RU2780026C2 - Specially developed liposomes for treatment of bacterial infections - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to the field of medicine and pharmaceutics, namely to empty liposomes for use in the treatment or prevention of a bacterial infection, where liposomes contain cholesterol in the amount of 40 wt.% or more and sphingomyelin; as well as to the use of the specified liposomes for the treatment or prevention of a bacterial infection; and to a method for the treatment or prevention of a bacterial infection, including the administration to a patient who needs it of therapeutically effective amount of the specified empty liposomes.

EFFECT: group of inventions provides for the expansion of the arsenal of antibacterial agents.

7 cl, 10 dwg, 3 tbl

Description

Technical field

The present invention relates to the use of empty liposomes or mixtures of liposomes and to the use of other lipid bilayers or monolayers of a defined lipid composition for the treatment and prevention of bacterial infections. The present invention also relates to the treatment of such bacterial infections, comprising the administration of empty liposomes or mixtures of liposomes alone or in combination with standard antibiotic treatment. In addition, the present invention relates to the new mixtures of liposomes themselves.

State of the art

Bacterial infections remain one of the main threats to human life. As bacterial resistance to even the strongest antibiotics grows, so must efforts to find new strategies to fight bacteria. Along with other virulence factors, many pathogenic bacteria secrete toxins that kill eukaryotic cells by damaging their cell membrane. Bacterial pore-forming toxins are active on the cell surface, which causes pore formation and damage to the cell membrane, followed by lysis or apoptosis of target host cells, while bacterial phospholipases induce host cell death by enzymatic degradation of plasma membrane phospholipids.

Bacterial membrane-destabilizing toxins such as cholesterol-dependent cytolysins (CCS: pneumolysin O, streptolysin O, tetanolysin), α-hemolysin or bacterial phospholipases (phospholipase C, sphingomyelinase) play a critical role in the development and progression of infectious diseases. These diseases are pneumonia, the leading cause of death among all age groups and the leading cause of death in children in low-income countries; bacteremia - a serious complication after infections or surgery, which is characterized by high mortality as a result of sepsis and septic shock; and meningitis, a life-threatening disease that also leads to serious long-term consequences such as deafness, epilepsy, hydrocephalus, and cognitive impairment.

To target host cells, bacterial membrane-destabilizing toxins either bind to individual membrane lipids (lipid head groups) or exploit the heterogeneous nature of the lipid bilayer of the cell membrane of eukaryotic cells by interacting with microdomains enriched in certain types of lipids (Gonzales M.R. et al., Cell Mol Life Sci 2008, 65:493-507). In vivo conditions do not favor heterogeneous distribution of lipids in the bilayer, since transmembrane proteins and the presence of a large number of individual lipid species with different lengths and degrees of saturation of the acyl chain prevent lipid exfoliation and, thus, the formation of stable lipid microdomains (Simons K. and Gerl M.J., Nat Rev Mol Cell Biol 2010, 11:688-99). However, lipid exfoliation can be taken to extremes in artificial protein free liposomes derived from a limited number of carefully selected lipid species in which longer stable lipid microdomains can be generated (Klose C. et al., J. Biol. Chem. 2010 , 285:30224-32). Moreover, artificial liposomes provide much higher relative concentrations of a particular lipid than would be encountered in vivo. Therefore, liposomes with stable lipid microdomains, having certain biochemical properties and containing high relative concentrations of specific lipids, can be obtained. Liposomes are currently used in the space and pharmaceutical industries as carriers for local and systemic drug delivery and are considered non-toxic.

Brief description of the invention

The present invention relates to the use of lipid-defined empty liposomes or mixtures of lipid-defined empty liposomes for the treatment and prevention of bacterial infections, in particular skin lesions, bacteremia, meningitis, respiratory tract infections such as pneumonia, and abdominal infections such as peritonitis.

Moreover, the present invention relates to lipid bilayers or lipid monolayers of defined lipid composition covering non-lipid surfaces for use in the treatment and prevention of bacterial infections.

Likewise, the present invention also relates to the treatment of such bacterial infections, comprising administering to a patient in need thereof a therapeutically effective amount of lipid-defined empty liposomes or mixtures of lipid-defined empty liposomes, and to a method of preventing such bacterial infections in a subject at risk. Moreover, the present depiction relates to the treatment of bacterial infections comprising administering to a patient in need thereof a therapeutically effective amount of lipid-defined empty liposomes, or mixtures of formulated empty liposomes, before, after, concomitantly, or in parallel with standard antibiotic treatment against a bacterial infection.

Moreover, the present invention relates to novel mixtures of empty liposomes of defined lipid composition.

Brief description of graphic materials

Fig. 1. Liposomes consisting of cholesterol and sphingomyelin protect monocytes from cholesterol-dependent cytolysins, α-hemolysin and phospholipase C.

A-D) Liposomes (mixtures 1:1 w/w) containing cholesterol in combination with phosphatidylcholine (PC) (3), sphingomyelin (Sm) (4) or phosphatidylserine (PS) (5), but not with phosphatidylethanolamine (PE ) (6) protected THP-1 cells from cholesterol-dependent cytolysins: pneumolysin O (A), streptolysin O (B), tetanolysin (C), and also from phospholipase C (D).

E) Liposomes (1:1 w/w) containing cholesterol in combination with Sm (4) but not with PC (3), PS (5) or PE (6) protected TPH-1 cells from α- hemolysin.

F) Liposomes without cholesterol (7-9) were ineffective.

c (o.e.) = number of cells kept in the presence of toxin (1, 3-9) relative to the number of cells kept in the absence of toxin (2), given in relative units (o.e.). PLY = pneumolysin O; SLO = streptolysin O; TL = tetanolysin; PLC=phospholipase C; HML = α-hemolysin. 1 = Control (no liposomes); 2=Control (no toxin), 3=liposomes cholesterol (Ch):PC (1:1 w/w); 4 = Ch:Sm liposomes (1:1 w/w); 5=liposomes Ch:PS (1:1 w/w); 6 = Ch:PE liposomes (1:1 w/w); 7=PC:Sm liposomes (1:1 w/w); 8 = Sm liposomes; 9 = PC liposomes. Ch = cholesterol; PC = phosphatidylcholine; PS = phosphatidylserine; Sm = sphingomyelin; PE = Phosphatidylethanolamine.

Fig. 2. Liposomes composed of cholesterol and sphingomyelin (1:1 w/w) protect monocytes from cholesterol-dependent cytolysins in microgram amounts, while protection from Staphylococcus aureus α-hemolysin and Clostridium perfringens phospholipase C requires 25 -100 micrograms of liposomes.

A) Protection against 0.2 micrograms of PLY. B) Protection against 0.4 microgram SLO. C) Protection against 0.2 microgram TL. D) Protection against 1.2 micrograms HML. E) Protection against 4.5 micrograms PLC.

c (o.e.) = number of cells kept in the presence of the toxin relative to the number of cells kept in the absence of the toxin, given in relative units. X-axis: LP (µg) = number of liposomes in micrograms. PLY = pneumolysin O; SLO = streptolysin O; TL = tetanolysin; HML = α-hemolysin; PLC = phospholipase C.

Fig. 3. To protect monocytes from pneumolysin, tetanolysin or α-hemolysin by liposomes consisting of cholesterol and sphingomyelin, cholesterol is required at concentrations greater than 30% (w/w)

A) protection against 0.2 micrograms of PLY. B) Protection against 0.2 microgram TL. C) Protection against 1.2 micrograms HML. c (o.e) = number of cells kept in the presence of the toxin, relative to the number of cells kept in the absence of the toxin, given in relative units. Ch (%) = percentage of cholesterol (w/w) in liposomes composed of cholesterol and sphingomyelin. PLY = pneumolysin; TL = tetanolysin; HML = α-hemolysin.

Fig. 4. Liposomes composed of cholesterol and sphingomyelin protect monocytes from a combination of cholesterol-dependent cytolysins and S. aureus α-hemolysin.

A) Ch:Sm liposomes (1:1 w/w) (6) provided complete protection against the combined action of α-hemolysin (HML; 1.2 micrograms), streptolysin O (SLO: 0.4 micrograms) and tetanolysin (TL; 0.2 micrograms), while Ch:PC liposomes (1:1 w/w) were ineffective (7).

c (o.e.) = number of cells kept in the presence of toxins (2-7) relative to the number of control cells kept in the absence of toxins (1), in relative units. 1 = Control (no toxins); 2 = SLO, no liposomes; 3=TL, no liposomes; 4 = HML, no liposomes; 5=SLO+TL+HML, no liposomes; 6=SLO+TL+HML, Ch:Sm liposomes; 7=SLO+TL+HML, Ch:PC liposomes. Ch = cholesterol; PC = phosphatidylcholine; Sm = sphingomyelin.

B) Full protective effect against the combined action of HML (1.2 micrograms), SLO (0.4 micrograms) and TL (0.2 micrograms) was observed at 25 micrograms of liposomes composed of Ch:Sm (1:1 w/w .). LP (µg) = number of liposomes in micrograms.

C) Centrifugation experiments confirmed that all three toxins bind directly to Ch:Sm liposomes (1:1 w/w). Toxins were preincubated together with Ch:Sm liposomes (6) or without them (2-5). After centrifugation, the supernatants were added to the cells. 1 = Control (no toxins); 2 = SLO, no liposomes; 3=TL, no liposomes; 4 = HML, no liposomes; 5=SLO+TL+HML, no liposomes; 6=SLO+TL+HML, Ch:Sm liposomes.

Fig. 5. Liposomes consisting of cholesterol and sphingomyelin (1:1 w/w) protect monocytes from Streptococcus pyogenes toxins.

A, B) THP-1 cells proliferate in the presence of brain heart extract (BHI) broth (squares; dotted line in (A)). Cultured supernatants of 5 strains of Streptococcus pyogenes (GAS 1-5 = clinical isolates; all grown in BHI broth) effectively killed THP-1 cells (triangles), but these cells were protected from exposure to streptococcal toxins by liposomes composed of cholesterol and sphingomyelin (circles ). 10 ⁵ s = number of cells × 10 ⁵ . t (d) = treatment time (days).

C) Liposomes composed of cholesterol and sphingomyelin protect monocytes from culture supernatants of Streptococcus pyogenes in microgram amounts. c (%) = percentage of cells maintained in the presence of bacterial supernatants, maintained in the absence of bacterial supernatants (100%). LP (µg) = number of liposomes in micrograms.

Fig. 6. Liposomes consisting of cholesterol and sphingomyelin (1:1 w/w), in combination with liposomes containing only sphingomyelin, completely protect monocytes from Streptococcus pneumoniae toxins.

A, B) THP-1 cells proliferate in the presence of BHI broth (squares). Culture supernatants of 2 strains of Streptococcus pneumoniae (Pneumo 1 = clinical isolate and Pneumo 2 = strain D39: both grown in BHI broth) effectively killed THP-1 cells (triangles), however, these cells were partially protected from the effects of Streptococcus pneumoniae toxins by liposomes consisting from cholesterol and sphingomyelin (1:1 w/w) (circles). 10 ⁵ s = number of cells × 10 ⁵ . t (d) = time after treatment (days).

C) A mixture of cholesterol-containing and cholesterol-free liposomes containing only sphingomyelin completely protected against Streptococcus pneumoniae toxins. The graph shows the protective effect of liposomal mixtures consisting of a constant (400 μg) amount of cholesterol:sphingomyelin liposomes (1:1 w/w) and varying amounts (0-400 μg) of sphingomyelin-only liposomes.

c (o.e.) = number of cells kept in the presence of bacterial packing liquid, relative to the number of cells kept in the absence of packing liquid, given in relative units (o.e.). Sm LP (μg) = number of sphingomyelin-only liposomes in micrograms.

Fig. 7. Liposomes cholesterol:phosphatidylcholine (1:1 w/w) and a mixture of liposomes cholesterol:phosphatidylcholine (1:1 w/w) and sphingomyelin liposomes protect monocytes from toxins secreted by Staphylococcus aureus strain MRSA 2040.

THP-1 cells proliferate in the presence of BHI broth (squares). Culture supernatants of Staphylococcus aureus (grown in BHI broth) effectively killed THP-1 cells in the absence of liposomes (triangles). (A) 900 micrograms (diamonds) cholesterol:phosphatidylcholine liposomes (1:1 w/w) provided significant protection against bacterial toxins, while only limited protection was observed at 600 micrograms (circles). (B) Complete protection was observed for a mixture of 600 micrograms of cholesterol:phosphatidylcholine liposomes (1:1 w/w) with 75 micrograms of Sm liposomes (diamonds). 900 micrograms of Sm liposomes used alone were ineffective (circles). 10 ⁵ c = number of cells × 10 ⁵ . t (d) = time after treatment (days).

Fig. 8. Cholesterol-free liposomes containing sphingomyelit and a mixture of cholesterol:phosphatidylcholine liposomes (1:1 w/w) and sphingomyelin-only liposomes protect monocytes from toxins secreted by strains of Doppelhof Staphylococcus aureus.

A, B) THP-1 cells are proliferated in the presence of BHI broth (squares). Culture supernatants of Staphylococcus aureus (grown in BHI broth) effectively kill THP-1 cells in the absence of liposomes (triangles). (A) 1200 micrograms (diamonds) of sphingomyelin liposomes provided significant protection against bacterial toxins. (B) When used at 600 micrograms, the most potent protection was observed for a mixture of sphingomyelin and sphingomyelin:phosphatidylcholine (1:1 w/w) (diamonds), while sphingomyelin liposomes alone (circles) and sphingomyelin:phosphatidylcholine liposomes (1:1) 1 w/w) alone (asterisks) were less effective. C) A mixture of cholesterol-containing and cholesterol-free liposomes containing only sphingomyelin completely protected against toxins secreted by Doppelhof strains of Staphylococcus aureus. The graph shows the protective effect of liposomal mixtures consisting of a constant (600 µg) amount of cholesterol:phosphatidylcholine liposomes (1:1 w/w) and varying amounts (0-1200 µg) of sphingomyelin-only liposomes.

10 ⁵ c = number of cells × 10 ⁵ . t (d) = time after treatment (days). c (oe) = number of cells kept in the presence of bacterial supernatant relative to the number of cells kept in the absence of supernatant, given in relative units (oe) Sm LP (μg) = number of liposomes containing only sphingomyelin, in micrograms.

Fig. 9. 4-component mixture of liposomes cholesterol:sphingomyelin (1:1 w/w), liposomes containing only sphingomyelin, liposomes sphingomyelin:phosphatidylcholine (1:1 w/w) protects monocytes from toxins secreted by strains of Staphylococcus aureus both Doppelhof and MRSA 2040.

THP-1 cells proliferate in the presence of BHI broth (squares). Supernatant culture fluids of Staphylococcus aureus strains MRSA 2040 (A) or Doppelhof (B) (grown in BHI broth) effectively kill THP-1 cells in the absence of liposomes (triangles), however 1200 micrograms of 4-component liposome mixture (1:1:1: 1) fully protect said cells from either MRSA 2040 (A) or Doppelhof (B) toxins.

10 ⁵ c = number of cells × 10 ⁵ . t (d) = time after treatment (days).

Fig. 10. Liposomes protect mice from bacteremia caused by Staphylococcus aureus, from pneumonia caused by Streptococcus pneumonia, and from bacteremia caused by Streptococcus pneumoniae.

A) Laboratory mice were intravenously injected with a lethal dose of the Doppelhof strain of Staphylococcus aureus. At 1, 5 and 24 hours after bacterial injection, mice were injected with either 25-50 microliters of isotonic saline (diamonds) or 25 microliters (1 mg) cholesterol:sphingomyelin liposomes (1:1) w/w. (squares), or 50 microliters (2 mg) of a mixture of liposomes cholesterol:sphingomyelin (1:1 w/w) + liposomes containing only sphingomyelin + liposomes sphingomyelin:phosphatidylcholine (3:1 w/w) in a ratio of 1 :2:2 (triangles).

B) Mice were intranasally infected with the D39 strain of Streptococcus pneumoniae. 30 minutes after bacterial injection, mice received either an injection of 50 microliters of isotonic saline (diamonds) or a single intranasal injection of 50 microliters (2 mg) of a mixture of cholesterol:sphingomyelin liposomes (1:1 w/w) + cholesterol:phosphatidylcholine liposomes (1 :1 wt./mass.) + liposomes containing only sphingomyelin + liposomes sphingomyelin:phosphatidylcholine (3:1 wt./mass.) in a ratio of 1:1:1:1 (triangles).

C) Mice were injected intravenously with a lethal dose of S. pneumoniae strain D39. At 8 and 12 hours after bacterial injection, mice received intravenously 75 microliters/injection (3 mg) of the following liposomes: 1) a mixture of cholesterol liposome:sphingomyelin (1:1 w/w) + sphingomyelin-only liposomes in a ratio of 1: 1 (triangles); 2) cholesterol:sphingomyelin liposomes (1:1 w/w) (squares); 3) liposomes containing only sphingomyelin (circles), or 4) isotonic solution (diamonds).

S (%) = percentage of surviving mice. t (d) = time after infection (days).

Detailed description of the invention

Empty liposomes and mixtures of liposomes for inhalation, intravenous or infusion administration are provided, designed to provide increased affinity for membrane-acting toxins as compared to said affinity in the body. Said empty liposomes and mixtures of liposomes act as traps for bacterial toxins present in the blood or respiratory tract of infected patients, enabling a new antibiotic therapy to isolate the toxins.

The present invention relates to the use of lipid-defined empty liposomes or mixtures of lipid-defined empty liposomes for the treatment and prevention of bacterial infections, in particular bacteremia, meningitis, bacterial skin infections, skin infections, respiratory tract infections such as pneumonia, and abdominal infections, such as peritonitis.

Liposomes according to the present invention are empty liposomes, i. liposomes that do not encapsulate any antibiotic or other drug. If desired, they can be used in combination with known or novel drug-containing liposomes.

The research underlying the present invention shows that artificial liposomes having a well-defined lipid composition, or mixtures of liposomes having a well-defined lipid composition, effectively isolate purified pore-forming toxins and phospholipase C, and thereby prevent their binding to target cells. Therefore, the use of liposomes or mixtures thereof prevents the lysis of cultured epithelial cells and monocytes induced by the use of purified toxins or culture supernatants of Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus aureus, and protects laboratory mice from death caused by experimentally induced bacteremia or pneumonia.

The present invention relates to the use of lipid-defined empty liposomes or mixtures of lipid-defined empty liposomes for the treatment and prevention of bacterial infections.

In addition, the present invention relates to lipid bilayers or lipid monolayers of defined lipid composition that coat non-lipid surfaces for use in the treatment and prevention of bacterial infections. Contemplated non-lipid surfaces are, for example, medical devices, biodegradable granules and nanoparticles.

The liposomes in question are artificial liposomes from 20 nm to 10 μm, preferably from 20 to 50 nm, containing lipids or phospholipids selected from the group of sterols, sphingolipids and glycerolipids, in particular selected from the group consisting of cholesterol, sphingomyedins, ceramides, phosphatidylcholines , phosphatidylethanolamines, phosphatidylserines, diacylglycerols and phosphatidic acids containing one (lyso-) or two (diacyl-) saturated or unsaturated fatty acids with a length of more than 4 carbon atoms and up to 28 carbon atoms.

The composition of the considered lipid bilayers or lipid monolayers is the same as indicated for liposomes.

Fatty acids containing from 4 to 28 carbon atoms are, for example, saturated linear alkanecarboxylic acids, preferably with an even number of carbon atoms, for example, 12-26 carbon atoms, for example, lauric, myristic, palmitic, stearic, arachidic or behenic acid, or unsaturated linear alkenecarboxylic acids preferably with an even number of carbon atoms from 12 to 26 and one, two or more preferably up to six double bonds in trans or preferably cis configuration, for example oleic acid, linoleic acid, alpha-linoleic acid, arachidonic acid or erucic acid.

The term "empty liposomes" means that the limosomes of the present invention do not contain antibiotics or other drugs. As used herein, the term "enclosed" means encapsulated in the cavity of a liposome, within a potential bilayer of a liposome, or as part of a membrane layer of a liposome. As used herein, the term "liposomes" also excludes liposomes modified with binding agents such as antibodies and mono- or oligosaccharides, such as in glycolipids. However, polyethylene glycol (PEG) modified liposomes are considered part of the present invention. PEG is known to alter the circulation time of the liposome.

In particular, the present invention relates to the use of empty liposomes containing cholesterol and sphingomyelin, and mixtures of empty liposomes containing cholesterol and sphingomyelin, or phosphatidylcholine and sphingomyelin, with other empty liposomes of a particular lipid composition, such as liposomes containing lipids or phospholipids selected from the group consisting of sterols, sphingolipids and glycerolipids, in particular selected from the group consisting of cholesterol, sphingomyelins, ceramides, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, diacylglycerols and phosphatidic acids containing one or two saturated or unsaturated fatty acids with a length of more than 4 carbon atoms and up to 28 carbons, to treat and prevent bacterial infections.

In one embodiment, the present invention relates to the use of empty liposomes containing sphingomyelin and 30% (w/w) or more cholesterol, and mixtures of empty liposomes containing sphingomyelin and 30% (w/w) or more cholesterol, with other empty liposomes as defined herein for the treatment and prevention of bacterial infections. In particular, the present invention relates to the use of empty liposomes consisting of sphingomyelin and 30% (w/w) or more cholesterol, and mixtures of empty liposomes consisting of sphingomyelin and 30% (w/w) or more cholesterol, with other empty liposomes as defined herein for the treatment and prevention of bacterial infections. In particular, the present invention relates to the use of empty liposomes consisting of sphingomyelin and from 35% to 65% (w/w) cholesterol, preferably from 40% to 55% (w/w) cholesterol, in particular from 45% to 55% (w/w) cholesterol, e.g. about 50% (w/w) cholesterol, and mixtures of empty liposomes consisting of sphingomyelin and 35%-65% or 40% to 55% , or from 45% to 55%, for example, about 50% (wt./mass.) cholesterol, with other empty liposomes defined in the present description, for the treatment and prevention of bacterial infections.

In a particular embodiment, the present invention relates to the use of a liposomal mixture of empty liposomes containing or consisting of cholesterol and sphingomyelin with other empty liposomes containing or consisting of sphingomyelin for the treatment and prevention of bacterial infections.

In another embodiment, the present invention relates to the use of a liposomal mixture of empty liposomes containing or consisting of phosphatidylcholine and sphingomyelin with other empty liposomes as defined herein for the treatment and prevention of bacterial infections. In particular, the present invention relates to the use of a liposomal mixture of empty liposomes containing or consisting of phosphatidylcholine and sphingomyelin with other empty liposomes containing or consisting of sphingomyelin for the treatment and prevention of bacterial infections.

In a specific embodiment, the present invention relates to the use of a ternary liposomal mixture of empty liposomes containing or consisting of cholesterol and sphingomyelin, with other empty liposomes containing or consisting of phosphatidylcholine and sphingomyelin, and with empty liposomes consisting of sphingomyelin, for the treatment and prevention of bacterial infections.

In another specific embodiment, the present invention relates to the use of a quaternary liposomal mixture of empty liposomes containing or consisting of cholesterol and sphingomyelin, with other empty liposomes containing or consisting of phosphatidylcholine and sphingomyelin, with empty liposomes consisting of sphingomyelin, and with empty liposomes, containing or consisting of cholesterol and phosphatidylcholine, for the treatment and prevention of bacterial infections.

The specific composition of the lipid bilayers or lipid monolayers in question is the same as indicated for liposomes, and they preferably contain cholesterol and sphingomyelin, and possibly also phosphatidylcholine.

Clearly, empty liposomes as defined above, mixtures of empty liposomes as defined above, and lipid bilayers or lipid monolayers can be used in conjunction with other compounds. For example, you can add components to obtain standard pharmaceutical compositions. Also contemplated is the addition of drugs or drug-like compounds, or the addition of additional liposomes that enclose drugs or drug-like compounds within the interior of the liposomes.

The drugs in question are, in particular, antibiotics. Such antibiotics are, for example, carbapenems such as imipenem/cilastatin, meropenem, ertapenem and doripenem; 1st generation cephalosporins such as cefadroxil and cephalexin; 2nd generation cephalosporins such as cefuroxime, cefaclor and cefprozil; 3rd generation cephalosporins such as ceftazidime, ceftriaxone, cefixime, cefdinir, cefditoren, cefotaxime, cefpodoxime and ceftibuten; 4th generation cephalosporins such as cefepime; 5th generation cephalosporins such as ceftaroline fosamil and ceftobiprole; glycopeptides such as vancomycin, teicoplanin and telavancin; macrolides such as clarithromycin, azithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin and spiramycin; penicillins such as amoxicillin, flucloxacillin, oxacillin, carbenicillin and piperacillin; penicillin combinations such as amoxicillin/clavulanate, piperacillin/tazobactam, ampicillin/sulbactam, and ticarcillin/clavulanate; quinolones such as ciprofloxacin (eg liposomal ciprofloxacin Aradigm) and moxifloxacin; mycobacterial drugs such as rifampicin (rifampin in the US), clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifabutin, rifapentine, and streptomycin; other antibiotics such as metronidazole, arsfenamin, chloramphenicol, fosfomycin, fusidic acid, linezolid, mupirocin, platesensimycin, quinupristin/dalfopristin, rifaximin, thiamphenicol, tigecycline and tinidazole; aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin, netidmycin, tobramycin (eg fluidosomes™ Axentis tobramycin) and paromomycin; sulfonamides such as mafenide, sulfonamidochrysoidine, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim and trimethoprim-sulfamethoxazole (cotrimoxazole, TMP-SMX), tetracyclines such as demeclocycline, doxycycline, minotracycline, and tetracycline; lincosamides such as clindamycin and lincomycin; and lipopeptides such as daptomycin.

Other contemplated drugs are anti-cancer agents, eg vincristine, sulfate, vincristine, cytarabine, daunorubicin and doxorubicin.

Other soluble drugs are anti-inflammatory drugs, e.g. corticosteroids (glucocorticoids) such as hydrocortisone (cortisol), cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA), aldosterone , budesonide, desonide and fluocinonide; non-steroidal anti-inflammatory drugs such as salicylates such as aspirin (acetylsalicylic acid), diflunisal and salsalat; propionic acid derivatives such as ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin and loxoprofen; acetic acid derivatives such as indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac and nabumetone; enolic acid derivatives (oxicam) such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam; fenamic acid derivatives (fenamates) such as mefenamic acid, meclofenamic acid, flufenamic acid and tolfenamic acid; selective COX-2 inhibitors (coxibs) such as celecoxib; and others such as licofelone.

Other drugs considered are vasoconstrictors and vasoconstrictors, eg vasopressin, oxymetazoline, phenylephrine, propylhexedrine, pseudoephedrine, epinephrine, norepinephrine, dopamine and antihistamines.

Other types of medicines are also considered, such as paracetamol (pain reliever), amphotericin B (for fungal infections), bupivacaine (for postoperative pain), hepatitis A, influenza, tetanus, evasive MRSA, whooping cough, diphtheria, meningococcus, cholera, typhoid, anthrax, pneumococcus (eg Prevnar 13®) and other antibacterial vaccines, morphine (pain reliever), verteporfin (ophthalmic disorders), estradiol (menopausal disorders), aganocide® compounds, eg auriclosen (auriclosene) (NVC- 422, N,N-dichloro-2,2-dimethyltaurine (antibacterials), M Bio Technology liposomal particles containing specific lipid bacterial antigens, such as bacterial mimic particles as vaccines (mycoplasmic infections including pneumonia) and bacteriophages.

Other drugs under consideration are antitoxins, for example, tetanus antitoxin such as tetanus immunoglobulin, nanosponges, polymeric nanoparticles, a core of biomimetic polymeric nanoparticles surrounded by host cell membranes (such as red blood cell membranes), toxin-targeting monoclonal antibodies, and fragments antibodies, natural compounds that inhibit the production of specific toxins, inhibitors of the bacterial toxin secretion system such as T3SS inhibitors, mucin-type toxin-binding fusion proteins, soluble T-cell receptors that act as a bait to neutralize toxins, and peptides that inhibit toxin processing.

Moreover, the present invention relates to novel mixtures of empty liposomes of defined lipid composition. In particular, the present invention relates to mixtures of empty liposomes containing cholesterol and sphingomyelin, or phosphatidylcholine and sphingomyelin, with other empty liposomes of a particular lipid composition, such as liposomes containing lipids or phospholipids selected from the group of sterols, sphingolipids and glycerolipids, in particular, selected from the group consisting of cholesterol, sphingomyelins, ceramides, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, diacylglycerols and phosphatidic acids containing one or two saturated or unsaturated fatty acids greater than 4 carbon atoms and up to 28 carbon atoms, for the treatment and prevention of bacterial infections.

Particular contemplated mixtures are mixtures of empty liposomes containing or consisting of sphingomyelin and cholesterol with other empty liposomes as defined herein, such as mixtures of empty liposomes containing or consisting of cholesterol and sphingomyelin with other empty liposomes containing or consisting of sphingomyelin.

Other specific mixtures contemplated are mixtures of empty liposomes containing or consisting of phosphatidylcholine and sphingomyelin with other empty liposomes as defined herein, such as mixtures of empty liposomes containing or consisting of phosphatidylcholine and sphingomyelin with other empty liposomes containing or consisting of from sphingomyelin.

Other specific mixtures contemplated are ternary liposomal mixtures of empty liposomes containing or consisting of cholesterol and sphingomyelin, with other empty liposomes containing or consisting of phosphatidylcholine and sphingomyelin, and with empty liposomes containing or consisting of sphingomyelin.

Other specific mixtures contemplated are quaternary liposome mixtures of empty liposomes containing or consisting of cholesterol and sphingomyelin, with other empty liposomes containing or consisting of phosphatidylcholine and sphingomyelin, with empty liposomes consisting of sphingomyelin, and with empty liposomes containing or consisting of cholesterol and phosphatidylcholine.

In liposomes, the components may be present in varying amounts depending on the propensity to form liposomes, the stability of liposomes having different compositions, and the intended use. Examples are liposomes consisting of two components in a composition of approximately 1:1, 2:1, 3:1, 4:1 or 5:1 (w/w). Additional components can be mixed in amounts of approximately 10, 20 or 25% (wt./mass.).

In preferred liposomes according to the invention, cholesterol is present in an amount of 30-70%, preferably 40-60%, for example 45-55%, in particular about 50% (w/w), phosphatidylcholine is present in an amount of 10-60%, preferably 20-60%, preferably 40-60%, eg 45-55%, more preferably about 50% (w/w); and sphingomyelin is present in an amount of 10-100%, preferably 20-60% or 100%, preferably 40-60%, such as 45-55%, more preferably about 50% (w/w) or 100%, cholesterol ratio: sphingomyelin is from 5:1 to 1:2, preferably from 2:1 to 1:2, in particular about 1:1 (w/w), and the ratio of cholesterol:phosphatidylcholine or phosphatidylcholine:sphingomyelin is from 5:1 up to 1:5, preferably from 2:1 to 1:2, in particular about 1:1 (w/w).

In liposomal mixtures, individual components of liposomes having different compositions are mixed in proportions determined by the needs of the treatment. Examples are approximately 1:1, 2:1, or 3:1 (w/w) for 2-component mixtures; approximately 1:1:1, 2:1:1 or 2:2:1 (w/w) for 3-component mixtures; and approximately 1:1:1:1, 2:1:1:1, 2:2:1:1, or 2:2:2:1 (w/w) for 4-component mixtures.

The liposomes in question are composed of one or more phospholipid bilayers. Large monolamellar vesicles (LUV) and multilamellar vesicles (MLV) are preferred. Most preferred are small monolamellar vesicles (SUVs).

Liposomes are made according to extrusion or sonication or microfluidization (eg, high pressure homogenization) techniques known in the art. For example, lipids are mixed in an organic solvent such as chloroform. The chloroform is evaporated off and the dry lipid film is hydrated in an aqueous solution such as isotonic solution (0.9 NaCl), Krebs' solution or Tyrods' solution and further sonicated to form liposomes. If necessary, the size of liposomes can be controlled by extrusion through membrane filters with a fixed pore diameter. Individually obtained liposomes having different lipid compositions are mixed in the required proportions immediately before use.

Epithelial cells constitute a physical barrier to pathogens. Artificial liposomes are able to protect human mesonephros epithelial cells (HEK 293) from streptolysin O (SLO) induced lysis. The resulting SLO pores in the cell membrane are large enough to induce efflux of cytoplasmic proteins with _Mg up to 100 kDa. Direct binding of SLO to cholesterol-containing liposomes was confirmed by pre-incubation of the liposomes together with the toxin followed by centrifugation. After centrifugation, liposomes isolated in the pellet were discarded, and liposome-free packing liquids were added to the cells. The supernatants did not damage unprotected cells in any way, suggesting that the toxin was effectively removed from the solution by its binding to liposomes.

Liposomes protected not only epithelial cells, but also cells of the innate immune system from various pore-forming toxins (PFTs). The effect of PFT on the proliferation of the human monocyte line THP-1 was evaluated in the presence or absence of liposomes having different lipid compositions. Proliferation of THP-1 cells was completely inhibited in the presence of 200 ng pneumolysin (PLY), 400 ng streptolysin O (SLO), 200 ng tetanolysin (TL), 1.2 μg S. aureus α-hemolysin (HML), or 4.5 μg phospholipase C Clostridium perfringens. As shown in FIG. 1 A-D, liposomes containing cholesterol in combination (1:1 w/w) with either phosphatidylcholine (PC), sphingomyelin (Sm), or phosphatidylserine (PS), but not phosphatidylethanolamine (PE), protected THP- 1 from cholesterol-dependent cytolysins (PLY, SLO, TL) or phospholipase C (PLC), while liposomes that did not contain cholesterol were ineffective (Fig. 1F).

In contrast, Ch:PC liposomes (1:1 w/w) and Ch:PS liposomes (1:1 w/w), in contrast, showed no protective effect against S. aureus α-hemolysin, which belongs to the group of small pore-forming toxins (Fig. 1E). Liposomes that did not contain cholesterol were also ineffective (FIG. 1F). However, liposomes containing Ch:Sm (1:1 w/w) could exert the full protective effect of α-hemolysin (FIG. 1E). Thus, only liposomes composed of cholesterol and sphingomyelin could protect THP-1 cells from any of the tested toxins.

In FIG. 2 shows that the full protective effect of Ch:Sm liposome (1:1 w/w) from 200 ng PLY was observed at 3 μg; from 400 ng SLO at 1.5 mcg; from 200 ng TL at 3 μg; from 1.2 µg HML at 25-50 µg and from 4.5 µg PLC at 100 µg.

In FIG. 3 shows that Ch:Sm liposomes required cholesterol at concentrations equal to or greater than 30% (w/w) to protect monocytes from PLY, TL, or HML. Maximum protection was observed at 50% (w/w) cholesterol, corresponding to 66 mole % cholesterol.

Since liposomes composed of cholesterol and sphingomyelin could protect cells from either cholesterol-dependent cytolysins or α-hemolysin, it was investigated whether these liposomes are effective against a combination of both classes of toxins. Indeed, 25 µg of Ch:Sm liposomes (1:1 w/w) showed full protective effect against the combined action of α-hemolysin (1.2 µg), SLO (400 ng) and TL (200 ng), while as liposomes composed of Ch:PC (1:1 w/w) had no effect (Fig. 4A, B). Centrifugation experiments confirm that all three toxins bind directly to Ch:Sm liposomes (FIG. 4C).

Ch:Sm liposomes can protect cultured cells from the full range of toxins secreted by clinically relevant strains of bacterial pathogens. The proliferation of THP-1 cells was assessed in the presence of lytic concentrations of bacterial culture seed liquids and in the presence or absence of liposomes.

As shown in FIG. 5A, B, Ch:Sm liposomes (1:1 w/w) protected cells from the action of toxins secreted by Streptococcus pyogenes. Full protective effect against Streptococcus pyogenes toxin(s) was observed at microgram amounts of liposomes (FIG. 5C). These amounts are similar to those required to neutralize lytic concentrations of purified cholesterol-dependent cytolysins, but are much less than the amounts required to neutralize either purified phospholipase C (Figure 2), suggesting that only cholesterol-dependent cytolysins are responsible for the cytolytic action of Streptococcus. pyogenes.

Ch:Sm liposomes also protected cells from exposure to toxins secreted by Streptococcus pneumoniae (FIGS. 6A-C). Whereas cholesterol-containing liposomes (1:1 w/w) (FIGS. 6A-C) achieved only limited protection, a mixture of cholesterol-containing (400 μg) and cholesterol-free liposomes containing only Sm (400 μg) completely protected against this pathogen (Fig. 6C).

Staphylococcus aureus is known for its resistance to the most powerful antibiotics. The isolation of toxins by liposomes provides protection even against this pathogen. Ch:Sm liposomes (1:1 w/w) showed only limited protection against toxins secreted by methicillin-resistant Staphylococcus aureus (MRSA 2040). Similar results were obtained for Ch:PC liposomes (1:1 w/w): up to 900 μg of Ch:PC liposomes were required to achieve significant protection, while 600 μg of these liposomes showed only a weak effect (Fig. 7A). However, a detailed analysis of various liposomal mixtures showed that the addition of up to 75 μg of sphingomyelin-only liposomes to 600 μg of Ch:PC liposomes (1:1 w/w) achieved complete protection against this pathogen (Fig. 7B). Sm liposomes alone or PC liposomes alone were not protective at amounts up to 900 μg (FIG. 7B).

The liposome treatment was also effective against a clinically significant "Doppelhof" strain of Staphylococcus aureus isolated from a patient with sepsis. Neither Ch:Sm liposomes (1:1 w/w) or Ch:PC liposomes (1:1 w/w) nor their combination with Sm-only liposomes were effective when used at concentrations which were protective against the MRSA 2040 strain. However, a detailed analysis of the protective effect of various liposomal formulations and combinations thereof showed that, unlike the MRSA 2040 strain, the cytolytic toxins secreted by the Doppelhof strain were effectively sequestered by Sm-only liposomes (Fig. 8A). While 1200 µg of Sm liposomes alone showed significant protection against the Doppelhof strain, at lower concentrations the mixture containing Sm liposomes and Sm:PC liposomes was more effective than the same amounts of either Sm liposomes or Sm:PC liposomes (Fig. .8B). A mixture of cholesterol-containing (1:1 w/w; 600 μg) and cholesterol-free liposomes containing only sphingomyelin (1'200 μg) completely protected against toxins secreted by the Doppelhof strain of Staphylococcus aureus (FIG. 8C).

Thus, not only do Staphylococcus aureus or Staphylococcus pneumoniae secrete numerous cytolytic toxins, but also the relative amounts of secreted toxins vary greatly among different strains, requiring the use of complex liposomal mixtures to achieve high affinity binding of toxins for their complete neutralization. However, due to the non-ideal selectivity of toxin-liposome interactions, significant partial protection can already be achieved with single liposomes if their concentration is high enough to promote low-affinity toxin binding.

A detailed analysis of various liposomal mixtures showed that 1'200 μg (total lipids) of a mixture of liposomes Ch:Sm (1:1 w/w) + Ch:PC (1:1 w/w) + liposomes containing only Sm + Sm:PC liposomes (1:1 w/w), in a 1:1:1:1 ratio, were required for protection against both MRSA 2040 and Doppelhof strains of 'Staphylococcus aureus (FIG. 9). Importantly, due to the presence of cholesterol-containing liposomes, said 4-component mixture also protects against streptococcal toxins (see FIGS. 1-5). Thus, a four-component liposomal mixture (total lipids 1200 μg) can protect cultured cells from the combined action of streptococcal and staphylococcal toxins. A two-component mixture consisting of cholesterol-containing liposomes (50% w/w cholesterol) and sphingomyelin-only liposomes also protected against all bacterial supernatants tested (FIGS. 6-8), but somewhat more (total lipid 1 '800 μg) of this mixture was required for complete protection against toxins secreted by the Doppelhof strain of Staphylococcus aureus (Fig. 8C).

Tested bacterial species (Streptococcus pneumoniae, Staphylococcus aureus and Streptococcus pyogenes) are known to induce or contribute to life-threatening conditions such as bacteremia. The preferred 3- or 4-component mixture of liposomes can protect laboratory mice from experimentally induced bacteremia or pneumonia.

Mice were intravenously injected with a lethal dose of the Doppelhof strain of Staphylococcus aureus, a clinical isolate from a patient with sepsis. At 1, 5 and 24 hours after bacterial injection, mice were injected intravenously with either an isotonic solution (control), 1 mg/injection of Ch:Sm liposomes (1:1 w/w), or 2 mg/injection of a mixture of Ch liposomes: Sm (1:1 mass/mass); liposomes containing only Sm, and liposomes Sm:PC (1:1 wt./mass.) in a ratio of 1:2:2. Control mice did not survive after 7 days, with 90% death occurring within 36 hours (FIG. 10A). Mice treated with cholesterol-sphingomyelin liposomes lived 2-3 days longer than controls but did not recover from bacteremia. However, treatment with the 3-component liposomal mixture resulted in complete recovery in 6 out of 8 mice.

In a pneumococcal pneumonia model, mice were intranasally infected with S. pneumoniae strain D39. 30 minutes after bacterial injection, mice received a single intranasal injection of 2 mg of a mixture of Ch:Sm liposomes (1:1 w/w) + Ch:PC liposomes (1:1 w/w) + Sm-only liposomes, + Sm:PC liposomes (3:1 w/w) in a ratio of 1:1:1:1. In FIG. 10B shows that said liposomal mixture provided protection against pneumonia.

In a pneumococcal bactremia model, mice were intravenously injected with a lethal dose of S. pneumoniae strain D39. At 8 and 12 hours after bacterial injection, mice received intravenously 3 μg/injection of the following liposomes: 1) a mixture of Ch:Sm liposome (1:1 w/w) + Sm-only liposomes in a ratio of 1:1; 2) Ch:Sm liposomes (1:1 w/w); 3) liposomes containing only Sm, or 4) isotonic solution. In FIG. 10C shows that control and Sm-only mice did not survive beyond 32 hours. However, 6 of 8 mice treated with the Ch:Sm liposomal mixture + Sm-only liposomes and 3 of 8 mice treated with Ch:Sm liposomes were still alive after 56 hours of bacteremia.

), которые вводят внутривенно путем инфузии пациентам, страдающим дисфункциями метаболизма жирных кислот, содержат, в дополнение к 2,7 г/кг жирных кислот, приблизительно 300 мг/кг фосфолипидов яйца; т.е. фосфолипидов, используемых в липосомальных препаратах согласно настоящему изобретению. Таким образом, можно безопасно вводить липосомы для лечения бактериальных инфекций у пациентов, представляющих собой людей, и липосомы не будут вызывать нежелательных явлений.The doses of liposomes (50-150 mg/kg) required to protect mice from staphylococcal bacteremia are known to be non-toxic when used as vehicles for intravenous antibiotic delivery in rats (400 mg/kg; Bakker-Woudenberg IAJM et al., Antimicrobial Agents and Chemotherapy, 2001, 45:1487-1492). In addition, the recommended doses of lipid emulsions (for example, Intralipid,

), which are administered intravenously by infusion to patients suffering from dysfunctions of fatty acid metabolism, contain, in addition to 2.7 g/kg of fatty acids, approximately 300 mg/kg of egg phospholipids; those. phospholipids used in liposomal preparations according to the present invention. Thus, liposomes can be safely administered for the treatment of bacterial infections in human patients, and the liposomes will not cause adverse events.

The efficiency of isolation of toxins by liposomes can be further improved. Since the liposomes used in this study were mainly multilamellar liposomes and therefore at least half of their lipid content was not available for toxin binding, it can be predicted that the ability of monolamellar liposomes to isolate toxins will be at least twice as high. Liposomes composed of selected synthetic lipids containing uniform acyl chains and additional lipid species (e.g., ceramide), which are known to greatly enhance lipid bilayer delamination, are a better target for bacterial toxins than liposomes made from natural lipids, which used in this study. With the use of PEG-derivatives of phosphatidylethanolamine, the circulation time of liposomes and thus their efficiency can also be significantly increased.

The lipid surface (bilayer) of liposomes forms spontaneously in aqueous solvents and therefore "traps" water and other water-soluble inorganic and organic molecules that may be present during liposome preparation, within the liposome. Empty liposomes used in the present study are liposomes prepared in buffers containing water and simple organic or inorganic molecules (eg NaCl, KCl, MgCl ₂ , glucose, HEPES and/or CaCl ₂ ). However, complex organic molecules (antibiotics, vitamins, adjuvants, and others) can also be included in the production of liposomes (loaded liposomes). It is assumed that these complex organic molecules do not interfere with the ability of liposomes to isolate toxins; however, they will provide additional therapeutic effects.

The present invention also relates to the treatment of bacterial infections comprising administering to a patient in need thereof a therapeutically effective amount of lipid-defined empty liposomes or mixtures of lipid-defined empty liposomes as described above.

The present invention also relates to the prevention of bacterial infections comprising administering to a subject at risk of infection a prophylactic amount of empty liposomes of a particular lipid composition or mixtures of empty liposomes of a certain lipid composition effective for protection.

Bacterial infections considered are those of the respiratory tract, gastrointestinal tract, genitourinary tract, cardiovascular system, or skin, as well as systemic infections caused by bacteria producing pore-forming toxins and phospholipases, such as those caused by Aeromonas hydrophila, Arcanobacterium pyogene, Bacillus thurgiensis, Bacillus anthracis , Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium septicum, Clostridium sordellii, Clostridium tetani, Corynebacterium diphtheriae, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus (including methicillin-resistant Staphylococcus, Streptococcus pneumoniae), Streptococcus pneumoniae (MRSA) (also known as group A streptococcus (GAS), Streptococcus equisimilis, Streptococcus agalactiae, Streptococcus suil, Streptococcus intermedius, or Vibrio cholera.

Other bacterial infections considered are infections of the nasopharyngeal system, CNS system, meninges, vagina, bones (eg, osteomyelitis) and joints, kidney, skeletal muscle, external ear (eg, otitis externa), and eyes, eg, infectious conjunctivitis, bacterial keratitis and infections of the inside of the eyes.

Specific bacterial infections considered as a target for treatment with liposomes described above are bacteremia, bacterial skin infections, meningitis, respiratory tract infections such as pneumonia, and abdominal infections such as peritonitis.

Contemplated doses for treating or preventing infections are 1 mg to 300 g of liposomes (total lipids) per inhalation/injection/infusion once or several times a day, preferably 100 mg to 10 g once to three times a day. The human equivalent dose (HED) is from 100 to 1000 mg/m ² , preferably about 300 mg/m ² or about 8 mg/kg in humans.

Liposomes can be administered as an aerosol for the treatment of respiratory tract infections. Obtaining aerosols from liposomes, such as empty liposomes according to the present invention, is known in the art. For example, a liquid suspension of liposomes can be delivered using a metered dose inhaler (MDI), i. a device that delivers a specific amount of a drug to the airways or lungs in the form of a short burst of aerosolized drug that is inhaled by the patient.

For the treatment of bacterial infections of the skin, the use of liposomes according to the present invention is contemplated in the form of topical pharmaceutical compositions such as liquid suspensions and the like. Preparation of a suspension from liposomes, such as empty liposomes according to the present invention, is known in the art. For example, a liposomal suspension prepared in isotonic saline or any other aqueous solution can be applied directly to the skin.

For the treatment of bacterial infections, the empty liposomes of the present invention are administered in the form of intravenous, intramuscular, or subcutaneous injections. Injectable solutions are prepared by standard methods known in the art, for example, as suspensions of liposomes in sterile isotonic saline. Such suspensions can be directly administered by injection. Also contemplated is the use of the liposomes of the present invention in a formulation suitable for sublingual or buccal administration. For the treatment of peritonitis, the possibility of intraperitoneal application is considered. Eye drops can be used for bacterial eye infections.

The empty liposomes of the present invention will isolate bacterial toxins and thus prevent bacteria from entering the host epithelium or spreading systemically. Thus, it is possible to prevent or slow down the development of a systemic disease; and pathogens can be effectively eliminated by the cells of the host's innate immune system, which are also protected from toxins by liposomes.

Liposomes themselves are not cytotoxic, nor are they bactericidal. Therefore, they are unlikely to exert selective antibacterial pressure that would favor the emergence of drug-resistant bacteria. The empty liposomes of the present invention mimic structures that already exist in host cells to lure bacterial toxins. It is therefore excluded that bacteria will adapt to the introduction of liposomes: every attempt to avoid the bait by reducing toxins to liposomes inevitably leads to the appearance of toxins that are also ineffective against host cells.

Liposome-based chemotherapy is an attractive alternative to both antibiotic therapy and toxin-separating antibody treatment.

The present invention also relates to the treatment of bacterial infections, comprising administering to a patient in need thereof a therapeutically effective amount of empty liposomes before, after, together with or in parallel with standard antibiotic treatment of a bacterial infection. In combination with antibiotic treatment, in addition to neutralizing actively secreted bacterial toxins during an active infection, liposome treatment will provide additional benefits to the patient by isolating toxins released during antibiotic treatment with lysed bacteria, a condition known to be unfavorable, e.g. , during meningitis, with Streptococcus pneumonia infection (sudden release of pneumolysin) and infection with Streptococcus pyogenes (sudden release of streptolysin O).

In this combined treatment, empty liposomes and mixtures of liposomes can be considered as adjuvants, and the corresponding treatment method as adjuvant treatment.

A limitation of liposome therapy is its limited efficacy in immunocompromised individuals, since bacteria elimination does not depend on the chemotherapeutic agent, but on the host's own immune system. However, even in immunocompromised patients, treatment with liposomes in combination with bactericidal chemotherapy will have the beneficial effect of slowing the progression of systemic disease and thus allowing the body much-needed time to allow the antibiotics to "unfold" their full bactericidal action.

Contemplated antibiotic treatment in conjunction with empty liposome treatment according to the present invention is, for example, treatment with cephalosporins and other β-lactam antibiotics, glycopeptides, lincosamides, lipopeptides, macrolides, penicillins and combinations of penicillins, quinolones, sulfonamides, chloramphenicol and chloramphenicol analogs, tetracyclines , clindamycin, and the folate inhibitors listed above. Particular antibiotics considered in the treatment in conjunction with empty liposomes according to the present invention are carbapenems such as imipenem, cilastatin and meropenem, 2nd generation cephalosporins such as cefuroxime, 3rd generation cephalosporins such as ceftazidime and ceftriaxone, cephalosporins 4 2nd generation such as cefepime, glycopeptides such as vancomycin, macrolides such as clarithromycin, penicillins such as amoxicillin and flucloxacillin, penicillin combinations such as amoxicillin/clavulanate and piperacillin/tazobactam combinations, quinolones such as ciprofloxacin and moxifloxacin, and fluoroquinolones such as levofloxacin and gemifloxacin.

All components of empty liposomes according to the present invention are substances that occur naturally in the human body. Therefore, these liposomes are well tolerated and excreted from the body in physiological ways. Liposomal aerosols should be used in humans to prevent pneumonia and other respiratory illnesses during seasonal influenza epidemics. Most importantly, preventive measures based on liposomal aerosols or other liposomal applications will be useful in preventing MRSA pneumonia or bacteremia in hospitals, Pseudomonas aeruginosa, S. aureus or S. pneumonia infections, and in other settings that contribute to the spread of infectious diseases.

Examples

toxins

(Берн) и E. Gulbins (Эссен).Streptolysin O (SLO) from Streptococcus pyogenes, α-hemolysin from Staphylococcus aureus, tetanolysin (TL) from Clostridium tetani and phospholipase C from Clostridium perfringens were purchased from Sigma. Pneumolysin was obtained from Prof. Kadioglu (Cruse G. et al., J. Immunol. 2012; 184:7108-7115). Other toxins include Panton-Valentine leukocidin (PVL) from S. aureus, listeriolysin O (LLO) from Listeria monocytogenes, perfringolysin O (PFO) from Clostridium perfringens, suilisin (SLY) from S. suis, intermedilysin (ILY) from S. intermedius , cereolysin O (CLO) from B. cereus, thuringiolysin O (TLO) from B. thuringiensis, botulinum lysin (BLY) from C. botulinum, sordellilysin (SDL) from C. sordelli, pyolysin (PLO) from Arcanobacterium pyogenes. Culture supernatants of Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus were obtained from Professors K.

(Bern) and E. Gulbins (Essen).

Cell culture

A human mesonephros cell line (HEK 293) was maintained as described by Monastyrskaya K et al., Cell Calcium. 2007, 41:207-219. The human acute monocytic leukemia (THP-1) cell line was maintained in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine and 100 U/ml penicillin, 100 μg/ml streptomycin.

Transfections

Blue fluorescent protein (CFP) was transiently expressed in HEK 293 cells (Monastyrskaya et al., cited above). HEK 293 cells expressing CFP were used for laser scanning module (LSM) imaging experiments 2 days after transfection.

Liposomes

Cholesterol (Ch) (C-8667), sphingomyelin (Sm) from hen's egg yolk (S0756), phosphatidylcholine (PC) from soybeans (P7443), phosphatidylethanolamine (PE) from bovine brain (P9137), and phosphatidylserine sodium salt ( PS) from bovine brain (P5660) was purchased from Sigma. The lipids were separately dissolved in chloroform at concentrations of 1 mg/ml and stored at -20°C. To obtain liposomes, solutions of individual lipids in chloroform were mixed in the composition and proportions given in the text, usually obtaining 50-500 μl of the final solution. Chloroform was completely evaporated within 20-50 minutes at 60°C. 50 µl or 100 µl Tyrode's buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 10 mM HEPES: pH=7.4) containing 2.5 mM CaCl ₂ was added to tubes containing films of dried lipids, and vigorously mixed by vortexing. Lipid suspensions were incubated for 20-30 minutes at 45°C in an Eppendorf thermomixer with vigorous shaking. To obtain liposomes, the final lipid suspensions were sonicated for 3×5 seconds at 6° C. in a Bandelin Sonopuls sonicator at 70% power. Liposomal preparations were left at least 1 hour at 6°C before using them in the experiments. The concentration of individual lipids in liposomes is always given as a weight ratio (w/w). In liposomes containing cholesterol and sphingomyelin, the ratio of 1:1 (wt./mass.) corresponds to 50% (wt./mass.) or 66 mol% of cholesterol. The amounts of liposomes are given as the total amount of lipids used for their preparation.

In an alternative method, approximately 25 ml of each composition was obtained by ethanol hydration and extrusion. The final formulations were sterilized by filtration and placed in glass vials with autoclaving serum (final concentration: 40 mg/ml). Liposomal particle sizes are in the range of 80-150 nm with a good polydispersity index (PDI). Osmolality measurements are also included. All of them are in the range of about 400 mmol/kg, which is quite close to the desired physiological level.

Toxin-induced cell lysis and the protective effect of liposomes

In human mesonephros epithelial cells (HEK 293), toxin-induced lysis was observed as a decrease in cytoplasmic fluorescence due to pore-induced efflux of intracellular CFP. Confluent HEK 293 cells seeded on 15 mm glass coverslips (2.5×10 ⁵ cells per coverslip) were placed in a perfusion chamber at 25°C in Tyrode's buffer containing 2.5 mM CaCl ₂ and their fluorescence was recorded under microscope Axiovert 200 M with a laser scanning module LSM 510 META (Zeiss, Germany) using a ×63 immersion objective (Monastyrskaya et al., in the above source). At time = 0 buffer was replaced with 100 μl or 200 μl of the same buffer additionally containing a cytolytic amount of a particular toxin (eg 120 ng SLO from Streptococcus pyogenes) and 20 mM/l dithiotrenthol (DTT). To study the protective effect of liposomes against toxin-induced cell lysis at time = 0, cells were infected in the usual way with 100 μl of a mixture containing toxin/DTT and liposomes at different concentrations having different lipid compositions. A mixture of toxins and liposomes was prepared immediately before being added to the cells (with a manipulation delay of 20 to 30 seconds). In some cases, 100 μl of a solution containing only liposomes was first added to the cells, followed by (with a manipulation delay of 20 to 30 seconds) 100 μl of a solution containing the toxin was added. The protective effect of liposomes was similar under all experimental conditions. The images were analyzed using the Physiology evaluation software package (Zeiss, Germany).

The effect of purified PFT or bacterial culture supernatants on the proliferation of the human monocinar cell line (THP-1) was evaluated in the presence or absence of liposomes having different lipid compositions. 100-600 µl of a solution containing the toxin (Buffer Ca ²⁺ -Tyrode or BHI broth) was added in the usual way to 100 µl (5×10 ⁴ cells) of cells maintained in culture medium and pre-mixed with 50-150 µl of liposomes having different lipid composition. After incubation for 3 hours, 1-2 ml of fresh culture medium was added to the tubes. Cells were counted every day or every other day for 8-12 days. Toxins and liposomes were present throughout the experiment. The data presented in the graphs correspond to day 5 or day 6, when cell growth was still in the linear phase.

Comparison of "empty" and "filled" liposomes

Protection against S. aureus or S. pneumoniae culture supernatants with a mixture of empty Ch:Sm+ liposomes containing only Sm was compared to protection with a mixture of Ch:Sm+ liposomes containing only Sm loaded with a fluorescent dye such as fluorescence, Oregon Green 488, rhodamine or Texas red.

Protection against culture supernatants of S. aureus or S. pneumoniae with a mixture of empty Ch:PC liposomes was compared with protection with a mixture of Ch:PC liposomes loaded with a fluorescent dye such as fluorescein, Oregon Green 488, rhodamine, or Texas red.

Protecting surfaces coated with lipids

Cholesterol and sphingomyelin coated beads were tested for their toxin isolation activity against culture supernatants of S. aureus or S. pneumoniae.

Effects in vivo in combination with antibiotic treatment on bacteremia induced by S. aureus or S. pneumoniae

2-component mixture of Ch:Sm liposomes and Sm-only liposomes; 3-component mixture of Ch:Sm liposomes; liposomes containing only Sm, and liposomes Sm:PC (1:2:2) and a 4-component mixture of liposomes Ch:Sm, liposomes containing only Sm, liposomes Sm:PC (1:1:1:1) were tested in models bacteremia in mice induced by either penicillin-susceptible Streptococcus pneumomonia or methicillin-resistant Staphylococcus aureus (MSSA). Two types of MSSA strains are considered, characterized by the ability to secrete or not to secrete the toxin - Panton-Valentine leukocidin (PVL). In addition, a 2-component mixture of PEGylated Ch:Sm and Sm-only liposomes (2% PEG or 5% PEG) was also tested.

Laboratory mice were infected by intraperitoneal (i/p), intravenous (i/v) or intranasal (i/i) injection of approximately 10 ⁷ or 10 ⁸ cfu/ml of bacteria.

For each bacterial strain, each route of infection, and for each liposomal mixture (LP mixtures), intravenous injections of two different doses (2 mg/kg or 6 mg/kg) of LP mixtures were started at either six hours (t=6), twelve hours (t =12), eighteen hours (t=18) or twenty-four hours (t=24) after bacterial challenge (in each case, injection of the LP mixture was followed by either one additional injection at 12 hours or two additional injections at 4 hours and 24 hours after the first injection) with or without penicillin treatment (30 mg/kg). Antibiotic treatment was started at the same time as liposome treatment. Two types of controls were performed: infection without treatment and infection treated with antibiotics alone (at t=6, t=12, t=18 or t=24).

For each strain of bacteria and for each mixture of liposomes, and for each dose of liposome and each route of infection, there were 10 groups of animals.

In group 1, a survival rate of 50% of the group was observed for at least 8 days, 25% of the group was euthanized one hour after bacterial infection, and the remaining 25% 6 hours after bacterial infection.

The number of bacteria was determined in the blood and some organs such as lungs, spleen and kidneys.

Data obtained: survival, signs of infections. metabolism (sequential measurements of weight loss and recovery, O ₂ consumption rate and CO ₂ production rate measured by indirect calorimetry, resting energy expenditure (REE) calculated using the modified Weir formula); characterization of inflammatory cytokines (enzyme-linked immunosorbent assay (ELISA) was performed in serum from subject tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-2 and IL-1b).

Minimum bactericidal concentration (MBC)

Lack of activity of sphingomyelin/cholesterol liposomes against conventional strains:

Minimum bactericidal concentration (MBC) testing was performed according to the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI).

For microliter broth dilution tests, 96-well plates supplemented with 50 µl of 0.5-16 mg/ml liposomes were inoculated with 50 µl of Mueller-Hinton broth containing a bacterial cell suspension of 1-5×10 ⁵ colony-forming units (CFU) per ml S aureus. The plates were incubated for 24 hours at 36°C. IBC was determined by transferring 10 μl aliquots from the wells of microtiter broth dilution plates onto Columbian blood agar (Oxoid, Wesel, Germany). The inoculated plates were further incubated for 24 hours at 36° C. and then colonies were counted.

Claims (7)

1. Empty liposomes for use in the treatment or prevention of bacterial infection, containing cholesterol in an amount of 40 wt.% or more and sphingomyelin. 2. Empty liposomes according to claim 1, characterized in that said empty liposomes are modified with polyethylene glycol. 3. Empty liposomes according to claim 1 or 2, characterized in that the bacterial infection is selected from bacteremia, bacterial infections of skin lesions, meningitis and infections of the respiratory tract. 4. The use of empty liposomes according to claim 1 or 2 for the treatment or prevention of a bacterial infection. 5. The use of empty liposomes according to claim 4, characterized in that the bacterial infection is selected from bacteremia, bacterial infections of skin lesions, meningitis and infections of the respiratory tract. 6. A method of treating or preventing a bacterial infection, comprising administering to a patient in need thereof a therapeutically effective amount of empty liposomes according to any one of paragraphs. 1-3. 7. The method of claim 6, wherein said therapeutically effective amount of empty liposomes is administered to said patient before, after, concomitantly, or in parallel with standard antibiotic treatment against a bacterial infection.

Images