Liposomal compositions for treatment of inflammation

Abstract

The invention relates to a pharmaceutical composition consisting of a liposomal formulation of a phospholipid component and an aqueous phase, wherein the phospholipid component is selected from the group consisting of phosphatidylcholine, and sphingomyelin, and wherein the phospholipid component comprises a saturated or mono-unsaturated (Z / cis) fatty acid having 14 to 20 carbon atoms. The invention further relates to the use of the pharmaceutical composition in treatment of a condition associated with presence of lipopolysaccharide (LPS), particularly endotoxemia.

Description

[0001] Liposomal Compositions for Treatment of Inflammation

[0002] This application claims the benefit of European patent application 24211761.2, with filing date 8thNovember 2024, which is fully incorporated by reference herein.

[0003] Field

[0004] The present invention relates to compositions containing comprising specific fatty acids, namely 15:0, 16:0, 16:1 and 18:1 , and in particular, liposomal compositions comprising sphingomyelin and phosphatidylcholine species of 15:0, 16:0, 16:1 and 18:1 fatty acids, and further to the application of these compositions in the treatment of inflammation and conditions associated with inflammation, both aseptic and inflammation caused by bacterial infections.

[0005] Background

[0006] Inflammation is a critical biological process initiated by the immune system in response to harmful stimuli. Inflammatory response involves activation of immune cells, release of cytokines, and vascular changes aimed at eliminating harmful stimuli and initiating tissue repair. Inflammation can be categorized based on duration (acute vs. chronic) or etiology (sterile vs. non-sterile). Both forms share common effector mechanisms but differ significantly in their initiating signals, involved receptors, and downstream responses. While acute inflammation is essential for healing, chronic inflammation can contribute to various pathological conditions including autoimmune and cardiovascular diseases, diabetes, neurodegeneration and cancer.

[0007] Non-sterile inflammation arises in response to pathogens such as bacteria, viruses, fungi, and parasites. The immune system detects these threats (e.g., lipopolysaccharide (LPS), flagellin or viral RNA) through Pathogen-Associated Molecular Patterns (PAMPs), which are recognized by Pattern Recognition Receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-l-like receptors (RLRs). The inflammatory process promotes pathogen clearance, recruitment of neutrophils and macrophages, and activation of the adaptive immune response. If unresolved, it may contribute to chronic infection or sepsis.

[0008] Sterile inflammation is initiated in the absence of microbial infection, often due to endogenous danger signals released from injured or stressed cells. These Damage-Associated Molecular Patterns (DAMPs) include ATP, HMGB1 , uric acid, DNA, and mitochondrial components. DAMPs are recognized by the same families of PRRs that detect pathogens, and downstream signaling promotes local and systemic inflammation. Examples of sterile inflammation are ischemia-reperfusion injury in organs such as the kidney, liver, or heart, trauma and surgery without infection, and chronic inflammatory diseases. Conditions like rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis result from immune dysregulation, where inflammation becomes self-sustaining and destructive; chronic vascular inflammation plays a key role in atherosclerotic plaque formation and cardiovascular events; microglial activation and chronic neuroinflammation contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, whereas inflammatory mediators can promote tumor initiation, progression, and metastasis.

[0009] Non-sterile and sterile inflammation go hand by hand in pathologies since chronic, non-sterile inflammation weakens the anti-microbial immune defense, whereas tissue damage induced by invading pathogens drives sterile inflammation supporting a vicious cycle of uncontrolled immune reaction to alternating (or simultaneous) external and internal insults.

[0010] Antibiotic therapy applied to bacterial infections leads to the disruption of bacterial cell integrity. When antibiotics are administered, they effectively inhibit vital processes such as cell wall synthesis, protein synthesis, or DNA replication thus inhibiting bacterial proliferation or ultimately resulting in bacterial cell death and lysis.

[0011] Lipopolysaccharides (LPS), also referred to as endotoxins, are a critical constituent of the outer membrane of Gram-negative bacteria, characterized by a complex structure comprising lipid A, core oligosaccharide, and O-antigen. This intricate configuration allows LPS to maintain bacterial cell wall integrity and serves as a barrier against environmental threats.

[0012] The peril associated with administration of antibacterial drugs in systemic infections lies in the subsequent release of cellular contents, including LPS, from lysed Gram-negative bacterial cells. LPS molecules can then enter the bloodstream, where they interact with pattern recognition receptors on immune cells, primarily Toll-like receptors (TLRs), particularly TLR4 in the case of lipid A from LPS. Upon binding to TLR4, LPS triggers a signalling cascade that culminates in the activation of nuclear factor-kappa B (NF-KB) and the production of pro- inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 P). These cytokines play pivotal roles in the immune response to infection, but when released in excessive quantities due to a surge in LPS, they can incite an overwhelming and dysregulated inflammatory response. This uncontrolled inflammatory response can manifest as sepsis, a life-threatening condition characterized by systemic inflammation, vasodilation, endothelial dysfunction, organ dysfunction, and, in severe cases, multiple organ failure. Endotoxemia may also originate from commensal enteric bacteria during Gram-positive infections or surgical interventions, since regional hypo-perfusion and mucosal ischemia might promote the translocation of endotoxin from the intestinal lumen to the systemic circulation.

[0013] Phospholipids are amphipathic molecules that constitute the fundamental architecture of cellular membranes. Beyond their structural role, they are pivotal in signal transduction, inflammation, and immune regulation. Through enzymatic cleavage, phospholipids generate numerous bioactive lipid mediators that orchestrate the initiation, propagation, and resolution of inflammation. Phospholipids and their metabolites constitute a complex regulatory network that modulates inflammation in both infectious and sterile settings. They are not only initial triggers of inflammation through PAMP and DAMP signaling but also critical players in its resolution. Dysregulated phospholipid metabolism contributes to various diseases, including infections, autoimmunity, atherosclerosis, and cancer.

[0014] Phospholipids are composed of a glycerol backbone linked to two fatty acid chains and a phosphate group, which is further substituted with head groups such as choline, ethanolamine, serine, or inositol, etc.

[0015] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to address inflammation, inflammatory processes and conditions associated with inflammatory responses, both in aseptic and septic inflammation. One such condition is the negative consequences of LPS release in patients suffering from sepsis, or undergoing treatment with antibiotic drugs.

[0016] This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0017] The inventors set out to identify liposomes composed of neutral lipids that might protect cells from LPS-induced cytostatic activity and even neutralize LPS. In the course of their investigations, they surprisingly found that formulations that helped to address LPS-induced release of cytokines, also allowed to impact cellular pathways associated with aseptic inflammation.

[0018] In the literature, there is a consensus that negatively charged lipids can prevent LPS-induced cellular activation, since they bear same structure and charge as LPS. However, negatively charged lipids activate the coagulation cascade in blood.

[0019] Some data indicate that neutral phospholipids can also neutralize LPS by a two-step mechanism in which LPS is transferred to soluble CD14 (sCD14), and then from LPS-sCD14 complexes to HDL [Wurfel et al. J Immunol (1997) 158 (8): 3925-3934],

[0020] Therefore, the inventors started with phosphatidylcholine containing identical acyl chains with 14 carbon atoms each (14:0 PC), which has acyl chains of the same length as most acyl chains of LPS. It turned out to be quite potent as LPS inhibitor, however doubling the concentration of 14:0 PC at which complete neutralization of LPS was achieved already resulted in some pronounced cytotoxicity. Therefore, the inventors decided to investigate other lipids that they hoped would also be protective but less toxic. Summary of the Invention

[0021] In one aspect, the invention relates to a pharmaceutical composition consisting of a formulation composed of a phospholipid component selected from phosphatidylcholine and sphingomyelin or mixtures of phosphatidylcholine and sphingomyelin, and an aqueous phase. The phospholipid component comprises a fatty acid selected from tetradecanoic acid, pentadecanoic acid, hexadecenoic (palmitic) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid.

[0022] In another aspect, the invention relates to a liposome comprised of a phospholipid bilayer consisting of a phospholipid component, the bilayer surrounding an aqueous phase (the liposomal content), the phospholipid component being selected from the group consisting of phosphatidylcholine and sphingomyelin or mixtures thereof, wherein the phospholipid component comprises a fatty acid selected from tetradecanoic acid, pentadecanoic acid, hexadecenoic (palmitic) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid.

[0023] The invention further relates to the use of the compositions as disclosed herein in treatment of inflammatory conditions and processes, and to methods of treatment wherein the compositions as disclosed herein are administered to a patient diagnosed with an inflammatory condition.

[0024] Terms and definitions

[0025] General

[0026] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0027] The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

[0028] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0029] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0030] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0031] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0033] The term lipopolysaccharide, or its plural form lipopolysaccharides, abbreviated as LPS, relates to bacterial toxins that consist of a lipid and a polysaccharide. LPS are composed of an polysaccharide O-antigen, an oligosaccharide outer core, and an oligosaccharide inner core to which lipids are attached, all joined by covalent bonds. LPS are found in the outer membrane of Gram-negative bacteria. The term endotoxin is used synonymously with LPS herein.

[0034] The term phosphatidylcholine, abbreviated PC, in the context of the present specification relates to a phospholipid composed of a choline ([(CH3)3NCH2CH2OH]+) group linked to glycerophosphoric acid through the phosphodiester group, with two fatty acids being linked by ester bond to the 2 and 3 hydroxy groups of the glycerol. In particular embodiments, one of the two fatty acids is saturated, the other one unsaturated (cis / Z configuration).

[0035] R, R’ = fatty

[0036] 3acid residues , , .. , . , .. phosphatidylcholine

[0037] The most frequent natural PCs are asymmetric (in saturation and / or chain length), with Sn1 (the glycerin carbon distal to the phosphate) typically being a saturated chain, and Sn2 (the C2 of glycerol) an unsaturated chain (monounsaturated or polyunsaturated).

[0038] The term phosphatidylglycerol in the context of the present specification relates to a compound characterized by an L-glycerol 3-phosphate backbone with two fatty acids being linked by ester to the 2 and 3 hydroxy groups of the glycerol.

[0039] The term phospatidylserine, abbreviated PS, in the context of the present specification relates to a phospholipid composed of a serine moiety linked to glycerophosphoric acid through a phosphodiester bond, with two fatty acids being linked by ester bond to the 2 and 3 hydroxy groups of the glycerol. In particular embodiments, one of the two fatty acids is saturated, the other one unsaturated (cis / Z configuration).

[0040] The term sphingomyelin, abbreviated SM, in the context of the present specification relates to a compound composed of a phosphocholine (or phosphoethanolamine) head group, sphingosine (2-amino-4-trans-octadecene-1 ,3-diol), and a fatty acid linked to the amino group of the sphingosine.

[0041] The term tatty acid in the context of the present specification relates to an aliphatic carboxylic acid of chain length of four to 24 carbon atoms. Particular embodiments of a fatty acid as recited herein include a C12, C14, C15, C16, C18 or C20 saturated, mono-unsaturated (Z / cis) or di-unsaturated (Z / cis) fatty acid.

[0042] The term PBS in the context of the present specification relates to Phosphate Buffered Saline: NaCI: 8.0 g / L; KCI: 0.2 g / L; Na2HPO4: 1.44 g / L KH2PO4: 0.24 g / L, pH 7.4.

[0043] As used herein, the term treating or treatment of any disease or disorder as addressed by the present invention (e.g. a bacterial infection, sepsis or as otherwise indicate) refers in one embodiment to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and / or prevention of disease are generally known in the art, unless specifically described hereinbelow.

[0044] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0045] Detailed Description of the Invention

[0046] Bacterial endotoxin, lipopolysaccharide (LPS), a major component of the outer cell membrane of Gram-negative bacteria, is a principal mediator of Gram-negative bacterial sepsis [Opal et al., J Infect Dis. 1999 Nov; 180(5): 1584-9]. LPS preferentially targets CD14-bearing inflammatory cells such as macrophage-monocyte lineage and circulating neutrophils. These cells are activated by LPS through a family of Toll-like receptors (TLR-4) and subsequently release pro-inflammatory mediators, including tumor necrosis factor (TNF-a), interleukins (IL- 1 P, IL-8, IL-12), cyclic endopeptidases, platelet-activating factor and other inflammatory products. Functioning in concert, these host-derived mediators might over-respond to induce the systemic inflammatory response syndrome, shock and multiple organ failure.

[0047] Endotoxin is frequently found in the systemic circulation in the presence of sepsis, surprisingly, regardless of the infecting microorganism [Opal et aL, ibid.]. Endotoxemia may have originated from unrecognized gram-negative infections in some patients or from enteric bacteria within the gastrointestinal tract, since regional hypo-perfusion and mucosal ischemia are thought to promote the translocation of endotoxin from the intestinal lumen to the systemic circulation.

[0048] Likewise, the peril associated with administration of antibacterial drugs in systemic infections lies in the subsequent release of cellular contents, including LPS, from lysed Gram-negative bacterial cells. (Lepper PM et aL, Intensive Care Med. 2002 Jul;28(7):824-33. doi: 10.1007 / S00134-002-1330-6).

[0049] The degree of cellular responsiveness to endotoxin is critically dependent on the presence of plasma constituents. The principal plasma protein responsible for transporting endotoxin to immune effector cells is lipopolysaccharide-binding protein (LBP). LBP is recognized as a part of the innate host defence against systemic microbial challenge. It is a hepatically synthesized, acute-phase protein that shuttles endotoxin molecules to its effector cells. However, the role of LBP is far from straightforward. In addition to transferring LPS to cells bearing CD14 on their surface, it can also transfer endotoxin to high-density lipoproteins and to soluble CD14 (sCD14), which shuttles LPS to non-myeloid cell lines such as endothelial cells. These additional activities of LBP shunt endotoxin away from immune cells that are the most potent mediators of inflammatory responses, potentially relieving the inflammatory syndrome. However, they might also lead to the activation of detrimental effects conveyed by non-myeloid cells. Moreover, LBP-sCD14 complexes can bind and shuttle other hydrophobic agents, notably phospholipids (Wurfel et al., ibid) Therefore, phospholipids might antagonize the transfer of LPS.

[0050] The inventors ventured to address in detail the possibility of preventing LPS-induced cellular activation by antagonistic action of liposomes composed of different lipids, particularly neutral phospholipids, with the aim of finding the best candidates for further development as therapeutic agents against LPS-induced endotoxemia and shock.

[0051] Since LPS-mediated cellular responses depend on the presence of plasma components, our experiments were performed at 40% fetal calf serum (FCS; approximating serum concentrations in circulating blood) and at 10% fetal calf serum ((modelling a drop in serum concentration outside discontinuous capillaries of sinusoids (bone marrow, liver, spleen) and in the extracellular milieu outside the blood circulation (mucosa and submucosa of lungs, gut, skin, etc.)).

[0052] Subsequent to the realization that the compositions and liposomes disclosed herein are useful in the treatment of conditions of septic inflammatory disease, the inventors found when investigating the mechanistic basis of the septic response, that the compositions and liposomes of the present invention were also suitable for treatment of aseptic inflammatory conditions, greatly broadening the scope of possible utility of the compositions of the invention.

[0053] Pharmaceutical compositions

[0054] A first aspect of the invention relates to a pharmaceutical composition consisting of a formulation of a phospholipid component and an aqueous phase, wherein the phospholipid component is selected from the group consisting of phosphatidylcholine and sphingomyelin or mixtures thereof, and wherein the phospholipid component comprises a fatty acid selected from tetradecanoic acid, pentadecanoic acid, hexadecenoic (palmitic) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid.

[0055] The inventors’ observations indicate that phosphatidylcholine and sphingomyelin having an acyl chain shorter than a C18 saturated carbon chain is protective for cells exposed to stimuli mimicking, or resulting from, septic and aseptic inflammation. The inventors have not been able to discriminate between septic and aseptic processes, i.e. in all experiments, compositions that have resulted protective in conditions used as a surrogate in one, were also protective in conditions used as a surrogate in the other.

[0056] In general, C18 acyl chains, when complemented in PC by a shorter (14:0, 16:1 or 18:1 ) acyl chain, led to favourable effects. In sphingomyelin, which only have one acyl chain, the 15:0 and 16:1 species are preferred.

[0057] The inventors’ observations indicate that two separate mechanisms of action lead to protection against inflammatory pathways, on one hand, and yet lead to cytotoxicity on the other. Thus, a balance needs to be struck that combines optimal protection from inflammation with minimal toxicity. By some measures, shorter (14:0 and shorter) acyl chains have demonstrated a high degree of down-regulation of responses to inflammatory stimuli, however such short acyl chain containing phospholipids also have been shown to be relatively highly cytotoxic, making problematic their use in human patients.

[0058] In one alternative, the first aspect of the invention may be formulated as a pharmaceutical composition consisting exclusively of a liposomal formulation composed of a phospholipid component and an aqueous phase. The phospholipid component is selected from the group consisting of phosphatidylcholine and sphingomyelin. The phospholipid component comprises a saturated or mono-unsaturated (Z / cis) fatty acid having 14 to 20 carbon atoms.

[0059] In particular embodiments, the pharmaceutical composition is a liposomal formulation.

[0060] In more particular embodiments, the pharmaceutical composition does not comprise a further pharmaceutically active ingredient. These embodiments are essentially encompassing “empty” liposomes that only consist of the phospholipid component and an aqueous, pharmaceutically acceptable buffer.

[0061] In particular embodiments, the phospholipid component only comprises saturated or monounsaturated (Z / cis) fatty acids having 16 to 18 carbon atoms.

[0062] In particular embodiments, the phospholipid component only comprises saturated or monounsaturated (Z / cis) fatty acids having 16 to 20 carbon atoms.

[0063] In certain embodiments, the phospholipid component of the pharmaceutical composition according to invention is constituted entirely of phosphatidylcholine.

[0064] In more particular embodiments, the phosphatidylcholine that the phospholipid component consists of comprises a. one fatty acid selected from pentadecanoic (15:0) acid, hexadecenoic (palmitic; 16:0) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid, and b. one fatty acid selected from tetradecanoic (myristic; 14:0) acid, pentadecanoic (15:0) acid, hexadecenoic (palmitic; 16:0) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid, and octadecanoic (stearic) acid.

[0065] The examples provided herein demonstrate that shorter acid tail lengths both provide a potent anti-inflammatory effect, and seem to be toxic, with higher toxicity found with decreasing acyl chain length. Thus, the indicated fatty acids have shown to provide the best equilibrium of efficacy and lack of toxicity.

[0066] In certain embodiments, the phospholipid component comprises a saturated or monounsaturated (Z / cis) fatty acid having 16 to 20 carbon atoms. In certain embodiments, the phospholipid component comprises a saturated or monounsaturated (Z / cis) fatty acid having 16 to 18 carbon atoms.

[0067] In more particular embodiments, the 16:1 or 18: 1 monounsaturated fatty acid has a Z (cis) double bond.

[0068] In even more particular embodiments, the 16:1 or 18: 1 monounsaturated fatty acid is selected from the group consisting of an octadec-9-enoic acid, an octadec-6-enoic acid, a hexadec-9- enoic acid, an hexadec-6-enoic acid, and a hexadec-11-enoic acid.

[0069] In yet even more particular embodiments, the monounsaturated fatty acid is selected from the group consisting of 18:1 (Z)-9 (oleic acid), 18:1 (Z)-6 (petroselinic acid), 16:1 (Z)-9 (palmitoleic) acid, 16:1 (Z)-6 (sapienic) acid, and cis-11 -hexadecenoic acid.

[0070] In more particular embodiments, the phosphatidylcholine is selected from 18:1 / 14:0 phosphatidylcholine, 16:1 / 14:0 phosphatidylcholine, 18:1 / 18:1 phosphatidylcholine, and 16:0 / 16:0 phosphatidylcholine.

[0071] In particular embodiments, the phosphatidylcholine comprises two identical fatty acids.

[0072] All phosphatidyl species described herein have two identical acyl groups, unless explicitly designated otherwise.

[0073] In more particular embodiments, the phosphatidylcholine comprises two 16:1 monounsaturated fatty acid moieties.

[0074] In general, the phosphatidylcholine compositions provided herein may be administered formulated as liposomes, or as aggregates. The inventors found that in their hands, phosphatidylcholine comprising two A9(syn)-16:1 acyl chains did not show a significant effect when applied as aggregates, but were the most powerful agents when administered as liposomes. In general, the inventors found that unsaturated PC species did not perform as well in aggregate form as in liposomal form.

[0075] In other more particular embodiments, the phosphatidylcholine comprises two pentadecanoic fatty acid moieties.

[0076] Aggregates

[0077] In particular embodiments, the pharmaceutical composition is not a liposomal formulation, but comprises phosphatidylcholine of pentadecanoic acid or hexadecenoic acid in aggregated form.

[0078] While both the (15:0)2 PC and the (16:0)2 PC had significant effects in liposomal form, the inventors observed an even greater effect, when they were supplied as aggregates. This observation might be of particular importance for the provision of topical or enteral administration forms.

[0079] In particular embodiments, the formulation comprises phosphatidylcholine of pentadecanoic acid or hexadecenoic acid only (exclusively) in aggregated form.

[0080] In more particular embodiments, the pharmaceutical composition of aggregates comprises 1 ,2- bis(pentadecanoyl)-sn-glycero-3-phosphocholine.

[0081] In other more particular embodiments, the pharmaceutical composition of aggregates comprises 1 ,2-bis(hexaadecanoyl)-sn-glycero-3-phosphocholine.

[0082] In certain embodiments relating to the aggregate form of the pharmaceutical composition, the composition does not comprise a further pharmaceutically active ingredient.

[0083] In yet more particular embodiments, the composition comprises only a single phosphatidylcholine species. This is advantageous from a regulatory and quality control point of view.

[0084] Sphingomyelin compositions

[0085] In other particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is sphingomyelin.

[0086] In more particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is constituted entirely (exclusively) of sphingomyelin.

[0087] In even more particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is constituted entirely (exclusively) of a single sphingomyelin species, in contrast to naturally occurring mixtures such as egg sphingomyelin.

[0088] Synthetic sphingomyelin is deemed to be an advantageous alternative to egg sphingomyelin from a regulatory and developmental point of view.

[0089] In other more particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is 16:1 sphingomyelin.

[0090] In even more particular embodiments, the phospholipid component is (9Z) hexadecenoic sphingomyelin.

[0091] In other more particular embodiments, the phospholipid component is 15:0 sphingomyelin.

[0092] In yet other embodiments, the phospholipid component of the pharmaceutical composition according to invention is selected from the group consisting of phosphatidylcholine and sphingomyelin and the phospholipid comprises in position 2 or 3 of the glycerol I sphingosine backbone: a. a first fatty acid selected from a 16:0, 16:1 , 18:0 and 18:1 fatty acid, and b. a second fatty acid selected from a saturated or monounsaturated C14, C15 or C16 fatty acid.

[0093] In more particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is 16:1 phosphatidylcholine.

[0094] In yet more particular embodiments of the 16:1 phosphatidylcholine, both chains are palmitoleic acid.

[0095] In another aspect of the invention, the composition according to the first aspect of the invention is provided for use in medicine.

[0096] The aqueous phase essentially consists of water and the necessary buffer components to allow administration of the composition to a patient. In certain embodiments, the aqueous phase may consist of a phosphate buffer system. In certain embodiments, the aqueous phase may consist of an acetate buffer system. In certain embodiments, the aqueous phase may consist of a citrate buffer system.

[0097] In an alternative aspect of the invention, the pharmaceutical composition as described in the first aspect of the invention may comprise one additional component, which is an antibacterial (antibiotic) pharmaceutical drug selected from the group consisting of aminopenicillins, ureidopenicillins, cephalosporins, quinolone antibiotics, carbapenems, aminoglycoside antibiotics, and monobactams (aztreonam).

[0098] In principle, the localization of the Z double bond of the hexadecenoic acid moiety forming part of the 16:1 phosphatidylcholine can be at any position. In particular embodiments, the 16:1 phosphatidylcholine has two monounsaturated C16 acyl chains with the cis-double bond connecting the carbon at C8, C9, C10, C11 , or C12 with the next carbon situated towards the a>-terminal CH3 of the fatty acid chain.

[0099] In yet more particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is (9Z) hexadecenoic phosphatidylcholine.

[0100] In other more particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is 18:1 / 14:0 phosphatidylcholine.

[0101] In other more particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is 16:1 / 14:0 phosphatidylcholine.

[0102] In alternative particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is 16:1 sphingomyelin.

[0103] All naturally occurring sphingomyelins have the d-erythro-(2S, 3R) configuration of the sphingosine base. In more particular embodiments, the phospholipid component of the pharmaceutical composition according to invention is (9Z) hexadecenoic sphingomyelin.

[0104] Another aspect of the invention relates to a pharmaceutical composition as described in any of the aspects or embodiments, formulated for intravenous administration. Lv. administration may preferably be applied by drip or injection.

[0105] The invention also encompasses the use of any of the pharmaceutical compositions and liposomes disclosed herein, as a medicament.

[0106] The invention further provides the pharmaceutical composition described herein, comprising of a liposomal formulation composed of a phospholipid component selected from phosphatidylcholine and sphingomyelin, and an aqueous phase, for use in treatment of a condition associated with presence of bacterial endotoxin lipopolysaccharide (LPS), particularly endotoxemia. The phospholipid component comprises a saturated or monounsaturated (Z / cis) fatty acid having 14 to 20 carbon atoms. Particularly advantageous embodiments of the pharmaceutical composition for use in treatment of endotoxemia are described above.

[0107] In particular embodiments, the condition associated with presence of LPS is selected from the group of systemic inflammatory response syndrome, sepsis, septic shock and multiple organ failure.

[0108] Alternatively the invention provides the pharmaceutical composition as described herein for use in treatment of bacterial infection, particularly by gram-negative bacteria.

[0109] In particular embodiments, the pharmaceutical composition as described herein is administered prior to, together with or subsequent to the administration of an antibacterial drug or an antitoxin drug.

[0110] The term antitoxin is used here to describe a therapeutic agent that neutralizes the biological activity of a toxin produced by a microorganism, thereby preventing or mitigating toxin- mediated damage without necessarily affecting the viability of the toxin-producing organism. Antitoxins may include polyclonal antisera, monoclonal antibodies, antibody fragments, receptor analogs, or other binding molecules that specifically bind to and inhibit the toxin or its interaction with target cells. In some embodiments, antitoxin agents encompass toxinneutralizing liposomes as disclosed in WO 2013186286 A1. In contrast to antibacterial or antiviral agents, which act directly on the pathogen, antitoxins act on the pathogenic factor itself to block or reverse its toxic effects.

[0111] The term antibacterial drug is used here to describe a therapeutic compound that inhibits the growth of, or kills, bacteria by interfering with essential bacterial structures or metabolic processes. Such drugs act directly on the bacterial cell, targeting pathways including cell wall synthesis (e.g., p-lactams, glycopeptides), protein synthesis (e.g., aminoglycosides, macrolides, tetracyclines), nucleic acid synthesis (e.g., fluoroquinolones, rifamycins), or metabolic functions (e.g., sulfonamides, trimethoprim). Antibacterial drugs thereby eliminate or suppress the causative organisms of bacterial infections, in contrast to antitoxins, which neutralize the toxins produced by such organisms.

[0112] In more particular embodiments, the antibacterial drug is selected from the group consisting of aminopenicillins, ureidopenicillins, cephalosporins, quinolone antibiotics, carbapenems, aminoglycoside antibiotics, and monobactams (aztreonam).

[0113] Aminopenicillins include ampicillin, amoxicillin and bacampicillin. Ureidopenicillins include azlocillin, piperacillin and mezlocillin. Cephalosporins include the 1stgeneration drugs cefalotin, cefazolin, cefalexin, cefradine, and cefadroxil are drugs belonging to this group. 2ndgeneration cephalosporins include cefoxitin, cefuroxime, cefaclor, cefprozil, and cefmetazole. Ceftazidime, ceftriaxone, and cefotaxime are classed as 3rdgeneration cephalosporins. 4thgeneration cephalosporins include cefepime and cefpirome.

[0114] Quinolone antibiotics include ciprofloxaxin, gatifloxacin, gemifloxacin, levofloxacin, ofloxacin and moxifloxacin.

[0115] Carbapenems include imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem and tebipenem.

[0116] Empty liposomes

[0117] Yet another alternative aspect of the invention relates to a liposome comprised of a phospholipid bilayer consisting of a phospholipid component, the bilayer surrounding an aqueous phase (the liposomal content), the phospholipid component being selected from the group consisting of phosphatidylcholine and sphingomyelin or mixtures thereof, wherein the phospholipid component comprises a fatty acid selected from tetradecanoic acid, pentadecanoic acid, hexadecenoic (palmitic) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid.

[0118] These “empty” liposomes may be present in a pharmaceutically acceptable buffer, or in a solution -also formulated for pharmaceutical administration purposes- containing further pharmaceutically active ingredients.

[0119] Medical treatment

[0120] Similarly, within the scope of the present invention is a method or treating an inflammatory condition, aseptic or caused by a bacterial infection, in a patient in need thereof, comprising administering to the patient a pharmaceutical composition or liposome according to the above description. Method of Manufacture and Method of Treatment according to the invention

[0121] The invention further encompasses, as an additional aspect, the use of a composition as identified herein, for use in a method of manufacture of a medicament for the treatment or prevention of an inflammatory condition, aseptic or caused by a bacterial infection, associated with presence of bacterial endotoxin lipopolysaccharide (LPS), particularly endotoxemia; a condition selected from the group of systemic inflammatory response syndrome, sepsis, septic shock and multiple organ failure, and / or bacterial infection, particularly by gram-negative bacteria.

[0122] Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with an inflammatory condition, aseptic or caused by a bacterial infection, or associated with presence of bacterial endotoxin lipopolysaccharide (LPS), particularly endotoxemia; a condition selected from the group of systemic inflammatory response syndrome, sepsis, septic shock and multiple organ failure, and / or bacterial infection, particularly by gram-negative bacteria. This method entails administering to the patient an effective amount of a composition as specified in detail herein.

[0123] Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

[0124] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

[0125] THP-1 cells are a human monocytic cell line commonly used as a model in biomedical research, particularly in the fields of immunology, cancer biology, and infectious disease research. These cells were originally derived from the peripheral blood of a leukaemia patient and have since been widely employed in research including the following areas:

[0126] Immune System Studies: THP-1 cells are often utilized to investigate the innate immune response, including phagocytosis, cytokine production, and cellular signalling pathways.

[0127] Inflammation Research: THP-1 cells serve as a model to study inflammatory processes and the release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) and interleukins (IL).

[0128] This cell line has become a common model to estimate modulation of monocyte and macrophage activities. The THP-1 cell line is a suitable and reliable model to study monocyte and macrophage functions / responses, macrophage differentiation and possible effects from external stimuli in the surrounding environment. This suggests the option to use such cells for compound screening purposes, product development and quality controls (Chanput et al., Inti Immunopharmacol, 23(1 ), 2014; Qi Z., Atherosclerosis 221(1 ), 2014).

[0129] Further, our major findings were corroborated using human blood cells obtained from healthy donors.

[0130] The invention further encompasses the following items:

[0131] 1. A pharmaceutical composition consisting of a liposomal formulation of a phospholipid component and an aqueous phase, the phospholipid component being selected from the group consisting of phosphatidylcholine and sphingomyelin or mixtures thereof, wherein the phospholipid component comprises a saturated or mono-unsaturated (Z / cis) fatty acid having 14 to 20 carbon atoms, particularly 16 to 20 carbon atoms.

[0132] 2. The pharmaceutical composition according to item 1 , wherein the phospholipid component is phosphatidylcholine.

[0133] 3. The pharmaceutical composition according to any one of the preceding items, wherein the phospholipid component is selected from the group consisting of phosphatidylcholine and sphingomyelin and the phospholipid comprises: a. a first fatty acid selected from a 16:0, 16:1 , 18:0 and 18:1 fatty acid, and b. a second fatty acid selected from a saturated or monounsaturated C14, C15 or C16 fatty acid.

[0134] 4. The pharmaceutical composition according to any one of the preceding items, wherein the phospholipid component is selected from: a) 16:1 phosphatidylcholine; b) 16:1 sphingomyelin; or c) egg sphingomyelin.

[0135] 5. The pharmaceutical composition according to item 4, wherein the phospholipid component is (9Z) hexadecenoic phosphatidylcholine.

[0136] 6. The pharmaceutical composition according to item 1 , wherein the phospholipid component is 18:1 / 14:0 phosphatidylcholine.

[0137] 7. The pharmaceutical composition according to item 1 , wherein the phospholipid component is 16:1 / 14:0 phosphatidylcholine.

[0138] 8. The pharmaceutical composition according to item 4, wherein the phospholipid component is (9Z) hexadecenoic sphingomyelin.

[0139] 9. The pharmaceutical composition according to any one of the preceding items, formulated for intravenous administration. 10. A pharmaceutical composition comprising of a liposomal formulation of a phospholipid component and an aqueous phase, the phospholipid component selected from the group consisting of phosphatidylcholine and sphingomyelin, wherein the phospholipid component comprises a saturated or mono-unsaturated (Z / cis) fatty acid having 14 to 20 carbon atoms, for use in treatment of a condition associated with presence of lipopolysaccharide (LPS), particularly endotoxemia.

[0140] 11. The pharmaceutical composition for use according to item 10, wherein the pharmaceutical composition is a pharmaceutical composition as specified in any one of the preceding items 1 to 9.

[0141] 12. The pharmaceutical composition according to any one of the preceding items for use in treatment of a condition selected from the group of systemic inflammatory response syndrome, sepsis, septic shock and multiple organ failure.

[0142] 13. A pharmaceutical composition according to any one of the preceding items for use in treatment of bacterial infection, particularly by gram-negative bacteria.

[0143] 14. The pharmaceutical composition for use according to any one of the preceding items 12 to 13, wherein the composition is administered prior to, together with or subsequent to the administration of an antibacterial drug.

[0144] 15. The pharmaceutical composition for use according to item 14, wherein the antibacterial drug is selected from the group consisting of aminopenicillins, ureidopenicillins, cephalosporins, quinolone antibiotics, carbapenems, aminoglycoside antibiotics, and monobactams (aztreonam).

[0145] Also encompassed in the invention are the following embodiments, wherein definitions of terms are synonymous to those used for the pharmaceutical compositions defined earlier:

[0146] 1. A liposome comprised of a phospholipid bilayer consisting of a phospholipid component, the bilayer surrounding an aqueous phase (the liposomal content), the phospholipid component being selected from the group consisting of phosphatidylcholine and sphingomyelin or mixtures thereof, wherein the phospholipid component comprises a fatty acid selected from tetradecanoic acid, pentadecanoic acid, hexadecenoic (palmitic) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid.

[0147] 2. The liposome according to embodiment 2, wherein the aqueous phase consists of a pharmaceutically acceptable buffer and does not contain a pharmaceutically active ingredient. 3. The liposome according to embodiment 1 or 2, wherein the phospholipid component is constituted entirely of phosphatidylcholine.

[0148] 4. The liposome according to embodiment 3, wherein the phosphatidylcholine comprises a. one fatty acid selected from pentadecanoic (15:0) acid, hexadecenoic (palmitic; 16:0) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid, and b. one fatty acid selected from tetradecanoic (myristic; 14:0) acid, pentadecanoic (15:0) acid, hexadecenoic (palmitic; 16:0) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid, and octadecanoic (stearic) acid.

[0149] 5. The liposome according to embodiment 4, wherein the 16:1 or 18: 1 monounsaturated fatty acid has a Z (cis) double bond.

[0150] 6. The liposome according to embodiment 4 or 5, wherein the 16:1 or 18: 1 monounsaturated fatty acid is selected from the group consisting of an octadec-9-enoic acid, an octadec-6-enoic acid, a hexadec-9-enoic acid, an hexadec-6-enoic acid, and a hexadec-11-enoic acid.

[0151] 7. The liposome according to any one of embodiments 4 to 6, wherein the monounsaturated fatty acid is selected from the group consisting of 18:1 (Z)-9 (oleic acid), 18:1 (Z)-6 (petroselinic acid), 16:1 (Z)-9 (palmitoleic) acid, 16:1 (Z)-6 (sapienic) acid, and cis-11 -hexadecenoic acid.

[0152] 8. The liposome according to any one of embodiments 3 to 7, wherein the phosphatidylcholine is selected from 18:1 / 14:0 phosphatidylcholine, 16:1 / 14:0 phosphatidylcholine, 18:1 / 18:1 phosphatidylcholine, and 16:0 / 16:0 phosphatidylcholine.

[0153] 9. The liposome according to embodiment 3 to 7, wherein the phosphatidylcholine comprises two identical fatty acids.

[0154] 10. The liposome according to embodiment 9, wherein the phosphatidylcholine comprises two 16:1 monounsaturated fatty acid moieties.

[0155] 11. The liposome according to embodiment 8 or 10, wherein the phosphatidylcholine comprises two 16:1 (Z)-9 (palmitoleic) acid moieties.

[0156] 12. The liposome according to embodiment 9, wherein the phosphatidylcholine comprises two pentadecanoic fatty acid moieties.

[0157] 13. The liposome according to any one of embodiments 3 to 12, wherein the liposome comprises only a single phosphatidylcholine species. 14. The liposome according to any one of the preceding embodiments, wherein the liposome is formulated for intravenous administration.

[0158] 15. The liposome according to any one of the preceding embodiments, for use as a medicament.

[0159] 16. The liposome according to any one of the preceding embodiments, for use in the treatment of an inflammatory condition.

[0160] 17. The liposome for use according to embodiment 16, wherein the inflammatory condition is selected from the group of systemic inflammatory response syndrome, sepsis, septic shock and multiple organ failure.

[0161] 18. The liposome according to any one of the preceding embodiments 1 to 15, for use in treatment of a condition associated with presence of lipopolysaccharide (LPS), particularly endotoxemia.

[0162] 19. The liposome according to any one of the preceding embodiments 1 to 15, for use in treatment of bacterial infection, particularly by gram-negative bacteria.

[0163] 20. The liposome for use according to any one of the preceding embodiments 16 to 19, wherein the composition is administered prior to, together with or subsequent to the administration of an antibacterial drug or an antitoxin drug.

[0164] 21. The liposome for use according to embodiment 20, wherein the antibacterial drug is selected from the group consisting of aminopenicillins, ureidopenicillins, cephalosporins, quinolone antibiotics, carbapenems, aminoglycoside antibiotics, and monobactams (aztreonam).

[0165] Embodiments on specific sphingomyelin species

[0166] 22. The liposome according to any one of the preceding embodiments 1 to 3, wherein the phospholipid component is sphingomyelin.

[0167] 23. The liposome according to embodiment 22, wherein the phospholipid component is 16:1 sphingomyelin.

[0168] 24. The liposome according to embodiment 23, wherein the phospholipid component is (9Z) hexadecenoic sphingomyelin.

[0169] 25. The liposome according to embodiment 22, wherein the phospholipid component is 15:0 sphingomyelin.

[0170] 26. The liposome according to any one of the preceding embodiments 22 to 26, wherein the liposome is a liposomal formulation formulated for intravenous administration. 27. The liposome according to any one of the preceding embodiments 22 to 27 for use as a medicament.

[0171] 28. The liposome according to any one of the preceding embodiments 22 to 27, for use in the treatment of an inflammatory condition.

[0172] 29. The liposome for use according to embodiment 28, wherein the inflammatory condition is selected from the group of systemic inflammatory response syndrome, sepsis, septic shock and multiple organ failure.

[0173] 30. The liposome according to any one of the preceding embodiments 22 to 26, for use in treatment of a condition associated with presence of lipopolysaccharide (LPS), particularly endotoxemia.

[0174] 31 . The liposome according to any one of the preceding embodiments 22 to 26, for use in treatment of bacterial infection, particularly by gram-negative bacteria.

[0175] 32. The liposome for use according to any one of the preceding embodiments 27 to 31 , wherein the composition is administered prior to, together with or subsequent to the administration of an antibacterial drug.

[0176] 33. The liposome for use according to embodiment 32, wherein the antibacterial drug is selected from the group consisting of aminopenicillins, ureidopenicillins, cephalosporins, quinolone antibiotics, carbapenems, aminoglycoside antibiotics, and monobactams (aztreonam).

[0177] Description of the Figures

[0178] Figs 1 - 28 show the effects of LPS and / or liposomes composed of various lipids on the proliferation of THP-1 cells.

[0179] Liposomes alone (Figs 1 - 4) or LPS, in the presence or absence of liposomes (Figs 5 - 14), were added to cells in RPMI medium containing either 10% or 40% FCS (and the cells were left to proliferate in the resulting LPSZ(andZor) liposome suspensions (final volume = 1 ml) in a CO2 incubator at 37°C. In control experiments, cells were processed identically albeit in the absence of LPS / liposomes. After 5-7 days of proliferation, cells were counted either manually or using an automated cell counter. X-axes: amounts of liposomes in pg (correspond to concentrations = pg / ml); Y-axes: relative cell density on days 5-7 (averaged) in relation to the corresponding controls in %.

[0180] Fig. 1 shows the effect of liposomes composed of synthetic phosphatidylcholines (PC) containing identical acyl chains. The number in the graph titles represent the number of carbon atoms and number of double bonds per acyl chain (e.g. 18 atoms and no double bonds in 18:0 PC). Phase-transition temperatures for the corresponding lipid species are given in degrees of Celsius in brackets. Triangles: experiment was performed in the RPMI medium containing 40% FCS, solid circles: - 10% FCS. A - 18:0 PC, B - 16:0 PC, C - 18:1 PC, D - 16:1 PC; E - 14:0 PC, F - 14:1 PC, G - 12:0 PC, H - 08:0 PC, I - 06:0 PC. Data points are shown as the Mean ± SD.

[0181] Fig. 2 shows the differences in the effects of 14:0 PC (circles) vs 14:1 PC (triangles) liposomes. Panel on the left shows experiments in 10% FCS, right experiments in 40% FCS. Further details as in Fig. 1.

[0182] Fig. 3 shows the effects of liposomes composed exclusively of egg sphingomyelin (Sm) (panel A) or liposomes composed of egg Sm (= 33 mol / %) and cholesterol (Ch; ~ 66 mol / %) (panel B). Details as in Fig. 1.

[0183] Fig. 4 shows the effect of liposomes composed of negatively charged phosphatidylglycerol (18:1 PG). Details as in Fig. 1.

[0184] Fig. 5 shows the cytostatic effect of LPS (X-axis, point = 0 pg / ml) and protective effects of liposomes (X-axis, points 12.5 - 400 pg / ml) on the proliferation of THP-1 cells treated with LPS (triangles) at 4-6 days. Squares - duplicate the liposome toxicity data shown in Fig. 1 to highlight the effect of liposomal toxicity on the outcome of the LPS-neutralization experiments. A -18:0 PC, B - 16:0 PC, C - 14:0 PC, D - 12:0 PC; E - 24:1 PC, F - 18:1 PC, G - 16:1 PC, H - 14:1 PC, I - egg Sm, J - 18:1 PG, K - Ch:Sm. Further details as in Fig. 1 .

[0185] Fig. 6 shows the differences in the LPS-neutralization effects between liposomes composed of various PC species containing identical acyl chains. Panel A shows 24:1 PC (squares) vs. 18:0 PC (triangles), panel B shows 16:1 PC (circles) vs. 14:1 PC (squares) vs. 14:0 PC (triangles), panel C shows 18:1 PC (diamonds) vs. 16:0 PC (circles), Panels on the left show experiments in 10% FCS and panels on the right show experiments in 40% FCS. Details as in Figs. 1 and 5.

[0186] Fig. 7 highlights the LPS-neutralization superiority of 16:1 PC (circles) vs 18:1 PC (triangles) liposomes. The panel on the left shows the experiment in 10% FCS, whilst the panel on the right shows the experiment in 40% FCS. Details as in Figs. 1 and 5.

[0187] Fig. 8 highlights the absence of differences in the LPS-neutralization between PC liposomes and Sm liposomes. Panel A shows 16:1 PC (circles) vs 16:1 Sm (triangles). Panel B shows 16:0 PC (circles) vs egg Sm (triangles). Panels on the left show experiments in 10% FCS and panels on the right show experiments in 40% FCS. Details as in Figs. 1 and 5. Fig. 9 highlights the absence of the LPS-neutralization effects by Ch:Sm liposomes (triangles). The results for 16:1 PC liposomes (circles) are shown as the reference. Details as in Figs. 1 and 5.

[0188] Fig. 10 shows the direct comparison of LPS-neutralization between neutral 16:1 PC liposomes (circles) and negatively charged 18:1 PG liposomes (triangles). Panels on the left show experiments in 10% FCS and panels on the right show experiments in 40% FCS. Details as in Figs. 1 and 5.

[0189] Fig. 11 compares effects of liposomes with identical and mixed acyl chains. Panel A: 16:1 PC (circles) vs 18:1 PC (triangles); Panel B: 16:1 PC (circles) vs 14:0 PC (triangles); Panel C: 16:1 PC (circles) vs 18:1 / 14:0 PC (triangles). Panels on the left show experiments in 10% FCS and panels on the right show experiments in 40% FCS. Details as in Figs. 1 and 5.

[0190] Fig. 12 shows the behavior of saturated fatty acid PC of different chain length (even number of carbon atoms vs odd number of carbon atoms): 14:0 PC (circles); 15:0 PC (triangles); 16:0 PC (squares). Panels on the left show experiments in 10% FCS and panels on the right show experiments in 40% FCS. Details as in Figs. 1 and 5.

[0191] Fig. 13 shows the behavior of mono- compared to bi-unsaturated fatty acid PC: 18:1 PC (circles); 18:2 PC (triangles). Panels on the left show experiments in 10% FCS and panels on the right show experiments in 40% FCS. Details as in Figs. 1 and 5.

[0192] Fig. 14 shows neutralization of LPS by CalO2 which is a 1 :1 mixture of Ch (66 mol / %):egg Sm (33 mol%) liposomes with egg Sm-only liposomes. 10% FCS (circles); 40% FCS (triangles). Details as in Figs. 1 and 5.

[0193] Fig. 15 shows that PC liposomes inhibit LPS-induced production of interleukin 8 by THP-1 cells that were differentiated to macrophages. Panel on the left show experiments in 10% FCS and panel on the right show experiments in 40% FCS.

[0194] Fig. 16 shows that PC liposomes inhibit LPS-induced production of pro-inflammatory cytokines by human PBMC (peripheral blood mononuclear cells). IL-8 = interleukin 8 (panel A). TNF = Tumor necrosis factor (panel B). Bars give amount of mRNA (determined by PCR) in PBMC treated with LPS. Data for IL-8 and TNF -alpha are given. Fold increase observed in the presence of LPS-only is set at 100%

[0195] Fig. 17 proliferation of THP-1 cells over time in presence or absence of septic (LPS) or aseptic (TNF) inflammation stimuli. A+B X-axis shows days

[0196] Fig 18 proliferation of THP-1 cells on day 5, in presence of egg PC. A+B X-axis is the concentration of egg PC liposomes Fig. 19 proliferation of THP-1 cells on day 5, in presence of egg and synthetic PC (same acyl chain on sn1 , sn2). X-axis is the concentration of PC liposomes

[0197] Fig. 20 proliferation of THP-1 cells on day 5, in presence of synthetic PC of different acyl composition.

[0198] Fig. 21 proliferation of THP-1 cells on day 5, in presence of synthetic PC of different acyl composition.

[0199] Fig 22 proliferation of THP-1 cells over time; X-axis is hours.

[0200] Fig. 23 proliferation of THP-1 cells over time; X-axis is hours.

[0201] Fig. 24 proliferation of THP-1 cells over time; X-axis is hours.

[0202] Fig. 25 proliferation of THP-1 cells over time in presence of different concentrations of

[0203] (13:0)2 PC; X-axis is days.

[0204] Fig. 26 proliferation of THP-1 cells over time in presence of different formulations of PC; squares: aggregated form; spheres: liposomes; X-axis is hours.

[0205] Fig. 27 proliferation of THP-1 cells over time in presence of different formulations of PC; squares: aggregated form; spheres: liposomes; X-axis is hours.

[0206] Fig. 28 A, B, C: proliferation of THP-1 cells over time (days) in response to different treatment regimes; proliferation of THP-1 cells at day 5 after TNF stimulus in presence of different amounts of 16:1 PC.

[0207] Examples

[0208] Methods

[0209] Cell Culture

[0210] The human acute monocytic leukemia cell line (THP-1 ; ATCC TIB-202) was maintained at ~106cells / ml concentration in RPMI 1640 medium (Gibco, Life Technologies) supplemented with 10% heat-inactivated Fetal Calf Serum (FCS), 2 mM L-glutamine, and 100 U / mL penicillin / 100 pg / mL streptomycin in a CC>2-incubator at 37°C.

[0211] Liposome preparations

[0212] Lipids (Avanti Polar Lipids) in powder form, were dissolved in PBS at the concentration of 20 mg / ml, heated to 40°C for 20 min, and sonicated on ice for 30 min with 5 x 10% duty cycles using a sonotrode sonicator at maximal power (Bandelin Sonoplus, Germany). Liposomes, stored at -80°C, were re-sonicated for 1 min immediately before the experiment. Liposomes (mean diameter = 120-150 ± 30-50 nm) were present mostly in uni- and bi-lamellar form. The diameter of the liposomes was measured by NanoSight NS300 (Malvern Panalytical, Malvern, UK).

[0213] Reagents

[0214] Lipopolysaccharide (LPS) from E. coli strain 055:B5 (Sigma), suspended in PBS at the concentration of 1 mg / ml, aliquoted and stored at -20°C. TNF-a was prepared similarly.

[0215] Proliferation / viability assays

[0216] THP-1 cells were pelleted at room temperature (1 ,100 rpm, 5 min), resuspended in PBS (if not indicated otherwise), counted and, after intermediate centrifugation, resuspended at 5 x 105cells / mL concentration in RPMI medium containing either 10% or 40% of FCS.

[0217] The effect of LPS or TNF-a on the proliferation of THP-1 cells was assessed in the presence or in the absence of liposomes. The cytotoxicity of liposomes was assessed in the absence of LPS / TNF-a.

[0218] For each condition, 800 pL of cell suspension (100 pL cells + 700 pL medium) were mixed with 100 pL of serially two-fold diluted liposomes in PBS and / or 100 pL of LPS or TNF-a diluted in PBS. PBS was added instead of reagent to obtain liposome- / LPS- / TNF-a-free controls.

[0219] The final volume was 1 mL and the final cell density at t = 0 was 5 x 104cells / mL. Unless stated otherwise, LPS was used at 500 ng / mL and TNF-a at 5 ng / mL; liposome concentrations are indicated in the figures (X-axes). LPS, TNF-a and / or liposomes were present for the whole duration of an experiment.

[0220] The cells displayed linear growth curve between 4 and 8 days of culturing. Data obtained on days 4-6 were used for analysis. Results are presented as % normalization (cells grown in the absence of liposomes, LPS or TNF-a in the same experiment are used as controls = 100%).

[0221] THP-1 differentiation

[0222] When indicated, THP-1 monocytes were differentiated into macrophages by 24 h incubation with 150 nM phorbol 12-myristate 13-acetate (PMA) in RPMI medium supplemented with 10% FCS, 2 mM L-glutamine and 100 U / mL penicillin / 100 pg / mL streptomycin followed by 48 h incubation in the same medium without PMA.

[0223] PCR and RT-qPCR.

[0224] For gene expression analysis, THP-1 cells (or differentiated cells, or PBMC) were treated with LPS and / or liposome essentially as described above, albeit the experiments were performed either in FCS-free RPMI medium or in RPMI medium supplemented with 40% FCS, LPS concentration was 1 ng / ml and the assay incubation time was 3 hours. Total RNA was isolated using the RNeasy Micro Kit (Qiagen, Hilden, Germany). For real-time RT-PCR analysis the SYBR Green detection system was used (Quanta Biosciences, Beverly, MA). All primers were designed using the Primer3 design tool. Expression profiling was performed in a 96-well format on the StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression of target genes was normalized to GAPDH. Results are presented as % normalization (cells treated with LPS in the absence of liposomes in the same experiment are used as controls = 100%).

[0225] PBMC

[0226] Peripheral blood mononuclear cells (PBMC) were isolated from human buffy coats obtained from healthy volunteers (RedCross Switzerland, Bern, Switzerland). PBMC were incubated overnight in RPMI medium supplemented with 40% FCS. Treatment with LPS / liposomes and RT-PCR was performed as described above for differentiated macrophages.

[0227] Example 1: Toxicity of liposomes

[0228] The effect of liposomes on the proliferation of THP-1 cells was assessed.

[0229] The following conclusions were drawn from the results of experiments shown in Fig. 1 :

[0230] Liposomes composed of 18:0 PC, 16:0 PC, 18:1 PC or 16:1 PC were not cytotoxic. Liposomes composed of 14:0 PC, 14:1 PC or 12:0 PC were cytotoxic.

[0231] 08:0 PC that was present mostly in the non-liposomal, micellar form was cytotoxic. Soluble, non-liposomal 06:0 PC was slightly cytostatic.

[0232] Liposomes composed of toxic PC species and 08:0 PC micelles were less toxic in the presence of 40% FCS compared to 10% FCS.

[0233] Liposomes composed of unsaturated PC species with acyl chain lengths > 14 carbon atoms (18:1 PC, 16:1 PC and 14:1 PC) and saturated 14:0 PC slightly potentiated proliferation of THP-1 cells. For liposomes composed of 14:0 PC and 14:1 PC species, the potentiating effect was observed only at low concentrations due to their toxicity at higher concentrations.

[0234] Figure 2 highlights the role of acyl chain saturation and acyl chain length in the toxicity of 12- 14 carbon PC species.

[0235] Liposomes composed of PC containing short monounsaturated acyl chains (14:1 PC) were more toxic than those containing fully saturated acyl chains of the same length (14:0 PC).

[0236] Liposomes composed of saturated PC species with shorter acyl chains (12:0 PC) were more toxic than those with longer ones (14:0 PC) (data not shown). Remarkably, non-liposomal 06:0 PC and 08:0 PC species were less toxic than liposomal 12:0 PC (see Fig. 1 ).

[0237] Overall, 12:0 PC was the most toxic among all tested PC species, followed by 14:1 PC (Fig. 1-2). Figure 3 relates to similar experiments performed with Sm-containing liposomes. Data for egg Sm are shown. Data for synthetic sphingomyelins are discussed below (Fig. 8). Arsov et al. (Chemistry and Physics of Lipids, Vol 213, 2018, 102-110) reported that egg SM (ESM) is widely regarded as the most homogeneous of the natural SMs; it has predominantly 86% N- palmitoyl (16:0) acyl chain (Filippov et al., Biophysical Journal 90(6), 2006, 2086-2092) with 93% of the sphingosine (18:1 ) long-chain base (Ramstedt et al., Biophys. J 77(3), 1999, 1498- 1506).

[0238] Liposomes composed exclusively of egg Sm or Ch:Sm liposomes composed of 66 mol / % cholesterol (Ch) and 33 mol / % egg Sm were neither toxic nor displayed pro-proliferative activity.

[0239] Figure 4 highlights the high toxicity of negatively charged liposomes (18:1 PG), which is in contrast to the corresponding PC species (18:1 PC, see Fig. 1 ).

[0240] Example 2: Interference of liposomes in LPS effect

[0241] Next, we addressed the potency of liposomes to antagonize the effects of LPS on proliferation of THP-1 cells.

[0242] Monocyte to macrophage maturation occurring in THP-1 cells upon 24 hours of LPS exposure results in downregulation of a number of proteins associated with cell division, DNA replication, centrosomal, microtubule and kinetochore regulation, which leads to a marked decrease in the proliferation of LPS-treated THP-1 cells (Mulvay et al., 2021 , Nat Commun. 2021 ; 12: 5773).

[0243] As expected, LPS displayed potent cytostatic activity towards THP-1 cells (Figs. 5 - 14), point “0” (X-axes)).

[0244] Figure 5 shows that liposomal protection over a range of liposomal concentrations was defined by the combination of the LPS-neutralizing activity of a particular liposome and its intrinsic toxicity. In the LPS graphs, the initial ascending slope reflects the efficacy of the liposomes against LPS, whereas the following descending slope reflects the liposomal toxicities.

[0245] Figure 6 shows that: liposomes composed of long-chain PC (18:0 PC and 24:1 PC) showed very little protection against LPS (panel A).

[0246] Liposomes composed of 16:1 PC, 14:1 PC and 14:0 PC displayed similar, highly efficient protection against LPS, however, two later PC-species were highly toxic, which is in contrast to the non-toxic 16:1 liposomes (panel B).

[0247] Two other non-toxic liposomal species (18:1 PC and 16:0 PC, panel C) were less efficient compared to 16:1 PC. Liposomes composed of 12:0 PC or of non-liposomal PC species (08:0 PC, 06:0 PC) possessed no protective activity against LPS (data not shown).

[0248] The superiority of 16:1 PC liposomes over 18:1 PC and 16:0 PC liposomes is highlighted in Fig. 7.

[0249] Figure 8 shows that Sm liposomes displayed toxicities and LPS-protection abilities that were very similar to that of corresponding PC species.

[0250] Figure 9 shows that Ch:Sm liposomes displayed no protection against LPS.

[0251] Negatively charged liposomes are known to inhibit LPS; however, they are pro-coagulative and therefore cannot be used as therapeutic agents against endotoxemia. Direct comparison of LPS-neutralizing effects between 18:1 PG liposomes and 16:1 PC liposomes demonstrated that negatively charged liposomes were not superior to the 16:1 PC (Fig. 10) Furthermore, 18:1 PG liposomes were highly cytotoxic towards THP-1 cells, which is in contrast to non-toxic 16:1 PC liposomes (Figs. 4, 10). The unique combination of high efficiency and low cytotoxicity highlights the superiority of 16:1 PC as a therapeutic agent against LPS.

[0252] Figure 11 reiterates that 18:1 / 18:1 PC liposomes are not toxic but they are less efficient than 16:1 / 16:1 PC liposomes (panel A). 14:0 / 14:0 PC liposomes are as efficient as 16:1 / 16:1 PC liposomes but they are cytotoxic (panel B). Panel C shows that liposomes composed of PC with mixed acyl chains (18:1 / 14:0 PC) possessed low toxicity of 18:1 PC and high efficacy of 14:0 PC which makes them almost as efficient as 16:1 / 16:1 PC liposomes.

[0253] Figure 12 demonstrates that liposomes composed of lipids with odd number of carbon atoms (15:0 PC) behave similar to the ones with even numbers. In this particular example, the efficacy and toxicity of 15:0 PC fits between 14:0 PC and 16:0 PC.

[0254] Figure 13 shows that liposomes composed of polyunsaturated lipids (18:2 PC) do not display higher efficiencies than monounsaturated ones (18:1 PC). Moreover, polyunsaturated liposomes are highly toxic which is in contrast to monounsaturated ones.

[0255] Figure 14 shows that CalO2 is protective against LPS.

[0256] Example 3: Interference of liposomes in LPS-induced production of proinflammatory cytokines

[0257] Next, we tested the efficacy of liposomes to inhibit production of cytokine IL-8 by THP-1 cells that were differentiated into macrophages by PMA treatment. Similar to the inhibition of the LPS-induced cytostatic activity, 16:1 PC and 14:0 PC displayed the highest efficacy for the inhibition of the LPS-induced IL-8 production (Fig. 15). The differences in the potency of different liposomes to inhibit IL-8 production were more pronounced at 40% FCS compared to 10% FCS (Fig. 15). Finally, we show that PC liposomes were able to neutralize LPS-induced production of proinflammatory cytokines by human peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers (Fig.16)

[0258] The inventors draw the following conclusions from the data provided herein:

[0259] Liposomes composed of saturated or monounsaturated PC species containing identical long acyl chains (> 16 carbon atoms) were not toxic.

[0260] Liposomes composed PC species containing saturated or unsaturated short acyl chains (< 16 carbon atoms) were cytoxic.

[0261] Liposomes composed of mixed PC species containing at least one fully saturated or monounsaturated fatty acid chain with 16 or more carbon atoms were not toxic. Liposomes composed of polyunsaturated PC species were toxic.

[0262] The most efficient protection against LPS was observed within a cluster of lipids containing acyl chains of 14 carbon atoms (irrespective of their saturation) or 16:1- species.

[0263] Among non-toxic PC liposomes with identical acyl chains only those composed of 16:0 / 16:0 PC, 16:1 / 16:1 PC and 18:1 / 18:1 PC inhibited cytostatic effect of LPS. Among them, 16:1 / 16:1 PC possessed the highest efficacy of LPS neutralization. Liposomes composed of PC species containing at least one saturated or monounsaturated polycarbon chain with 14, 15 or 16 carbon atoms were protective against LPS.

[0264] Liposomes composed of PC species in which one saturated or monounsaturated acyl chain contained 14, 15 or 16 carbon atoms and another saturated or monounsaturated acyl chain contained 16 or more carbon atoms were non-toxic and protective against LPS.

[0265] Liposomes composed of Sm species or PC species demonstrated similar toxic and LPS-protection properties.

[0266] Example 4: Inflammatory responses to stimuli resembling septic and aseptic inflammation

[0267] Upon tissue entry, monocytes undergo striking morphologic remodeling, functional reprogramming, and proteomic / transcriptomic rewiring that stabilize macrophage identity. During monocyte-to-macrophage differentiation cells acquire strong substrate adherence and high secretory output of cytokines / chemokines (e.g., TNF, IL-i p, IL-6, IL-10, TGF-p, CCL2 / 3 / 4 / 5).

[0268] Downregulation of genes associated with cell division, cell cycle arrest and upregulation of inflammatory genes were apparent in LPS-induced macrophage-like maturation of cultured THP-1 monocytic cells (Mulvay et al., 2021 ). Accordingly, in LPS-treated THP-1 cells, we observed transient substrate adherence and cell cycle arrest (Fig. 17A), accompanied by upregulation of multiple inflammatory genes (discussed in detail below).

[0269] To further substantiate these findings, THP-1 monocytes were exposed to a defined set of stress and inflammatory stimuli representing both aseptic and septic conditions. Nutrient deprivation during medium exchange served as short-term starvation stress, transiently reducing proliferation and increasing reactive oxygen species (ROS), thereby promoting inflammasome activation (White et al., 2020). LPS and TNF-a were used as prototypical bacterial and cytokine mediators of inflammation, respectively. In parallel, culture supernatants from Pseudomonas aeruginosa were applied to mimic pathogen-derived virulence factors. To assess the modeling capacity of these stimuli, several parameters were measured following exposure, including cell proliferation, inflammasome activation, cytokine production, NF-KB- dependent gene expression (SOD2, COX2, CASP1 ), and surface-marker modulation involved in antigen presentation, adhesion, and immune signaling (CD-80, ICAM-1 , CD-40, CD-14, CD- 86).

[0270] LPS is largely responsible for the initiation of the inflammatory response during Gram-negative infections, whereas TNF-a orchestrates a wide range of inflammatory responses in both sterile and infectious contexts. Similar to the LPS-exposure, the transient cell cycle arrest was apparent in cells treated with TNF-a (Fig. 17B).

[0271] The most prominent differences in proliferation between control and LPS- or TNF-treated cells were observed on days 4-6 (Fig. 17A, B). Therefore, averaged cell counts taken on these days were used to monitor integral, long-term biological activities of phosphatidylcholines, in particular, their cytotoxicity and modulatory effects on the inflammation-induced monocyte-to- macrophage transition.

[0272] Across all stimuli, overlapping inflammatory modules were detected, encompassing inflammasome signaling (IL-1 P), cytokine production (IL-8, IL-6, TNF-a, CASP-4), N F-KB- dependent genes (SOD2, COX2, CASP1 ), and surface markers linked to adhesion and immune co-stimulation (CD-80, ICAM-1 , CD-40, CD-14, CD-86).

[0273] These results collectively demonstrate that the applied triggers converge on shared inflammatory pathways, supporting their predictive value for modeling both aseptic and septic inflammation.

[0274] Example 5: The protective component in egg phosphatidylcholine

[0275] Phosphatidylcholines from natural sources are most often used in research. In control, non- LPS-stimulated cells, liposomes composed from egg PC slightly potentiated cellular proliferation at low concentrations (50 pg / ml, p = 0.02), whereas at higher concentrations they displayed pronounced cytostatic activity (400 pg / ml, p < 0.001) (Fig. 18). In LPS-treated cells, egg PC liposomes abrogated cell cycle arrest in a concentrationdependent manner, - the proliferative activity being fully restored at 50-100 pg / ml of egg PC (Fig. 18, p < 0.05 for any points with non-overlapping error bars). Increased cellular proliferation at low PC concentrations and the cytostatic activity at its high concentrations was not affected by LPS.

[0276] Our data support our previous findings describing anti-inflammatory PC activity. We show that egg PC liposomes might display anti-inflammatory activity by preventing monocyte-to- macrophage maturation caused by pro-inflammatory stimuli.

[0277] Natural PCs are complex mixtures of PC molecules whose acyl chain composition displays species- and organ- specificity. Egg PC is composed of 18:1 (32%), 16:0 (33%), 18:2 (17%) and 18:0 (12%) acyl chain species.

[0278] Experiments with liposomes composed of synthetic PCs with identical acyl chains in both sn1 and sn2 positions showed that 18:1 PC (18:1 / 18:1 PC) was the dominant species responsible for the potentiation of THP-1 cell proliferation in the absence of LPS (Fig. 19 A) and for the blocking of the LPS-induced cell cycle arrest (Fig. 19 B). 16:0 PC (16:0 / 16:0 PC) liposomes displayed similar biological effects, albeit with lower efficacy (Fig. 19 C,D), whereas 18:0 PC (18:0 / 18:0 PC) displayed slight anti-proliferative activity (< 10%, p < 0.05 for 12.5-400 pg points) in the absence of inflammatory stimulus (Fig. 19 E,F). Liposomes composed of 18:2 PC (18:2 / 18:2) were cytotoxic, being largely responsible for the cytostatic activity of egg PC (Fig. 19 G,H). However, at its lower, non-toxic concentrations, 18:2 PC displayed anti-LPS activity comparable to that of 18:1 PC (Fig. 19 H).

[0279] Experiments with liposomes composed of 18:1 PC species with identical acyl chains that differed in steric configuration (cis vs trans) of the double bond (18:1 / 18:1 PC / 9Z vs 18:1 / 18:1 PC / 9T) or in its position within acyl chains (18:1 / 18:1 PC / 9Z vs 18:1 PC / 18:1 PC / 6Z) demonstrated that both parameters had little (if any) effect on the anti-inflammatory properties of 18:1 phosphatidylcholine (Fig. 20 A,B).

[0280] Natural PCs generally contain different acyl chains in sn1 and sn2 positions. Experiments with liposomes composed of synthetic PCs with non-identical acyl chains in sn1 and sn2 positions showed that their anti-inflammatory activity roughly corresponded to the vectorial sum of activities of corresponding PCs with identical acyl chains, reflecting the decrease in the concentration of the most active acyl chain species in the mixed PC (Fig. 21 A-C). The exact position of non-identical acyl chains (sn1 vs sn2) did not influence the biological activity of liposomes (Fig. 21 D).

[0281] We conclude that anti-inflammatory activity of synthetic PCs offers itself for use in therapeutic interventions in patients experiencing dysregulated immune (over)responses. Our data also point out to the detrimental cytotoxicity of certain PCs species. Further, our findings imply that by selecting the most active anti-inflammatory PC species and by avoiding the most toxic ones the efficacy of synthetic PCs might be significantly enhanced. We show that the emphasis in the selection process should lie with the length and the saturation status of acyl chains, parameters that fundamentally define the anti-inflammatory properties and the toxicity of PCs, whereas the steric configuration of double bonds and their position in acyl chains does not seem to play a defining role. Furter, we show that the anti-inflammatory activity of liposomes composed of PCs with two identical most active acyl chains may be superior to liposomes composed of PCs with mixed acyl chains.

[0282] Liposomes (formed by sonication; mostly unilamellar, diameter = 80-150 nm) composed of bilayer-forming, cylindric 18:1 PC prevented the LPS-induced cell cycle arrest and were nontoxic (Fig. 19A). However, 18:1 PC aggregates (mixed population of large empty-core or filled vesicles (diameter ~ 5-15 pm) and amorphous compact particles (diameter ~ 10- 40 pm), formed by suspension of solid PC in medium without sonication, were biologically inert (data not shown). Lipid particles composed of not bilayer-forming, conical 18:1 fatty acid (diameter < 0.5 pm, below resolution of light microscope), formed by suspension of liquid FA in medium without sonication, were likewise inert (data not shown), whereas lipid particles composed of not bilayer-forming, inverted conical 18:1 lysoPC (initially below resolution of light microscope vesicles, later vesicles ~ 5-10 pm), formed by suspension of solid lysoPC in medium without sonication, were highly cytotoxic.

[0283] These findings indicate the defining role of steric configuration of entire lipid molecules and their association forms, in which these molecules are presented to cells.

[0284] Having established the major role of individual acyl chains, overall shape of molecules and their association forms in the anti-inflammatory and cytotoxic activities of PC, we set to comprehensively investigate effects of these factors using a broader range of synthetic PCs with defined acyl chain compositions.

[0285] Initially we assessed the effects of synthetic PCs on proliferation of THP-1 cells in the absence of inflammatory stimuli (Fig. 22). 15:0 PC, 16:0 PC, 16:1 PC and 18:1 PC significantly potentiated cell proliferation (p < 0.01 at 200 pg point for any lipid species and serum concentration condition). These lipids were not toxic. 13:0 PC, 14:0 PC and 14:1 PC also potentiated cell proliferation, albeit this effect was obvious only at low concentrations (p < 0.01 at 100 pg / 40% FCS point for any lipid species); at higher concentrations the potentiation activity was, most likely, masked by strong cytostatic / cytotoxic activity of these lipids (p < 0.0001 at 400 pg point for any lipid species and serum concentration condition). No potentiation activity was observed for 10:0 PC, 12:0 PC, 18:2 PC and 18:1 lyso PC (18:0 lyso PC), however, these lipids were the most toxic ones (p < 0.0001 at 200 pg point for any lipid species and serum concentration conditions), therefore it is possible that their pro-proliferative activity was fully offset by their cytotoxicity. Surprisingly, 18:0 PC, one of the most abundant lipids of mammalian cells, displayed slight cytostatic activity at both 10% FCS and 40 % FCS (-10%, p < 0.05 for 100-400 pg points at any serum concentration condition).

[0286] The activities of PCs containing acyl chains with 15 carbon atoms or above (including highly cytotoxic 18:2 PC and 18:1 lyso PC) were only slightly affected by the components of blood serum (10% FCS vs 40% FCS), (Fig. 22). However, at 40% FCS, the cytotoxicity of PCs with acyl chains containing less than 15 carbon atoms was strongly decreased compared to 10% FCS condition, suggesting that serum components can neutralize cytotoxic activities of shortchain lipids.

[0287] We further assessed the effects of synthetic PCs on proliferation of THP-1 cells in the presence of inflammatory stimuli (Fig. 23, gray-colored curves displaying cytotoxic activities in the absence of inflammatory stimuli are given as a reference). The integral effects of individual PCs species amounted to the sum of their capability to abolish the stimulus-induced cell cycle arrest and their intrinsic cytotoxic activity. Lipid cytotoxicities were not affected by either LPS or TNF-a. In general, the capability to abrogate cell cycle arrest caused by inflammatory stimuli was similar to the pro-proliferative PCs activities in the absence of the stimuli. Similar were also the effects of FCS (not shown).

[0288] Among the tested PC species, liposomes composed of 14:0 PC, 14:1 PC and 16:1 PC displayed the highest efficacy in abrogating the cell cycle arrest (Fig. 24, p < 0.05 for any points with non-overlapping error bars). However, 14:1 PC liposomes were highly cytotoxic. Liposomes, composed of PCs with 13 or less carbon atom chains displayed low efficacies and were highly toxic. Likewise, lysoPC and polyunsaturated PC were highly toxic.

[0289] Therefore, liposomes composed of 14:0 PC and 16:1 PC were considered as best candidates for future therapeutic applications. Also 15:0 PC liposomes were considered, since they were not toxic and their efficacy was only slightly lower than that of the best candidates.

[0290] The inhibition of cell cycle arrest by PC liposomes and their cytotoxic activity significantly differed in their kinetic properties and their integral effect on the cell proliferation was drastically dependent on PC concentrations (Fig. 25). For example, at 200 pg / ml the inhibition of cell cycle arrest by 13:0 PC was apparent already on day 2, whereas its cytostatic activity was clearly noticeable only on day 5. At 100 pg / ml, liposomes were fully protective and displayed no cytostatic / cytotoxic activities over the whole duration of the assay, whereas at 400 pg / ml, the strong cytotoxic activity was already present on day 1 completely overplaying the anti-cell cycle arrest effects of the liposomes. These findings emphasize the non-linearity of integral PC effects and their dependence not only on the concentration of lipids in the serum but also on the active dose of lipids acquired by cells over time. Effect dependence on formulation of lipids

[0291] Phospholipid uptake occurs by two different mechanisms, - whole donor particle uptake or selective uptake routes. By means of selective phospholipid transfer, specific phospholipids are extracted from extracellular donor particles and subsequently inserted into the extracellular leaflet of the plasma membrane. The selective uptake is expected to be critically dependent on the concentration of available lipids. In (unilamellar) liposomes around half of lipids are displayed at the surface. These lipids are available for the transfer. In contrast, in multilamellar aggregates, most lipid molecules are not available on the surface. Therefore, selective lipid transfer would favor liposomal versus aggregate PC.

[0292] Cells can also unspecifically incorporate entire lipid particles, including phospholipids, by endocytosis / phagocytosis. The whole particle uptake is expected to be more efficient for aggregated lipids compared to liposomes.

[0293] Therefore, we assessed the differences in the long-term efficacy of PCs presented to cells either in liposomal or aggregated form (Fig. 25). In contrast to their liposomal form, aggregates of 18:1 PC and 16:1 PC neither potentiated cell proliferation in the absence of inflammatory stimuli nor abrogated the stimulus-induced cell cycle arrest. Aggregates of 13:0 PC, 14:0 PC and 14:1 PC were as effective as the liposomal form of these lipids. Surprisingly, aggregates of 15:0 PC and 16:0 PC were more active than the liposomes. The anti-proliferative activity of 18:0 PC was also more prominent in aggregated form.

[0294] Biological activities of 15:0 PC and 16:0 PC, potentiated by application in their aggregated form, were comparable to the activities of the previously selected best liposomal candidates (Fig. 27). Therefore, aggregates of 15:0 PC and 16:0 PC were also considered as therapeutic candidates.

[0295] Mechanism of action in septic and aseptic inflammation

[0296] LPS acts via the CD14 / TLR4 / MD2 receptor complex whereas TNF-a binds to its specific cell surface receptors. Also, signaling events triggered by either CD14 / TLR4 / MD2 receptors or TNF-receptors initially propagate via different signaling cascades. Surprisingly, the PCs’ capabilities to abrogate cell cycle arrest caused either by LPS or by TNF-a were identical (Fig. 23). Moreover, the capability to abrogate cell cycle arrest caused by inflammatory stimuli was almost identical to the pro-proliferative PCs’ activities in the absence of the stimuli (Fig. 23).

[0297] Without wanting to be bound by theory, the inventors surmise that the pro-proliferative activity of PCs observed in the absence of added LPS or TNF was due to the abrogation of partial cell cycle arrest induced in their assays by a weak intrinsic inflammatory stimulus. For example, limited release of pro-inflammatory mediators by THP-1 cells, environmentally stressed by handling during execution of the experimental protocol, might be considered. The protocols used in the examples include an interim PBS-wash step during replacement of “used” cell culture medium by the freshly prepared one. During this period (15-20 minutes of handling time) cells remained without access to external nutrients and were exposed to the medium with different composition of inorganic ions (PBS vs RPMI / serum). Since cell proliferation stagnates under conditions of serum deprivation, the following experiments were performed, as an exception, without the PBS wash; - after centrifugation and aspiration of cell culture medium, cell pellets were directly resuspended in fresh medium.

[0298] In the absence of external inflammatory stimuli and in the absence of PC liposomes, this protocol resulted in the potentiation of cellular proliferation compared to the “PBS-wash” conditions (p < 0.05 for 4-7-day points) (Fig. 27A). The proliferation rate observed in this experiment (no PBS-wash, no added liposomes) became indistinguishable from that observed during potentiation of cellular proliferation obtained under the “PBS-wash” conditions in the presence of PC liposomal (Fig. 27 B,C). Moreover, in the “No PBS-wash” experiments, the PC-induced potentiation of cellular proliferation disappeared also in TNF-stimulated cells, which, in combination with anti-TNF effects of non-toxic 16:1 PC liposomes, resulted in proliferation rates that were identical to the PC- and TNF-free controls (Fig. 27 D).

[0299] Nutrient deprivation leads, among others, to the increased ROS production. Since environmental stresses (including ROS or ion disbalance) activate inflammasome-mediated inflammatory response, the inventors addressed effects of short-term serum deprivation, as effected in their typical protocol, on the expression levels of IL-1 p, which is the main effector of the inflammasome-mediated inflammation. The IL-1 expression was tested by RT-QPCR at 0 hours, 3 hours and 24 hours after transient (20 min) PBS-exposure in the absence of external inflammatory stimulus. Additionally, the effect of 16:1 PC liposomes on IL-i p expression was tested at 3 hours and 24 hours.

[0300] Environmental stress increased IL-1 p expression in a time-dependent manner. The effect of 16:1 PC on IL-i p expression was negligible after 3 hours of exposure (no statistical significance). However, strong inhibition was observed after 24 hours of exposure (p < 0.001 for 50-400 pg points), with IL-1 p expression levels returning to the values observed at 0-hour controls.

[0301] These findings suggest that even short-term environmental stress might lead to the development of mild, long-lasting inflammatory response. Therefore, the inventors concluded that PC-treatment abolished not only effects of potent inflammatory stimuli such as LPS or TNF, but also inflammasome-mediated inflammatory responses caused by environmental stress.

[0302] Extracellularly administered PC completely blocked maturation of monocytes into more immunologically active macrophages. The inventors were able to demonstrate that certain PC were also capable of preventing the induction of a plethora of inflammatory mediators with broadly different modes of action that was triggered either by LPS (specifically, expression of IL-8, CD80, CD86, ICAM, TNFa, IL-1 R>, SOD-2, CD14 and CD40, which surged upon exposition to LPS, were all restored to control values when cells were exposed to (16:1 )2 PC) or by culture supernatants of a Gram-negative pathogen, Pseudomonas aeruginosa, for which results were essentially the same with all tested immune-response related transcript.

[0303] Taken together, the findings presented in Fig. 23 and the results on gene expression in relation to exposure to LPS and Pseudomonas supernatants, suggest that it is unlikely that the active PC species exert their anti-inflammatory activity by interfering with a particular inflammatory stimulus, by disrupting a particular stimulus-receptor interaction or by interfering with early downstream signaling. More likely, protective PC species interfere with universal, broadspectrum inflammatory regulator(s) or effect changes to general aspects of cellular homeostasis.

[0304] This warrants pharmacological application of synthetic PCs having the carbon-atom acyl chains disclosed herein as protective, as broad-spectrum generalized anti-inflammatory modulators.

[0305] To gain further insights into mechanisms that define long-term anti-inflammatory activity, the inventors assessed effects of synthetic PC species on IL-8 expression levels following 3 / 24- hour, short-term exposure of THP-1 cells to LPS.

[0306] Surprisingly, after 3-hour LPS-stimulation the anti-inflammatory activities of cytotoxic, shortchain PC species, that displayed no (12:0 PC) or limited (13:0 PC) protection against LPS- induced cell cycle arrest in the long-term proliferation assays, were active in short-term assay (data not shown). Likewise, 18:2 PC and lysol 8:1 PC, which were highly cytotoxic and not protective in the long-term assay, revealed a strong anti-inflammatory response. 16:0 PC and 18:1 PC were the least active in the short-term assay, whereas 18:0 PC showed a tendency to pro-inflammatory activity. Also, 18:2 PC tended to display pro-inflammatory activity, albeit only at its low concentrations.

[0307] Cited references:

[0308] Arsov et aL, 2018, Chemistry and Physics of Lipids, Vol 213, 102-110

[0309] Chanput et al., 2014, Int Immunopharmacol. 23(1 ): 37-45

[0310] Filippov et aL, 2006, Biophysical Journal 90(6), 2086-2092

[0311] Lepper PM et aL, Intensive Care Med. 2002 Jul;28(7):824-33.

[0312] Mulvay et aL, 2021 , Nat Commun. 2021 ; 12: 5773 Opal et aL, J Infect Dis. 1999 Nov;180(5):1584-9

[0313] Pretty et aL, 2022 Open Biol. 12: 210318

[0314] Qin Z., 2012, Atherosclerosis 221 (1 ):2-11

[0315] Ramstedt et aL, 1999, Biophys. J 77(3), 1498-1506 White et al, 2020, Sc / Rep 10, 12505

[0316] Wurfel et aL J Immunol (1997) 158 (8): 3925-3934

[0317] All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

Claims

Claims1 . A pharmaceutical composition consisting of a formulation of a phospholipid component and an aqueous phase, the phospholipid component being selected from the group consisting of phosphatidylcholine and sphingomyelin or mixtures thereof, wherein the phospholipid component comprises a fatty acid selected from tetradecanoic acid, pentadecanoic acid, hexadecenoic (palmitic) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid.

2. The pharmaceutical composition according to claim 1 , wherein the pharmaceutical composition is a liposomal formulation.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition does not comprise a further pharmaceutically active ingredient.

4. The pharmaceutical composition according to any one of the preceding claims, wherein the phospholipid component is phosphatidylcholine.

5. The pharmaceutical composition according to claim 4, wherein the phosphatidylcholine comprises c. one fatty acid selected from pentadecanoic (15:0) acid, hexadecenoic (palmitic; 16:0) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid, and d. one fatty acid selected from tetradecanoic (myristic; 14:0) acid, pentadecanoic (15:0) acid, hexadecenoic (palmitic; 16:0) acid, a 16:1 monounsaturated fatty acid and an 18:1 monounsaturated fatty acid, and octadecanoic (stearic) acid.

6. The pharmaceutical composition according to claim 5, wherein the 16:1 or 18: 1 monounsaturated fatty acid has a Z (cis) double bond.

7. The pharmaceutical composition according to claim 5 or 6, wherein the 16:1 or 18: 1 monounsaturated fatty acid is selected from the group consisting of an octadec-9-enoic acid, an octadec-6-enoic acid, a hexadec-9-enoic acid, an hexadec-6-enoic acid, and a hexadec-11-enoic acid.

8. The pharmaceutical composition according to any one of claims 5 to 7, wherein the monounsaturated fatty acid is selected from the group consisting of 18:1 (Z)-9 (oleic acid), 18:1 (Z)-6 (petroselinic acid), 16:1 (Z)-9 (palmitoleic) acid, 16:1 (Z)-6 (sapienic) acid, and cis-11 -hexadecenoic acid.

9. The pharmaceutical composition according to any one of claims 4 to 8, wherein the phosphatidylcholine is selected from 18:1 / 14:0 phosphatidylcholine, 16:1 / 14:0 phosphatidylcholine, 18:1 / 18:1 phosphatidylcholine, and 16:0 / 16:0 phosphatidylcholine.

10. The pharmaceutical composition according to claim 4 to 8, wherein the phosphatidylcholine comprises two identical fatty acids.

11. The pharmaceutical composition according to claim 10, wherein the phosphatidylcholine comprises two 16:1 monounsaturated fatty acid moieties.

12. The pharmaceutical composition according to claim 9 or 11 , wherein the phosphatidylcholine comprises two 16:1 (Z)-9 (palmitoleic) acid moieties.

13. The pharmaceutical composition according to claim 10, wherein the phosphatidylcholine comprises two pentadecanoic fatty acid moieties.

14. The pharmaceutical composition according to any one of claims 4 to 13, wherein the composition comprises only a single phosphatidylcholine species.

15. The pharmaceutical composition according to claim 1 , wherein the pharmaceutical composition is not a liposomal formulation, but comprises phosphatidylcholine of pentadecanoic acid or hexadecenoic acid in aggregated form.

16. The pharmaceutical composition according to claim 15, wherein the pharmaceutical composition comprises 1 ,2-bis(pentadecanoyl)-sn-glycero-3-phosphocholine.

17. The pharmaceutical composition according to claim 15, wherein the pharmaceutical composition comprises 1 ,2-bis(hexaadecanoyl)-sn-glycero-3-phosphocholine.

18. The pharmaceutical composition according to any one of claims 15 to 17, wherein the pharmaceutical composition does not comprise a further pharmaceutically active ingredient.

19. The pharmaceutical composition according to any one of the preceding claims 4 to 14, wherein the pharmaceutical composition is a liposomal formulation formulated for intravenous administration.

20. The pharmaceutical composition according to any one of the preceding claims 15 to 17, wherein the pharmaceutical composition is a liposomal formulation formulated for topical administration.21 . The pharmaceutical composition according to any one of the preceding claims, for use as a medicament.

22. The pharmaceutical composition according to any one of the preceding claims, for use in the treatment of an inflammatory condition.

23. The pharmaceutical composition for use according to claim 22, wherein the inflammatory condition is selected from the group of systemic inflammatory response syndrome, sepsis, septic shock and multiple organ failure.

24. The pharmaceutical composition according to any one of the preceding claims 1 to 20, for use in treatment of a condition associated with presence of lipopolysaccharide (LPS), particularly endotoxemia.

25. The pharmaceutical composition according to any one of the preceding claims 1 to 20, for use in treatment of bacterial infection, particularly by gram-negative bacteria.

26. The pharmaceutical composition for use according to any one of the preceding claims 23 to 25, wherein the composition is administered prior to, together with, or subsequent to, the administration of an antibacterial drug or an antitoxin drug.

27. The pharmaceutical composition for use according to claim 26, wherein the antibacterial drug is selected from the group consisting of aminopenicillins, ureidopenicillins, cephalosporins, quinolone antibiotics, carbapenems, aminoglycoside antibiotics, and monobactams (aztreonam).

28. The pharmaceutical composition according to any one of the preceding claims 1 to 3, wherein the phospholipid component is sphingomyelin.

29. The pharmaceutical composition according to claim 28, wherein the phospholipid component is 16:1 sphingomyelin.

30. The pharmaceutical composition according to claim 29, wherein the phospholipid component is (9Z) hexadecenoic sphingomyelin.

31. The pharmaceutical composition according to claim 28, wherein the phospholipid component is 15:0 sphingomyelin.

32. The pharmaceutical composition according to any one of the preceding claims 28 to 31 , wherein the pharmaceutical composition is a liposomal formulation formulated for intravenous administration.

33. The pharmaceutical composition according to any one of the preceding claims 28 to 32 for use as a medicament.

34. The pharmaceutical composition according to any one of the preceding claims 28 to 32, for use in the treatment of an inflammatory condition.

35. The pharmaceutical composition for use according to claim 34, wherein the inflammatory condition is selected from the group of systemic inflammatory response syndrome, sepsis, septic shock and multiple organ failure.

36. The pharmaceutical composition according to any one of the preceding claims 28 to 32, for use in treatment of a condition associated with presence of lipopolysaccharide (LPS), particularly endotoxemia.

37. The pharmaceutical composition according to any one of the preceding claims 28 to 32, for use in treatment of bacterial infection, particularly by gram-negative bacteria.

38. The pharmaceutical composition for use according to any one of the preceding claims 37 to 35, wherein the composition is administered prior to, together with or subsequent to the administration of an antibacterial drug or an antitoxin drug.

39. The pharmaceutical composition for use according to claim 38, wherein the antibacterial drug is selected from the group consisting of aminopenicillins, ureidopenicillins, cephalosporins, quinolone antibiotics, carbapenems, aminoglycoside antibiotics, and monobactams (aztreonam).

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