up to volume with purified water.

(4) Chloroform/methanol mixture

Fifty ml of chloroform and 100 ml of methanol were mixed in a glass bottle.

(5) Eritoran Solution

Sixty mg of eritoran was weighed into a glass bottle and 10 ml of chloroform/methanol was added. Complete dissolution of eritoran was ensured by shaking.

(6) DSPC solution

One point two tenths of a g of DSPC was weighed into a glass bottle and 50 ml of chloroform/methanol was added. Ensured complete dissolution of DSPC by shaking.

(7) Cholesterol solution

Fifty mg of cholesterol was weighed into a glass bottle and 25 ml of chloroform/methanol was added. Complete dissolution of cholesterol was ensured by shaking.

(8) DSPE-PEG2000 solution

Four hundred mg of DSPE-PEG2000 was weighed into a glass bottle and 20 ml of chloroform/methanol was added. Ensured complete dissolution of DSPE-PEG2000 by shaking.

(9) BHA solution

Four mg of BHA were weighed into a glass bottle and 10 ml of chloroform/methanol was added. Complete dissolution of BHA was ensured by shaking.

[0061] (1) Preparation of a thin film

To obtain the composition indicated in Table 3, 1.17 ml of erytoran solution, 12.13 ml of DSPC solution, 5.8 ml of cholesterol solution, 0.62 ml of DSPE-PEG2000 solution and 400 μl of BHA solution were mixed in a 50 ml round bottom flask . After making sure that all components were completely dissolved, the solvent was removed using a rotary evaporator to obtain a thin film from the components. The round bottom flask was placed in a vacuum chamber overnight to remove residual solvent.

[0062] (2) Preparation of liquid dispersion

The sucrose buffer solution was heated to 70° C. in a water bath, and 5 ml of the heated sucrose buffer solution was added to a round bottom flask containing a thin film. The mixture was subjected to ultrasonic treatment in an ultrasonic bath until the thin film was completely dispersed.

[0063] (3) Preparation of composition

The extruder was mounted with PC filters having a pore size of 0.1 µm, 0.2 µm, 0.4 µm, 0.8 µm stacked on top of each other, and the temperature was set to 70° C. in a circulating water bath. The extruder was attached to a No. ₂ canister with a regulator and safety valve. The assembled extruder was allowed to warm up and then loaded with the liquid dispersion. The extruder was closed and the liquid dispersion was allowed to equilibrate to 70°C. The N ₂ bottle piping was opened and the N ₂ pressure was slowly increased until a steady stream of liquid dispersion exiting the outlet hose was observed. The filtration steps were repeated 4 more times. The filtrate after the 5th filtration was collected in a clean tube, and after cooling to room temperature, the pH of the filtrate was adjusted to 6.5 using 0.1N HCl. NaOH and/or 0.1 N. HCl. The filtrate was sterilized using a syringe filter (Whatman PES filter material with a polypropylene body and a pore size of 0.2 μm) and stored in a refrigerator without freezing until further use in experiments. It was confirmed by dynamic light scattering analysis that the composition thus obtained contained liposomes.

[0064] Example 4

Obtaining a composition containing the components listed in table 4

Table 4

Example 4: 2.5 mol. % eritoran, 1 mol. % DSPE-PEG2000, 6 mol. % cholest. Material mm Mol. % in 5 ml lot Eritoran 1.00 2.50 7.01 mg* Cholesterol 2.40 6.00 4.64 mg DSPC 36.17 90.43 142.91 mg DSPE-PEG2000 0.40 1.00 5.61 mg BHA 0.03 0.07 0.03 mg Total 40.00 100.00 160.19 mg

*: mg eritoran as tetrasodium salt

[0065] Example 4 used the following reagents.

(1) 0.1 N NaOH

One ml 1 N. NaOH was added to a 10 ml volumetric flask and then made up to volume with purified water.

(2) 0.1 N HCl

One ml 1 N. HCl was added to a 10 ml volumetric flask and then made up to volume with purified water.

(3) Sucrose buffer solution

Twenty g of sucrose was weighed into a 200 ml glass beaker and approximately 180 ml of purified water was added. Then 24 mg of NaH ₂ PO ₄ was added and the pH was adjusted to 6.5 by adding 0.1N. NaOH and 0.1 N. HCl. The solution was added to a 200 ml volumetric flask and made up to volume with purified water.

(4) Chloroform/methanol mixture

Fifty ml of chloroform and 100 ml of methanol were mixed in a glass bottle.

(5) Eritoran Solution

Fifty mg of eritoran was weighed into a glass bottle and 10 ml of chloroform/methanol was added. Complete dissolution of eritoran was ensured by shaking.

(6) DSPC solution

Six hundred mg of DSPC was weighed into a glass bottle and 10 ml of chloroform/methanol was added. Ensured complete dissolution of DSPC by shaking.

(7) Cholesterol solution

Twenty-five mg of cholesterol was weighed into a glass bottle and 10 ml of chloroform was added. Complete dissolution of cholesterol was ensured by shaking.

(8) DSPE-PEG2000 solution

Eighty mg of DSPE-PEG2000 was weighed into a glass bottle and 10 ml of chloroform/methanol was added. Ensured complete dissolution of DSPE-PEG2000 by shaking.

(9) BHA solution

Six mg of BHA was weighed into a glass bottle and 10 ml of chloroform/methanol was added. Complete dissolution of BHA was ensured by shaking.

[0066] (1) Preparation of a thin film

To obtain the composition shown in Table 4, 1.40 ml of erytoran solution, 2.38 ml of DSPC solution, 1.86 ml of cholesterol solution, 0.70 ml of DSPE-PEG2000 solution and 40 μl of BHA solution were mixed in a 50 ml round bottom flask . After making sure that all components were completely dissolved, the solvent was removed using a rotary evaporator to obtain a thin film from the components. The round bottom flask was placed in a vacuum chamber overnight to remove residual solvent.

[0067] (2) Preparation of liquid dispersion

The sucrose buffer solution was heated to 70° C. in a water bath, and 5 ml of the heated sucrose buffer solution was added to a round bottom flask containing a thin film. The mixture was subjected to ultrasonic treatment in an ultrasonic bath until the thin film was completely dispersed.

[0068] (3) Preparation of composition

The extruder was mounted with PC filters having a pore size of 0.1 µm, 0.2 µm, 0.4 µm, 0.8 µm stacked on top of each other, and the temperature was set to 70° C. in a circulating water bath. The extruder was attached to a No. ₂ canister with a regulator and safety valve. The assembled extruder was allowed to warm up and then loaded with the liquid dispersion. The extruder was closed and the liquid dispersion was allowed to equilibrate to 70°C. The N ₂ bottle piping was opened and the N ₂ pressure was slowly increased until a steady stream of liquid dispersion exiting the outlet hose was observed. The filtration steps were repeated 4 more times. The filtrate after the 5th filtration was collected in a clean tube, and after cooling to room temperature, the pH of the filtrate was adjusted to 6.5 using 0.1N HCl. NaOH and/or 0.1 N. HCl. The filtrate was sterilized using a syringe filter (Whatman PES filter material with a polypropylene body and a pore size of 0.2 μm) and stored in a refrigerator without freezing until further use in experiments. It was confirmed by dynamic light scattering analysis that the composition thus obtained contained liposomes.

[0069] Examples 5-8

Preparation of formulations containing the components listed in tables 5 and 6.

Table 5

Example 5: 2.5 mol. % eritoran, 2.5 mol. % DSPE-PEG2000, 0 mol. % cholest. Material mm Mol. % in 5 ml lot Eritoran 1.00 2.50 7.01 mg* Cholesterol - - - DSPC 37.97 94.93 150.02 mg DSPE-PEG2000 1.00 2.50 14.03 mg BHA 0.03 0.07 0.03 mg Total 40.00 100.00 171.08 mg Example 6: 2.5 mol. % eritoran, 2.5 mol. % DSPE-PEG2000, 30 mol. % cholest. Material mm Mol. % in 5 ml lot Eritoran 1.00 2.50 7.01 mg* Cholesterol 12.00 30.00 23.20 mg DSPC 25.97 64.93 102.61 mg DSPE-PEG2000 1.00 2.50 14.03 mg BHA 0.03 0.07 0.03 mg Total 40.00 100.00 146.87 mg

*: mg eritoran as tetrasodium salt

Table 6

Example 7: 0.5 mol. % eritoran, 1 mol. % DSPE-PEG2000, 6 mol. % cholest. Material mm Mol. % in 5 ml lot Eritoran 0.25 0.50 1.75 mg* Cholesterol 3.00 6.00 5.80 mg DSPC 46.21 92.43 182.58 mg DSPE-PEG2000 0.50 1.00 7.01 mg BHA 0.04 0.07 0.03 mg Total 50.00 100.00 197.18 mg Example 8: 0.5 mol. % eritoran, 2.5 mol. % DSPE-PEG2000, 6 mol. % cholest. Material mm Mol. % in 5 ml lot Eritoran 0.25 0.5 1.75 mg* Cholesterol 3.00 6.00 5.80 mg DSPC 45.46 90.93 179.62 mg DSPE-PEG2000 1.25 2.50 17.53 mg BHA 0.04 0.07 0.03 mg Total 50.00 100.00 204.74 mg

*: mg eritoran as tetrasodium salt

[0070] Examples 5-8 used the following reagents.

(1) 0.1 N NaOH

One ml 1 N. NaOH was added to a 10 ml volumetric flask and then made up to volume with purified water.

(2) 0.1 N HCl

One ml 1 N. HCl was added to a 10 ml volumetric flask and then made up to volume with purified water.

(3) Sucrose buffer solution

Twenty g of sucrose was weighed into a 200 ml glass beaker and approximately 180 ml of purified water was added. Then 24 mg of NaH ₂ PO ₄ was added and the pH was adjusted to 6.5 by adding 0.1N. NaOH and 0.1 N. HCl. The solution was added to a 200 ml volumetric flask and made up to volume with purified water.

(4) Chloroform/methanol mixture

Fifty ml of chloroform and 100 ml of methanol were mixed in a glass bottle.

(5) Eritoran solution

Five mg of eritoran was weighed into a glass bottle and 5 ml of chloroform/methanol was added. Complete dissolution of eritoran was ensured by shaking.

(6) DSPC solution

Seven hundred and fifty mg of DSPC was weighed into a glass bottle and 50 ml of chloroform/methanol was added. Ensured complete dissolution of DSPC by shaking.

(7) Cholesterol solution

Forty-five mg of cholesterol was weighed into a glass bottle and 10 ml of chloroform was added. Complete dissolution of cholesterol was ensured by shaking.

(8) DSPE-PEG2000 solution

Sixty-five mg of DSPE-PEG2000 was weighed into a glass bottle and 5 ml of chloroform/methanol was added. Ensured complete dissolution of DSPE-PEG2000 by shaking.

(9) BHA solution

Six mg of BHA was weighed into a glass bottle and 10 ml of chloroform/methanol was added. Complete dissolution of BHA was ensured by shaking.

[0071] (1) Preparation of a thin film

1.39 ml of erytoran solution, 10.0 ml of DSPC solution, 1.07 ml of DSPE-PEG2000 solution and 40 μl of BHA solution were mixed in a 50 ml round bottom flask to obtain the composition of example 5 indicated in Table 5.

To obtain the composition of example 6 indicated in table 5, in a 50 ml round bottom flask were mixed 1.39 ml of erytoran solution, 4.98 ml of cholesterol solution, 6.84 ml of DSPC solution, 1.07 ml of DSPE-PEG2000 solution and 40 µl of BHA solution.

To obtain the composition of example 7 indicated in table 6, in a 50 ml round bottom flask were mixed 0.35 ml of erytoran solution, 1.25 ml of cholesterol solution, 12.18 ml of DSPC solution, 0.54 ml of DSPE-PEG2000 solution and 50 µl of BHA solution.

To obtain the composition of example 8 indicated in table 6, in a 50 ml round bottom flask were mixed 0.35 ml of erytoran solution, 1.25 ml of cholesterol solution, 11.98 ml of DSPC solution, 1.34 ml of DSPE-PEG2000 solution and 50 µl of BHA solution.

After making sure that all components were completely dissolved, the solvent was removed from each flask using a rotary evaporator to obtain a thin film from the components. Round bottom flasks were placed in a vacuum chamber overnight to remove residual solvent.

[0072] (2) Preparation of liquid dispersion

The sucrose buffer solution was heated to 70° C. in a water bath, and 5 ml of the heated sucrose buffer solution was added to each round bottom flask containing a thin film. The mixture was subjected to ultrasonic treatment in an ultrasonic bath until the thin film was completely dispersed.

[0073] (3) Preparation of composition

The extruder was mounted with PC filters having a pore size of 0.1 µm, 0.2 µm, 0.4 µm, 0.8 µm stacked on top of each other, and the temperature was set to 70° C. in a circulating water bath. The extruder was attached to a No. ₂ canister with a regulator and safety valve. The assembled extruder was allowed to warm up and then loaded with the liquid dispersion. The extruder was closed and the liquid dispersion was allowed to equilibrate to 70°C. The N ₂ bottle piping was opened and the N ₂ pressure was slowly increased until a steady stream of liquid dispersion exiting the outlet hose was observed. The filtration steps were repeated 4 more times. The filtrate after the 5th filtration was collected in a clean tube, and after cooling to room temperature, the pH of the filtrate was adjusted to 6.5 using 0.1N HCl. NaOH and/or 0.1 N. HCl. The filtrate was sterilized using a syringe filter (Whatman PES filter material with a polypropylene body and a pore size of 0.2 μm) and stored in a refrigerator without freezing until further use in experiments. It was confirmed by dynamic light scattering analysis that the formulations thus obtained contained liposomes.

[0074] Examples 9-26

Preparation of formulations containing the components listed in Table 7.

Table 7

Compound from Example No. Mol. % Eritoran DSPC Cholest. DSPE-PEG2000 BHA Compound from example 9 one 87.929 6 5 0.071 Compound from example 10 2.5 86.429 6 5 0.071 Compound from Example 11 5 83.929 6 5 0.071 Compound from Example 12 7.5 81.429 6 5 0.071 Compound from Example 13 5 86.429 6 2.5 0.071 Compound from Example 14 7.5 83.929 6 2.5 0.071 Compound from Example 15 5 87.929 6 one 0.071 Compound from Example 16 5 88.929 6 0 0.071 Compound from Example 17 5 81.429 6 7.5 0.071 Compound from Example 18 7.5 78.929 6 7.5 0.071 Compound from Example 19 one 75.429 6 17.5 0.071 Compound from Example 20 5 71.429 6 17.5 0.071 Compound from example 21 one 85.429 6 7.5 0.071 Compound from example 22 2.5 83.929 6 7.5 0.071 Compound from example 23 one 92.929 6 0 0.071 Compound from example 24 0 91.429 6 2.5 0.071 Compound from example 25 0 88.929 6 5 0.071 Compound from example 26 0 86.429 6 7.5 0.071

[0075] The compositions of examples 9-26 were prepared according to essentially the same procedure as used in example 1. The concentration of eritoran in the compositions of table 7 was about 1 mm. The total lipid concentration in the formulations without erytoran (Examples 24-26) was about 100 mM.

[0076] <Dynamic Light Scattering Analysis>

Analysis of the compositions of examples 1-26 was performed using the method of dynamic light scattering (DLS) to determine the average particle size (Z-ave) and coefficient of polydispersity (PdI). The following settings were used for DLS analysis.

[0077] (Experimental procedure)

System: Zetasizer Nano-ZS (Malvern Panalytical)

Measurement conditions:

Sample reflectance: 1.45

Sample adsorption rate; 0.01

Dispersion medium: Milli-Q water

Reflection coefficient of the dispersion medium: 1.330

Viscosity of the dispersion medium (cP): 1.0031

Measurement temperature: 20°C

Time to incubation: 5 min

Measurement time 60 sec, 3 times

Measuring point (distance): 4.65mm

Sample preparation:

fifty microliters of sample in 5 ml Milli-Q water

[0078] (DLS Analysis Result)

The Z-ave and PdI values for each formulation are summarized in Table 8. For formulations that differ from the formulations of Examples 18-20, the Z-ave was 100 nm or greater, so it was believed that erytoran-containing liposomes formed in these formulations. For formulations that differ from those of Examples 18-22 and 26, the PdI was 0.2 or less, so it was believed that the particle size distribution was sufficiently controlled in these formulations. The PdI for the erytorane-containing formulations of Examples 18-22 (i.e., greater than 0.2) indicates a possible problem with uniformity of the erytorane-based finely divided formulation.

Table 8

Compound from Example No. Z-ave (nm) PdI Compound from example 1 117 0.11 Compound from example 2 106.1 0.091 Compound from Example 3 112.5 0.185 Compound from Example 4 120.3 0.095 Compound from Example 5 116.5 0.046 Compound from example 6 119.7 0.034 Compound from Example 7 112.2 0.05 Compound from Example 8 114.3 0.04 Compound from example 9 107.6 0.091 Compound from example 10 118.5 0.095 Compound from Example 11 119.6 0.098 Compound from Example 12 100.4 0.188 Compound from Example 13 118.4 0.092 Compound from Example 14 104.8 0.126 Compound from Example 15 121.3 0.095 Compound from Example 16 107.9 0.145 Compound from Example 17 104.4 0.195 Compound from Example 18 49.8 0.243 Compound from Example 19 62.7 0.477 Compound from Example 20 44.2 0.337 Compound from example 21 110.9 0.253 Compound from Example 22 109 0.209 Compound from example 23 109.3 0.126 Compound from example 24 120.4 0.092 Compound from example 25 119.9 0.068 Compound from Example 26 104.8 0.209

[0079] <Micellized Eritoran and Placebo Used in In Vivo and In Vitro Assays>

The micellized eritoran and placebo used in the in vivo and in vitro assays were prepared from the lyophilized powder in a vial. The micellized erytoran vial contains erytoran (as basic tetrasodium salt 4.0 [Na]) 7.46 mg, lactose monohydrate and butylated hydroxyanisole. Sodium hydroxide and phosphoric acid are also contained in sufficient quantities in the micellized eritoran vial. The placebo vial contains lactose monohydrate. Sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate, and sodium hydroxide are also contained in adequate amounts in the placebo vial. The contents of each vial were reconstituted by adding 3.0 ml of sterile water for injection (solution concentration of 2.33 mg/ml or 1.66 mm). Reconstituted micellized erytoran was diluted with reconstituted placebo solution to obtain an appropriate concentration.

[0080] <In vivo efficacy of the formulations of the examples>

The in vivo efficacy of the formulations obtained in Examples 1-26 was evaluated through studies in mice. Female C57BL/6NCrl mice (Charles River Laboratories) with a body weight of approximately 20 g per mouse were used, 4 mice per group. Animals were given 9.1 mg/kg micellized eritoran or formulations of Examples 2 hours prior to and/or administration of LPS from Escherichia coli O111:B4 (0.5 μg/animal. Cat. No. 201, List Biological Laboratories, Inc.). Two groups of mice were injected with the same volume of sucrose buffer and placebo as a mock formulation control and a micellized eritoran control, respectively. Animals were sampled for cytokine levels 2 hours after LPS administration and plasma was collected (from the abdominal vein under anesthesia) in EDTA/3K tubes (cat. no. 499388, Greiner Bio-One GmbH). IL-6 cytokine levels were measured according to the ELISA kit protocol (Mouse IL-6 ELISA Set, cat. no. 555240; reagent kit B, pH 9.5, cat. no. 550534, BD Biosciences) except for the procedures described below. The binding antibody was diluted in carbonate-bicarbonate buffer (1 capsule in 50 ml Milli-Q water, cat. no. C3041-100CAP, Sigma-Aldrich Corporation) as an immobilization buffer. The wells were washed with phosphate buffered saline (p/n P3563-10PAK, Sigma-Aldrich Corporation) using a plate washer (ELx405 select, BioTek Instruments, Inc.) at each step. To remove residual wash buffer, the plate was centrifuged at 4000 rpm for 1 min upside down at each stage (HITACH CF16RXII; rotor: T5S32-0049). An absorption spectrophotometer was used to determine the optical density in each well (SpectraMax 190, Molecular Devices, LLC). Duplicate well absorbances were averaged and a standard curve was fitted by four parameter logistic (4-PL) curve fitting using SoftMax Pro 6.5.1 software (current version number 219831, Molecular Devices, LLC). Plot and median with interquartile range were plotted using GraphPad Prism 7.02 (GraphPad software, Inc.). Part of the results are shown in Figure 1, and additional results are shown in Table 9. These results show that erytoran-containing liposomes suppress IL-6 production compared to micellized erytoran.

[0081]

Table 9

Example Connection IL-6 level at optimal concentration (% of control) Compound from Example 1 11.54 Compound from Example 2 12.69 Compound from Example 3 9.74 Compound from Example 4 11.13 Compound from Example 5 5.54 Compound from example 6 8.46 Compound from example 9 11.81 Compound from Example 10 2.59 Compound from Example 11 6.52 Compound from Example 12 6.55 Compound from example 13 9.74 Compound from Example 14 15.84 Compound from Example 15 17.33 Compound from Example 16 13.60 Compound from Example 17 4.88 Compound from Example 18 6.01 Compound from example 20 82.23 Compound from example 21 7.63 Compound from example 22 2.39 Compound from Example 23 8.95 Compound from example 24 91.08 Compound from Example 25 69.84 Compound from Example 26 85.27 Micellized eritoran 59.26

[0082] <In vivo screening analysis of the formulations of the examples>

Screening analysis to clarify the number of the best compositions of examples 1-26 was carried out according to the above analysis with a partial change in its protocol ("The effectiveness of the compositions of examples in vivo"). Animals under the same conditions were injected iv with 1.1375 mg/kg of the formulations of the examples diluted (8-fold) with sucrose buffer, and a control was used 2 hours before ip administration of LPS from Escherichia coli O111:B4. Animals were selected to measure cytokine levels 2 hours after LPS administration and blood plasma was collected (from the abdominal vein under anesthesia) in EDTA/3K tubes. Cytokine IL-6 was measured by the same procedure as in the above analysis ("In vivo efficacy of the formulations of the examples"). Some of the results are shown in figures 2-1, 2-2 and 2-3.

[0083] Figure 2-1 shows that the diluted compound of Example 1 exhibited 78.8% inhibition of IL-6 production compared to the sham control (median IL-6 concentrations in the sham group and in the group of the diluted compound of Example 1 were 2851.8 pg/mL and 606.0 pg/mL, respectively; line indicates median value with interquartile range). IL-6 production suppression activity was not detected in the diluted compound of Example 11 and the diluted compound of Example 13 (mean concentrations of IL-6 in the groups of diluted compound of Example 11 and diluted compound of Example 13 were 3203.0 pg/ml and 4104, 6 pg/ml, respectively).

[0084] Figures 2-2 and 2-3 show that suppression of this condition was also observed with diluted Example 2, diluted Example 3, diluted Example 4, and diluted Example 5 (average concentrations IL-6 in the groups of sham control, diluted compound from example 2, diluted compound from example 3 and diluted compound from example 4 were 2605.8 pg/ml, 390.9 pg/ml, 357.8 pg/ml, 626.3 pg/ml, respectively). The diluted compound of Example 5, the cholesterol-modified formulation of Example 1 also showed high IL-6 suppression activity (96.7%, median IL-6 concentrations in the sham group and in the compound of Example 5 group were 2812.1 pg/mL). and 92.1 pg/ml, respectively).

[0085] A summary of the results for all diluted formulations is shown in Table 10.

Table 10

Diluted compound from the example IL-6 level at low concentration
(% control) 1st analysis 2nd analysis Compound from Example 1 21.25 26.26 Compound from Example 2 15.00 39.69 Compound from Example 3 13.73 42.66 Compound from Example 4 24.03 51.43 Compound from Example 5 0.98 3.27 Compound from example 6 271.35 75.56 Compound from Example 7 141.36 - Compound from example 8 76.83 - Compound from example 9 119.18 - Compound from Example 10 14.26 63.96 Compound from Example 11 112.32 37.10 Compound from Example 12 70.86 60.01 Compound from example 13 143.93 - Compound from Example 14 176.94 - Compound from Example 15 231.98 - Compound from Example 16 102.86 - Compound from Example 17 108.65 - Compound from Example 18 47.25 104.81 Compound from Example 20 129.72 - Compound from Example 21 95.63 - Compound from example 22 101.21 - Compound from example 23 190.15 -

[0086] <Human HDL Inactivation Assay>

The effect of high density lipoproteins (HDL) on the formulations of the examples was evaluated according to the following protocol. The formulations were diluted to 50 pM with saline (cat. no. 3311401A3111, Otsuka Pharmaceutical Co., Ltd.) and mixed with 1 mg/ml human HDL (cat. no. LP3-5MG, Merck KGaA). Mixed compositions were incubated at 37°C for 18 hours. Healthy human whole blood was collected in EDTA/2K test tubes (VENOJECT (registered trademark), cat. no. VP-H100K, TERUMO CORPORATION). The mixed formulations were mixed with 4-fold volumes of whole blood containing 10 ng/ml LPS at the final concentration. After incubation at 37°C for 3 hours, the supernatant was obtained by centrifugation (1000 x g for 5 min at 4°C). TNF-α was measured by ELISA (cat. no. STA00C, R&D Systems). ELISA analysis was performed in accordance with the attached instructions for use. The wells were washed with a plate washer (ELx405 select, BioTek Instruments, Inc.) at each stage. To remove residual wash buffer, the plate was centrifuged at 4000 rpm for 1 min upside down at each stage (HITACH CF16RXII; rotor: T5S32-0049). An absorption spectrophotometer was used to determine the optical density in each well (SpectraMax 190, Molecular Devices, LLC). Duplicate well absorbances were averaged and a standard curve was fitted by four parameter logistic (4-PL) curve fitting using SoftMax Pro 6.5.1 software (current version number 219831, Molecular Devices, LLC). Plot and median with interquartile range were plotted using GraphPad Prism 7.02 (GraphPad software, Inc.). The results are shown in Table 11 and Figure 3. These results show that the formulations of Examples 1-4 retain their activity even in the presence of human HDL.

Table 11

PBS control Micellized eritoran Compound from example 1 Compound from Example 2 Compound from Example 3 Compound from Example 4 TNF-α concentration (pg/ml) 6108.0 4351.8 210.3 437.2 273.7 215.1 TNF-α level (% of control) 100.0 71.2 3.4 7.2 4.5 3.5

Claims (24)

1. Composition to suppress the binding of lipopolysaccharides with TLR4 containing liposomes, where the liposomes contain from 0.7 to 3.0 mol. % eritoran or its pharmaceutically acceptable salt, from 0.5 to 3.0 mol. % pegylated phospholipid in terms of liposome and from 0 to 10 mol. % sterol based on liposome, where pegylated phospholipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. 2. The composition of claim 1 wherein the pharmaceutically acceptable salt is the tetrasodium salt. 3. The composition according to p. 1, where the liposomes contain from 1.0 to 2.5 mol. % eritoran or its pharmaceutically acceptable salt and from 1.0 to 2.5 mol. % of pegylated phospholipid in terms of liposome. 4. Composition according to claim 1, where the liposomes contain 1.0 mol. % eritoran or its pharmaceutically acceptable salt and 1.0 mol. % 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in terms of liposome. 5. Composition according to claim 1, where the liposomes contain 1.0 mol. % eritoran or its pharmaceutically acceptable salt and 2.5 mol. % 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in terms of liposome. 6. Composition according to claim 1, where the liposomes contain 2.5 mol. % eritoran or its pharmaceutically acceptable salt and 1.0 mol. % 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in terms of liposome. 7. Composition according to claim 1, where the liposomes contain 2.5 mol. % eritoran or its pharmaceutically acceptable salt and 2.5 mol. % 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in terms of liposome. 8. Composition according to claim 1, wherein the liposomes additionally contain phosphatidylcholine. 9. The formulation of claim 8 wherein the phosphatidylcholine is distearoylphosphatidylcholine. 10. Composition according to claim 1, wherein the liposomes additionally contain an antioxidant. 11. The composition of claim 10 wherein the antioxidant is butylated hydroxyanisole. 12. The composition of claim 1 wherein the sterol is cholesterol. 13. Composition according to claim 1, wherein the liposomes do not contain a sterol. 14. The formulation of claim 1 wherein the liposomes have an average particle size of 100 to 120 nm as measured by dynamic light scattering (DLS). 15. Composition according to claim 1, wherein the liposomes have a polydispersity index of 0.20 or less. 16. The method of obtaining the composition according to paragraphs. 1-15 containing liposomes, including obtaining a solution containing from 0.7 to 3.0 mol. % eritoran or its pharmaceutically acceptable salt, from 0.5 to 3.0 mol. % pegylated phospholipid and solvent, based on all liposome components to be used; evaporating the solvent from the solution to form a thin film; dispersion of a thin film in a buffer solution to form a liquid dispersion, and extruding the liquid dispersion through a filter to form a composition. 17. The method of claim 16 wherein the solution further comprises phosphatidylcholine and an antioxidant. 18. The method of claim 16 wherein the solvent is a combination of chloroform and methanol. 19. The method of claim 16 wherein the dispersion step is carried out by sonication. 20. The method of claim 16, wherein the method further comprises adjusting the pH of the composition formed during the extrusion step to 6.2-6.8.

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