Lymphatic-targeted anticoagulants for the prevention of lymphatic thrombosis

Abstract

Novel methods, compositions and kits for using lymphatic targeting anti-thrombosis (LTAT) molecules are provided herein. The LTAT molecules can be used to treat or prevent lymphatic thrombosis resulting particularly from infection or inflammation in the intestine.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 393,409, filed Jul. 29, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] License Information This invention was made with government support under Grant No. DK123528 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The subject matter of the present disclosure relates to antithrombotic molecules targeting intestinal lymph for treating or preventing intestinal lymph thrombosis. The subject matter of the present disclosure further provides methods, compositions, and kits for treating inflammation, coagulation, and cell aggregation that impair lymphatic function in the intestine.

Background Art

[0004] The lymphatic system is a vascular system that collects interstitial fluid and returns it to the bloodstream, transports immune cells, and transports fats absorbed from the intestine. Interstitial fluid is generated by leakage of fluid from the bloodstream into tissues and is then collected by the lymphatic system and returned to the bloodstream. Lymph fluid contains a large amount of molecular components of the coagulation cascade, and it has been previously known that lymph fluid can form fibrin clots in in vitro and ex vivo models of coagulation initiation. Lymph clots may be formed in association with infections involving lymphatic vessels, but such lymphatic thrombi are rare, and it is not clear whether they truly occur independently of blood and blood - derived cells.

[0005] The intestinal lymphatic network drains interstitial fluid and immune cells from the stomach, small intestine, and colon. Intestinal lymphatics also play a special role in the absorption of fat from the intestine via the transport of chylomicrons formed by the intestinal epithelium from dietary lipids. These chylomicrons are transported from the intestine to the lymphatic system together with lymphatic fluid and delivered to the bloodstream through lymphovenous junctions. Intestinal lymphatics play an important role in transporting intestinal immune cells that are constantly exposed to threats due to the intestine being constantly exposed to endogenous and exogenous bacteria and other threats. Intestinal immune monitoring may require patent lymphatics to transport and remove activated immune cells and regulate the duration and intensity of inflammation.

SUMMARY OF THE INVENTION

[0006] The objects and advantages of the disclosed subject matter are set forth in the description which follows, are apparent from the description, and are obtained by practice of the disclosed subject matter. Further advantages of the disclosed subject matter will be realized and attained by the techniques particularly pointed out in this specification and the claims thereof, as well as the appended drawings.

[0007] To achieve these and other advantages, and in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter includes lymphatic targeting antithrombotic (LTAT) molecules, as well as methods for using said molecules for treating inflammatory and thrombotic conditions that affect the intestinal lymphatic system. In addition to the treatment methods, the disclosed subject matter further provides pharmaceutical compositions and kits that include said molecules together with suitable pharmaceutical carriers.

[0008] In a first aspect, the present disclosure provides a lipid complex, the lipid complex including an active molecule for treating one or more lymphatic conditions, diseases or disorders; and a lipid or lipid-like molecule. In certain embodiments, the lipid complex further includes a linker region. In certain embodiments, the lipid complex further includes a lipid head group.

[0009] In certain embodiments, the treatment active molecule for one or more lymphatic states, diseases or disorders is an anticoagulant molecule. In certain embodiments, the anticoagulant molecule is an antithrombin, an anti-Xa molecule, or a thrombolytic agent. In certain embodiments, the anticoagulant molecule is an antithrombin, an anti-Xa molecule, or a thrombolytic agent. In certain embodiments, the anticoagulant molecule blocks thrombin or Xa enzyme activity.

[0010] In certain embodiments, the antithrombin molecule is hirudin, bivalirudin, ximelagatran, a derivative of dabigatran, or a derivative of a tripeptide-type thrombin inhibitor. In certain embodiments, the anti-Xa molecule is selected from the group consisting of edoxaban, rivaroxaban, apixaban, or combinations thereof.

[0011] In certain embodiments, the lipid comprises a long-chain fatty acid or a monoglyceride.

[0012] In certain embodiments, the lymphatic condition, disease or disorder active agent is selected from active agents that can treat a lymphatic condition, disease or disorder selected from the group consisting of sepsis, necrotizing enterocolitis, autoimmune disease, Crohn's disease, celiac disease, ulcerative colitis, rheumatoid arthritis, cardiovascular disease, bacterial infection, viral infection, viral hepatitis (including hepatitis C), alcoholic hepatitis, insulin resistance in adipocytes, pancreatitis, metabolic syndrome, trauma-induced inflammation, acute respiratory distress syndrome (ARDS), COVID-19-induced systemic inflammation, organ rejection after transplantation, amyloidosis, lymphangitis, obesity, primary or secondary lymphedema, congenital lymphatic insufficiency, lymphangiogenesis insufficiency, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), chronic granulomatous disease (CGD), malignant lymphoma (including, but not limited to, Hodgkin's disease, non-Hodgkin lymphoma and Castleman disease), Milroy disease, Meige disease, elephantiasis, lymphatic disorders secondary to tissue damage (e.g., infarction, surgical injury, organ or tissue transplantation, radiotherapy, chemotherapy and occlusion or blockage of lymphatic vessels (total or partial)), non-lymphatic malignancies, colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, sepsis, necrotizing enterocolitis, autoimmune disease, and Castleman disease), Milroy disease, Meige disease, elephantiasis, lymphatic disorders secondary to tissue damage, such as infarction, surgical injury, organ or tissue transplantation, radiotherapy, chemotherapy, and occlusion or blockage of lymphatic vessels (total or partial).

[0013] In certain embodiments, the present disclosure provides a method for treating intestinal lymphatic thrombosis, the method comprising administering to a subject in need of such treatment an effective amount of a lipid complex, wherein administering the effective amount of the lipid-based complex maintains hemostasis in the subject.

[0014] In certain embodiments, the lymphatic thrombosis is associated with infection or inflammation, at least in the intestine.

[0015] In certain embodiments, the lipid complex is selectively packaged with chylomicrons and transported from the intestine in lymphatic fluid. In certain embodiments, the lipid complex is not readily absorbed into the bloodstream.

[0016] In certain embodiments, the lipid complex is in the form of a tablet, capsule, sachet, suppository, liquid, oil, or a combination thereof.

[0017] In certain embodiments, the lipid complex is administered orally.

[0018] In certain embodiments, the lipid complex is free lipid within an oil solution, micelle, liposome, or solid lipid nanoparticle.

[0019] In certain embodiments, the present disclosure provides a method of preventing the formation of lymph clots using a lipid complex.

[0020] In certain embodiments, the present disclosure provides a method for reducing intestinal inflammation using a lipid complex.

[0021] In certain embodiments, the present disclosure provides a method of treating intestinal infections using a lipid complex.

[0022] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid complex.

[0023] In certain embodiments, the present disclosure provides a kit comprising a lipid complex.

[0024] In a first aspect, the present disclosure provides a method of treating lymphatic thrombosis, the method comprising administering to a subject in need of such treatment an effective amount of a lymph-targeted antithrombotic molecule (LTAT). In certain embodiments, the lymphatic thrombosis is associated with at least an infection or inflammation in the intestine.

[0025] In certain embodiments, unlike non-lipid-based antithrombotic molecules, LTAT molecules are selectively packaged with chylomicrons and transported from the intestine in the lymph rather than in the blood. In certain embodiments, LTAT molecules are not readily absorbed into the bloodstream compared to non-lipid-based antithrombotic molecules. In certain embodiments, LTAT molecules comprise a lipid or lipid-like molecule complexed to an active agent such as an anticoagulant.

[0026] In certain embodiments, LTAT molecules further comprise a linker region. In certain embodiments, LTAT molecules further comprise a lipid head group.

[0027] In certain embodiments, the anticoagulant molecule is an antithrombin, an anti-Xa molecule, or a thrombolytic agent. In certain embodiments, the anticoagulant molecule blocks thrombin or Xa enzyme activity.

[0028] In certain embodiments, the antithrombin molecule is hirudin, bivalirudin, ximelagatran, a derivative of dabigatran, or a derivative of a tripeptide-type thrombin inhibitor.

[0029] In certain embodiments, the anti-Xa molecule is selected from edoxaban, rivaroxaban, apixaban, or combinations thereof.

[0030] In certain embodiments, the lipid comprises a long-chain fatty acid or a monoglyceride.

[0031] In certain embodiments, LTAT is in the form of a tablet, capsule, sachet, suppository, liquid, oil, or combinations thereof.

[0032] In certain embodiments, LTAT molecules are administered orally. In certain embodiments, LTAT molecules are free lipids within an oil solution, micelle, liposome, or solid lipid nanoparticle.

[0033] In another aspect, the present disclosure provides a method for preventing, reducing, or treating the formation of lymph clots using the methods described herein. In certain embodiments, the present disclosure provides a method for reducing intestinal inflammation. In certain embodiments, the present disclosure provides a method for treating intestinal infections.

[0034] In another aspect, the present disclosure provides a pharmaceutical composition for carrying out the methods described herein.

[0035] In another aspect, the present disclosure provides a kit for carrying out the methods described herein.

[0036] The present disclosure further provides a bioconjugate. The bioconjugate includes an anticoagulant molecule and an active agent such as a lipid or lipid-like molecule. In certain embodiments, the bioconjugate further includes a linker region. In certain embodiments, the bioconjugate further includes a lipid head group. BRIEF DESCRIPTION OF THE DRAWINGS

[0037]

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[0038] The present disclosure relates to methods, compositions, and kits for preventing intestinal lymphatic thrombosis without interfering with hemostasis in a treated individual.

[0039] For the purpose of clarifying the present disclosure, but not by way of limitation, the detailed description of the subject matter of the present disclosure is divided into the following subsections: 5.1. Definitions; 5.2 Disorders; 5.3 Lipid Complexes 5.4. Methods of Use; 5.5. Compositions; and 5.6. Kit

[0040] 5.1. Definition The terms used in this specification generally have their ordinary meanings in the context of this disclosure and in the particular context in which each term is used. Specific terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners in describing the compositions and methods of this disclosure and their methods of making and using.

[0041] The use of the word "a" or "an" when used in combination with the term "comprising" in the claims and / or in this specification can mean "one", but is also consistent with the meanings of "one or more", "at least one" and "one or plural".

[0042] The terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)" and their variations used in this specification are intended to be open-ended transitional phrases, terms or words that do not exclude additional acts or structures. This disclosure contemplates other embodiments "comprising", "consisting of", and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly recited.

[0043] The terms "about" or "approximately" mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art and depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within or exceeding three standard deviations, according to the conventions of the relevant art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or even up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within one order of magnitude, within fivefold, or within twofold of a value.

[0044] The term "cell" as used herein refers to any cell suitable for use in the present disclosure, such as a eukaryotic cell. For example, without limitation, suitable eukaryotic cells include animal cells, such as mammalian cells. In certain embodiments, the suitable cell is a cultured cell. In certain aspects, the suitable cell is a host cell, a recombinant cell, and a recombinant host cell. In certain embodiments, the suitable cell is a cell line obtained from or derived from mammalian tissue that can grow and survive when placed in a medium containing suitable nutrients and / or growth factors.

[0045] As used herein, the terms "expression" or "expressing" refer to transcription and translation occurring within a cell, such as a mammalian cell. In certain embodiments, the expression level of a gene and / or nucleic acid in a cell can be determined based on either the amount of the corresponding mRNA present in the cell or the amount of the protein encoded by the gene and / or nucleic acid produced by the cell. For example, it is desirable that the mRNA transcribed from the gene and / or nucleic acid be quantified by Northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pages 7.3 - 7.57 (Cold Spring Harbor Laboratory Press, 1989). The protein encoded by the gene and / or nucleic acid can be quantified by assaying for the biological activity of the protein or by using an assay unrelated to such activity, such as Western blotting or radioimmunoassay using an antibody capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pages 18.1 - 18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0046] The term "thrombosis" as used herein refers to a thrombus or clot that forms within a lymphatic vessel and thereby impedes lymph flow. This term encompasses lymphatic vessels that are healthy but exposed to high thrombin levels due to intestinal bacteria and the immune cell environment, as well as lymphatic vessels in states of injury or disease, edema, fibrosis, immune disorders, malnutrition, and other conditions that can occur within the lymphatic system. Importantly, lymphatic thrombi are composed primarily of a fibrin meshwork and do not contain thrombus-promoting cells such as platelets and neutrophils that circulate in the blood but not in the lymph. This term is distinct from a "blood clot" that forms within a blood vessel lumen. Both are stimulated by thrombin proteolysis of fibrinogen to form cross-linked fibrin, but blood clots contain platelets and neutrophils that are generated by platelets and neutrophils and do not circulate in the lymph.

[0047] The term "disease, disorder, or condition" refers to a disease, disorder, or condition in which a patient is diagnosed with or suspected of having lymphatic thrombosis, particularly a disease, disorder, or condition associated with lymphatic thrombosis. Diseases, disorders, or conditions include, but are not limited to, pathogenic infections, inflammation-related conditions, side effects related to drugs or treatments, and idiopathic conditions characterized by symptoms including inflammation.

[0048] In some contexts, the term "lymphatic condition, disease or disorder" is intended to include all disorders characterized by insufficient or abnormal lymphatic function, including, but not limited to, viral or bacterial infections, wounds, cancer, amyloidosis, solitary cases, lymphangitis, obesity, primary or secondary lymphedema, congenital lymphatic insufficiency, lymphangiogenesis imperfecta, cardiovascular disease, heart disease, inflammatory bowel disease, ulcerative colitis, Crohn's disease, chronic granulomatous disease (CGD), malignant lymphoma (including, but not limited to, Crohn's disease, Hodgkin's disease, non-Hodgkin lymphoma, and Castleman disease), Milroy disease, Meige disease, elephantiasis, lymphatic disorders secondary to tissue damage, such as infarction, surgical injury, organ or tissue transplantation, radiation therapy, chemotherapy, and occlusion or blockage (total or partial) of lymphatic vessels.

[0049] In some contexts, the term "lymphatic endothelial cell" (also referred to as LEC) refers to the endothelial cells that line the inside of lymphatic vessels, which are distinct from, but related to, the endothelial cells that line the inside of blood vessels, referred to as vascular endothelial cells.

[0050] The term "effective amount" refers to the amount of an active ingredient that, when a compound is administered, is effective to alleviate or reduce to some extent one or more of the symptoms of a disease for which treatment is needed, or to delay the onset of a clinical marker or symptom of a disease for which prevention is needed. Thus, an effective amount refers to the amount of an active ingredient that exhibits an effect such as (i) reversing the rate of progression of a disease, (ii) suppressing to some extent further progression of a disease; and / or (iii) alleviating (or eliminating) one or more symptoms associated with a disease. An effective amount can be determined empirically by experimentation with the relevant compound in known in vivo and in vitro model systems of the disease for which treatment is needed. The context in which the phrase "effective amount" is used may indicate a particular desired effect. For example, "the amount of an LTAT molecule effective to prevent or treat lymphatic thrombosis" and similar phrases refer to the amount of an LTAT molecule that, when administered to a subject, causes a measurable improvement in the subject's state of lymphatic thrombosis. Unlike anticoagulation in the blood system, effective LTAT cannot be measured by laboratory evaluation of established coagulation parameters. This is because they are all based on blood and plasma. The effective amount can vary depending on the individual's body weight, gender, age and medical history, as well as the severity of one or more of the patient's conditions, the type of one or more diseases and the mode of administration. The effective amount can be readily determined using routine experimentation, for example, by titration (increasing the dose until an effective dose is found) and / or by reference to the amount that has been effective in previous patients.

[0051] A subject can be a human or a non-human animal, such as, but not limited to, a non-human primate, dog, cat, horse, rodent, cow, goat, rabbit, etc.

[0052] As used herein, the term "complex" refers to two or more components that are covalently bound, with at least one of the components being a biomolecule such as an enzyme, protein or antibody. For example, the complexes of the present disclosure can include a lipid or lipid-like molecule covalently bound to an anticoagulant molecule.

[0053] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with a therapeutic agent. Such physiological carriers are sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a suitable carrier. Physiological saline as well as aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. The composition can also contain, if necessary, small amounts of wetting or emulsifying agents, or pH buffering agents.

[0054] As used herein, the term "agent" means a substance that produces or can produce an effect, and includes, but is not limited to, chemical substances, pharmaceuticals, biological agents, organic small molecules, antibodies, nucleic acids, peptides and proteins.

[0055] As used herein, the term "inhibitor" refers to a compound or molecule (e.g., small molecule, peptide, peptidomimetic, natural compound, siRNA, antisense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, prevents, decreases, suppresses, eliminates, or blocks) the signaling function of a protein or pathway. An inhibitor can be a compound or molecule that alters the activity of a protein (a signaling molecule, a molecule involved in a specified signaling molecule, or a specified related molecule), e.g., RNF167, or a compound or molecule that interferes with the interaction of a protein, e.g., RNF167, with a signaling partner. Inhibitors also include molecules that indirectly modulate the biological activity of a specified protein, e.g., RNF167, by interfering with upstream signaling molecules.

[0056] 5.2 Disorder In certain embodiments, the lipid complexes, methods, compositions, and kits provided herein can be used to treat any condition or disease / disorder (a "disorder") associated with intestinal lymphatic thrombosis, including, but not limited to, infections, wounds, cancer, and inflammation, including, but not limited to, insufficient or abnormal lymphatic function.

[0057] In certain embodiments, lymphatic diseases include, but are not limited to, sepsis, necrotizing enteritis, autoimmune diseases, Crohn's disease, celiac disease, ulcerative colitis, rheumatoid arthritis, cardiovascular diseases, bacterial infections, viral infections, viral hepatitis (including hepatitis C virus hepatitis), alcoholic hepatitis, insulin resistance of adipocytes, pancreatitis, metabolic syndrome, trauma-induced inflammation, acute respiratory distress syndrome (ARDS), COVID-19-induced systemic inflammation, organ rejection after transplantation, amyloidosis, lymphangitis, obesity, primary or secondary lymphedema, congenital lymphatic insufficiency, lymphangiogenesis insufficiency, inflammatory bowel disease, chronic granulomatous disease (CGD), malignant lymphomas (including, but not limited to, Hodgkin's disease, non-Hodgkin lymphoma, and Castleman disease), Milroy disease, Meige disease, elephantiasis, lymphatic disorders secondary to tissue damage, such as infarction, surgical injury, organ or tissue transplantation, radiation therapy, chemotherapy, and occlusion or blockage (total or partial) of lymphatic vessels, and can be related to conditions causing enteritis. In certain embodiments, lymphatic conditions can also include non-lymphatic malignancies affecting the lymphatic system, including, but not limited to, colorectal cancer, liver cancer, gastric cancer, pancreatic cancer. Insufficient or abnormal lymphatic function can result from, for example, deficiencies or lack of any component of the lymphatic system, including valves, capillaries, ducts, etc. Modulation of the repair or growth of these and other lymphatic system components is contemplated using the methods described herein.

[0058] 5.3 Lipid Complexes In certain embodiments, the disclosure includes lipid complexes. In certain embodiments, the disclosure includes lipids or lipid-like components conjugated to an active agent such as an anticoagulant molecule. In certain non-limiting embodiments, the anticoagulant molecule is an antithrombotic molecule. In certain embodiments, the lipid antithrombotic molecule is a lymphatic-targeted antithrombotic (LTAT) molecule. In certain embodiments, the lipid anticoagulant molecule targets the intestinal lymphatic system. Lipid anticoagulants can be delivered as free lipids in an oil solution or as micelles, liposomes, or solid lipid nanoparticles to enhance the absorption and availability of the anticoagulant in the lymphatic system. The conjugation of the lipid or lipid-like component to the anticoagulant molecule prevents separation of the lipid and anticoagulant components upon administration and ensures their integrity and effectiveness for targeted delivery in the lymphatic system. Lipid anticoagulant molecules can be used for targeted anticoagulation treatment focused on the lymphatic system.

[0059] Non-limiting examples of lipids or lipid-based molecules include dipalmitoyl phosphatidylcholine (DPPC), cholesterol, distearoyl phosphatidylethanolamine (DSPE), phosphatidylcholine (PC), and phosphatidylethanolamine (PE).

[0060] In certain embodiments, the lipid includes long-chain fatty acids or monoglycerides. Non-limiting examples of long-chain fatty acids include palmitic acid (C16:0); stearic acid (C18:0); oleic acid (C18:1), linoleic acid (C18:2), α-linolenic acid (C18:3). Non-limiting examples of monoglycerides include monoolein, monostearin, monoglyceride, and monopalmitin.

[0061] In certain non-limiting embodiments, the anticoagulant is not orally available in the bloodstream when part of a lipid structure or alone.

[0062] In certain non-limiting embodiments, the anticoagulant is resistant to proteases of the digestive system.

[0063] In certain non-limiting embodiments, the anticoagulant molecule is an antithrombin, an anti-Xa molecule, or a thrombolytic agent. The anticoagulant molecule can block thrombin or Xa enzyme activity.

[0064] Non-limiting examples of antithrombin molecules include hirudin, bivalirudin, ximelagatran, derivatives of dabigatran, and derivatives of tripeptide-type thrombin inhibitors.

[0065] Non-limiting examples of anti-Xa molecules include edoxaban, rivaroxaban, apixaban, DX-9065a, and YM-60828.

[0066] In certain non-limiting embodiments, the lipid or lipid-like molecule is complexed to the anticoagulant molecule by reacting the lipid or lipid-like molecule with the anticoagulant molecule via click chemistry, an esterification reaction, an amidation reaction, or another conjugation reaction.

[0067] In certain non-limiting embodiments, the anticoagulant is covalently bound to the lipid head group, and the point of attachment is sufficiently far from the enzyme interface so as not to reduce the effectiveness of inhibition. Non-limiting examples of covalent bonds are terminal sulfhydryl, acid, hydroxyl, ester, aldehyde, and amine, etc.

[0068] In certain non-limiting embodiments, the lipid or lipid-based molecule contains a reactive moiety. In certain non-limiting embodiments, the anticoagulant molecule contains a reactive moiety. In certain non-limiting embodiments, the reactive moiety is a ligation moiety such as, but not limited to, trans-cyclooctene, tetrazine, cyclooctyne, alkyne or azide, alkene, tetrazole, photo-DIBO, or cyclopropenone.

[0069] In certain non-limiting embodiments, the conjugation of a lipid or lipid-like molecule with an anticoagulant molecule can be prepared by different reaction schemes known in the art. Examples of suitable reactions include, but are not limited to, the Diels-Alder reaction, azide-alkyne click reactions (e.g., Cu(I)-catalyzed azide-alkyne cycloaddition and metal-free azide-alkyne cycloaddition), Staudinger ligation, thiol-maleimide addition, oxime ligation, and thiol-ene reactions.

[0070] For purposes of illustration and not limitation, FIGS. 8A-9B schematically depict a process for making a lipid complex according to the subject matter of the present disclosure. The lipid formulation serves as a vehicle for the conjugation of the anticoagulant molecule and an anticoagulant-lipid nanoparticle (NP) is formed.

[0071] The lipid complexes of the subject matter of the present disclosure can further include one or more additional components. For example, in certain embodiments, a spacer can be placed between the various components of the lipid complex. Further, the spacer, if present, can be used to strengthen the bonds between the components of the lipid complex and to control the relative positions of the lipids or lipid-like molecules with the anticoagulant. In certain embodiments, the spacer can include a polymer or a biomolecule. In certain non-limiting examples, the polymer can be polyethylene glycol, polyethylene, polyethylene glycol, dendrimer, polyacrylic acid, hydroxyethyl starch (HES), polylactide-co-glycolide, poly-D,L-p-dioxanone polylactic acid-ethylene glycol block copolymer (PLA-DX-PEG), poly(ortho)esters, poly-glutamate, poly-aspartate, a-B-unsaturated monomers, such as polymers of (meth)acrylic acid, crotonic acid, maleic acid, maleic anhydride, fumaric acid, itaconic acid / anhydride, vinyl ethers, vinyl esters, vinyl amine amides, olefins, and / or diallyldialkylammonium halides, preferably vinyl ethers, poly(diethyl glycol adipate), polyethyleneimine, polyglycolide, polyureas, polylimonene (=polylimo(Polylimo)), poly(2-methyl-1,3-propylene adipate), and graft polymers and graft polymers, such as graft (block) polymers with other polymers.

[0072] For example, polyethylene glycol (PEG)n can be used as a spacer, but is not limited thereto.

[0073] In certain non-limiting embodiments, the size and use of the spacer can be selected to control the overall size of the lipid complex. Thus, the size and use of the spacer can be based, at least in part, on the size of the bioconjugate. In certain embodiments, the overall size of the lipid complex can be controlled to induce enhanced absorption and availability of the drug in the lymphatic system.

[0074] 5.4 Treatment method In certain embodiments, the present disclosure provides a method of treating an infectious, inflammatory, or autoimmune disorder affecting the intestinal lymphatic system, comprising administering to a subject in need of such treatment an effective amount of an LTAT molecule that prevents, reduces, or inhibits one or more signs or symptoms of such a disorder, without significantly altering the subject's blood clotting dynamics.

[0075] In certain embodiments, the present disclosure provides a method for treating or preventing lymphatic thrombosis associated with a viral or bacterial infection in a subject, comprising administering to a subject in need of such treatment an effective amount of an LTAT molecule to treat clotting in the subject's lymphatic system. In certain embodiments, the infection causes inflammation that leads to the formation of lymphatic clots. In certain embodiments, the present disclosure provides a method for treating and / or preventing lymphatic thrombosis in a subject.

[0076] In certain embodiments, the present disclosure provides a method for treating lymphatic thrombosis associated with an inflammatory disease in a subject, comprising administering to a subject in need of such treatment an effective amount of an LTAT molecule to treat clotting in the subject's lymphatic system. In certain embodiments, the inflammatory condition is inflammatory bowel disease, including but not limited to ulcerative colitis and Crohn's disease. In certain embodiments, the present disclosure provides a method for treating and / or preventing intestinal inflammation caused by lymphatic thrombosis.

[0077] In certain embodiments, the present disclosure provides methods for treating or preventing one or more lymphatic clotting associated with a viral or bacterial infection. In certain embodiments, the present disclosure provides methods for treating or preventing lymphatic clotting associated with an inflammatory condition, including, but not limited to, inflammatory bowel disease, such as ulcerative colitis and Crohn's disease.

[0078] 5.5 Pharmaceutical Compositions In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a lipid complex and a pharmaceutically acceptable carrier. Suitable carriers that can be used with the subject matter of the present disclosure have the characteristic of not interfering with the effectiveness of the active ingredient, such as the disclosed inhibitor / anticancer agent, and not being toxic to the patient. Non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, and sterile solutions. Further non-limiting examples of pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, implantable elements containing inhibitors and / or any other suitable vehicle, delivery or dispensing means or materials. Such pharmaceutically acceptable carriers can be formulated by conventional methods and administered to a subject. In certain embodiments, pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., but not limited to octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).In certain embodiments, suitable pharmaceutically acceptable carriers can include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof.

[0079] In certain non-limiting embodiments, the pharmaceutical compositions of the present disclosure can be formulated using pharmaceutically acceptable carriers well known in the art suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, slurries, suspensions, etc. for oral or nasal ingestion by the patient being treated. In certain embodiments, the pharmaceutical composition is formulated as a capsule. In certain embodiments, the pharmaceutical composition can be in a solid dosage form. In certain embodiments, the tablet can be an immediate release tablet. Alternatively or additionally, the tablet can be a sustained release or controlled release tablet. In certain embodiments, the solid dosage can include both an immediate release portion and a sustained release or controlled release portion.

[0080] The dosing regimen of the compounds of the invention will vary according to known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health status, medical condition, and weight of the recipient; the nature and degree of the symptoms; the type of concomitant treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the desired effect. A physician or veterinarian can determine and prescribe the effective amount of the drug required to prevent, counteract, or arrest the progression of a medical thromboembolic disorder.

[0081] Dosage forms (pharmaceutical compositions) suitable for administration can contain from about 1 milligram to about 1000 milligrams of the active ingredient per dosage unit. In these pharmaceutical compositions, the active ingredient is usually present in an amount of about 0.1 to 95% by weight based on the total weight of the composition.

[0082] As a general guideline, the daily oral dosage of each active ingredient, when used for the indicated effects, is in the range of about 0.001 to about 1000 mg / kg body weight. The compounds of the present invention may be administered in a once-daily dose or the total daily dose may be administered in divided doses of 2, 3, or 4 times a day.

[0083] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an LTAT molecule described herein in a suitable pharmaceutical carrier. The amount of the LTAT molecule present in the composition can be calculated to provide an effective amount of the LTAT molecule when administered to a subject in need of such treatment.

[0084] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of an LTAT molecule, for example, but not limited to, in combination with a pharmaceutical carrier such as water or other physiological solvents. The therapeutically effective amount prevents, reduces or inhibits the formation of lymph clots.

[0085] In certain non-limiting embodiments, the LTAT molecule can be included in an oil solution, micelle, liposome, or similar structure.

[0086] In certain embodiments, the pharmaceutical composition can be a liquid containing the LTAT molecule in a liquid pharmaceutical carrier, for example, water (aqueous carrier) or physiological saline. In certain embodiments, the liquid composition can optionally further contain one or more buffers or preservatives.

[0087] In certain other embodiments, the pharmaceutical composition of the present disclosure can be a solid, for example, in the form of a tablet, capsule, sachet or suppository, and contains an amount of the LTAT molecule that provides an effective amount of the LTAT molecule to a subject in need of such treatment when administered according to a dosing schedule. In certain embodiments, the solid pharmaceutical composition can further contain one or more excipients, for example, but not limited to, lactose, sucrose, mannitol, erythritol, carboxymethylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, starch, polyvinylpyrrolidone, etc.

[0088] In certain embodiments, the pharmaceutical composition can include additional agents having antibacterial and / or anti-inflammatory activity. In certain embodiments, such compounds include, but are not limited to, antibiotics, steroids or non-steroidal anti-inflammatory drugs. In certain other embodiments, the pharmaceutical composition can include an analgesic. In certain further embodiments, the pharmaceutical composition can include an agent that lyses existing blood clots, such as tissue plasminogen activator (tPA) or activated plasminogen.

[0089] Pharmaceutically acceptable salts are recognized in the art and include relatively non-toxic inorganic and organic acid addition salts of the compositions of the subject matter of the present disclosure, including, but not limited to, therapeutic agents, excipients, other materials, etc. Examples of pharmaceutically acceptable salts include salts derived from mineral acids such as hydrochloric acid and sulfuric acid, and salts derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Examples of inorganic bases suitable for salt formation include hydroxides, carbonates and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, etc. The salts may also be formed with suitable organic bases, including organic bases that are non-toxic and strong enough to form such salts. By way of illustration, classes of such organic bases may include mono-, di- and trialkylamines such as methylamine, dimethylamine and triethylamine; mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids such as arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; (trishydroxymethyl)aminoethane; etc.; see, for example, J. Pharm. Sci., 66:1-19 (1977).

[0090] 5.6 Kit The subject matter of the present disclosure further provides kits that include materials useful for practicing the methods and compositions disclosed herein. For example, without limitation, any combination of materials useful in the present disclosure can be packaged together as a kit for practicing either the methods or the compositions of the present disclosure.

[0091] In certain embodiments, the kits of the present disclosure can include LTAT molecules together with a suitable pharmaceutical carrier. In certain embodiments, the kit components can be packaged in a single-use form suitable for a single administration.

[0092] In certain embodiments, the kit further includes an accompanying document that describes instructions for use of the components provided in the kit. For example, the kits of the present disclosure can include an accompanying document that describes instructions for using the LTAT molecules provided in the kit.

[0093] Alternatively or additionally, the kit can include other materials that are desirable from a commercial and user perspective, including other buffers, diluents, and filters. In certain embodiments, the kit can include materials for preparing the LTAT molecules.

[0094] The kits can provide pre-measured amounts of reagents to simplify the practice of the methods of the invention and the administration of the compositions. Optionally, the kits of the present disclosure include instructions for practicing the method or administering the composition. Any other elements of the kits of the present disclosure include suitable buffers, reagents, packaging materials, and the like. The kits of the present disclosure can further include additional reagents necessary for practicing the disclosed methods and compositions. The reagents of the kit can be in containers in which they are stable, for example, in lyophilized form or a stabilized liquid.

Examples

[0095] The subject matter of the present disclosure will be better understood, without limitation, by reference to the following examples provided as illustrations of the subject matter of the present disclosure.

[0096] Results Antithrombotic properties of lymphatic endothelium. The present disclosure shows that lymphatic endothelium lining the inside of lymphatic vessels expresses a transcription factor family including FOXC2, PROX1, and GATA2, and these transcription factors play important roles in the development of the lymphatic system, particularly lymphatic valves, and are also associated with a strong antithrombotic phenotype. The antithrombotic phenotype is also observed in venous valve endothelium, indicated by the expression of FOXC2 and PROX1, and plays a role in preventing thrombus formation within the pockets of venous valves. The antithrombotic phenotype included downregulation of several thrombus-promoting and inflammation-promoting endothelial surface proteins (including von Willebrand factor and P-selectin), as well as upregulation of the antithrombotic endothelial surface proteins thrombomodulin (THBD) and endothelial protein C receptor (EPCR) (Figures 1A and 1B). Lymphatic endothelium, particularly the intestinal lymphatic network, shared this antithrombotic expression pattern characterized by high THBD and EPCR expression. Lymphatic vessel endothelium showed a synergistic effect, resulting in a decrease in thrombus-promoting proteins and an increase in antithrombotic proteins, which led to an additive response and a strong inhibition of clot formation.

[0097] The PAR1-Tango mouse model reports thrombin activity in vivo. The present disclosure provides a mouse model with the PAR1-Tango allele as a reporter of thrombin expression under normal and healthy conditions (Figures 2A and 2B). Mice expressing the PAR1-Tango allele showed strong thrombin activity in intestinal lymph (Figure 3B). On the other hand, when the PAR1 receptor was not exposed to thrombin, lack of reporter activity was observed in the lung, heart, skin, and liver (Figure 3A). The present disclosure shows that the intestinal lymphatic system environment is naturally procoagulant.

[0098] Lymph clot formation in response to gastrointestinal infection. This disclosure demonstrated that the formation of fibrin clots in lymph is an important part of the natural immune response of the intestine that serves to prevent the spread of pathogenic bacteria introduced into the intestine of mice (Figures 4A - 4E). As shown in Figures 4A and 4B, PAR1 - Tango activity occurs in coordination with the gut microbiota. Lymphatic vessels (LYVE1 - positive) were found to have negative PAR1 - Tango expression at P0 and P7, but showed expression at P14 (GFP - positive cells) and even stronger expression at P21. This disclosure demonstrated the presence of PAR1 - Tango - positive cells in the neonatal intestine.

[0099] When clots form within intestinal lymphatic vessels, the flow of body fluids, immune cells, and bacteria is blocked, preventing the spread of infection, increasing the time for activated immune cells to respond to invading bacteria, and preventing the spread of pathogens from the intestine to other parts of the body (i.e., sepsis). Similarly, in viral infections, the formation of intestinal lymph clots occurs, where it serves to prevent the spread of pathogens and concentrate locally activated immune cells in that area to eliminate the pathogens. In both cases, the coagulation of lymph fluid is beneficial to health. This disclosure shows the inhibitory effect of neonatal antibiotic ("ABX") treatment on PAR1 - Tango reporter activity and confirms that thrombin generation is associated with intestinal bacterial colonization. This disclosure established the relationship between bacteria in the intestinal lumen and thrombin activity in the intestinal lymphatic vessel lumen. This disclosure further demonstrated that bacterial infection generated lymph clots. As shown in Figures 5A - 5D, mature mice exposed to Salmonella or Shigella by oral gavage form fibrin clots in LYVE1 - positive lymphatic vessels. This disclosure shows that approximately 20% of intestinal lymphatic vessels had thrombosis after Salmonella infection (Figure 5C). This was further demonstrated in mice gavaged with GFP - expressing Salmonella bacteria, where GFP - expressing Salmonella bacteria were found at the sites of fibrin clot formation in intestinal lymphatic vessels (Figure 5D).

[0100] Inflammation of the intestine results in the formation of clots in intestinal lymph. The present disclosure has found that inappropriate induction of lymphatic clots can occur in the context of inflammation caused by non - pathogenic causes and in autoimmune or inflammatory diseases. In these cases, activated immune cells, which are known to be thrombotic, may activate intraluminal thrombin activity within lymphatic vessels, thereby inducing fibrin clot formation. Both a murine model of enteritis (induced by diet ingestion of DSS) and human samples from patients with chronic inflammatory bowel disease show significant amounts of lymphatic clot formation compared to healthy controls (Figures 6A - 6E). In these examples, the clots do not prevent the spread of pathogens, but rather prevent the clearance of activated immune cells, thereby exacerbating inflammation and contributing to tissue damage and symptoms. Clinical data from patients with chronic intestinal inflammation indicate that they can develop nutritional deficiencies consistent with dysregulation of dietary lipid uptake and consistent with disruption of intestinal lymphatic flow.

[0101] Natural anticoagulant molecules expressed by intestinal lymphatic vessels prevent the coagulation of intestinal lymph. The present disclosure has demonstrated in mice that genetic deletion of lymphatic endothelial THBD results in extensive intestinal lymphatic coagulation and disruption of intestinal lymphatic flow. Oral administration of the anticoagulant warfarin prevented lymphatic coagulation in this context (Figures 7A - 7C). This demonstrates that antithrombotic drugs can prevent lymphatic clot formation and result in the restoration of lymphatic function. However, administration of existing antithrombotic drugs results in systemic anticoagulation and establishes a significant risk of bleeding throughout the body, including the intestine and brain. The risk of systemic anticoagulation, i.e., anticoagulation that affects both the blood and the lymphatic system, is significantly higher in patients with chronic inflammation, especially due to the high clinical bleeding rate in the intestine. Therefore, there is clearly a need for functional antithrombotic drugs that can be delivered to intestinal lymphatic vessels at therapeutic doses and act preferentially. Furthermore, the amount of lymphatic fluid in the intestine is much less than the amount of blood in the body. This means that a therapeutic dose of a drug administered directly to intestinal lymphatic vessels is highly diluted to levels far below the therapeutic level when it reaches the bloodstream, thereby preventing systemic anticoagulation and inappropriate bleeding.

[0102] A lipid complex for targeting antithrombotic drugs to the intestinal lymphatics. The present disclosure demonstrated that lipid complex molecules having flexible linkers of various lengths can bind to orally active antithrombin molecules for the purpose of specific lymph-targeted antithrombotic drug delivery. Binding of the antithrombotic drug to the lipid head groups generates various LTAT molecules (FIGS. 8A-8D). Such molecules can be formulated as isolated lipids, as micelles, or as lipid nanoparticles including liposomes of solid lipid nanoparticles. Such molecules are used in clinical cases of small intestine inflammation (such as Crohn's disease) to reduce the intensity and duration of intestinal inflammation and also in viral or bacterial infections where lymph fluid clotting may prevent access of the drug to cells within the clot. The molecules can be orally delivered in oil capsules, specifically taken up into the intestinal lymph as chylomicrons, and the antithrombotic drug is exposed to the luminal lymph fluid environment where it can block the thrombin or factor Xa enzyme activity required for thrombus formation. LTAT molecules can be delivered as free lipids in oil solution, as micelles, or as liposomes to enhance drug absorption and availability in the lymphatic system (FIGS. 8A and 8B). LTAT molecules can be the only lipid used to form micelles or liposomes or can be the major component of micelles or liposomes. The unabsorbed drug can pass through the intestine more readily than non-lipid-based small molecules and is not readily absorbed into the bloodstream, thus reducing the possibility of the antithrombotic drug reaching a blood level high enough to cause bleeding.

[0103] The process of preparing LTAT molecules is shown in FIGS. 9A and 9B. First, base liposomes were prepared using a 2% molar concentration of DSPE-PEG-azide formulation. With this formulation, azide groups were located on the outer surface of the liposomes, facilitating the attachment of anticoagulant molecules. After liposome formation, PPACK-dibenzocyclooctyne, a very potent and selective irreversible inhibitor of thrombin, was attached to the outer surface of the liposomes. The resulting PPACK nanoparticles (NPs) retained antithrombotic activity, as demonstrated by the chromozym TH assay. PPACK NPs showed an IC50 value of approximately 0.8 nM, while stand-alone PPACK had an IC50 value of approximately 0.0002 nM. Thus, liposomes were proven to be very effective in loading PPACK, and the number of PPACK molecules per liposome was estimated to be approximately 400 (FIG. 9C). To mimic the effect of oral administration and examine changes in nanoparticle size, PPACK NPs were incorporated into mouse-derived chylomicrons (FIGS. 9D and 9E).

[0104] As shown in FIGS. 10A and 10B, oral administration of PPACK NPs resulted in thrombin inhibition in chylomicrons. Thrombin activity was evaluated for chylomicrons diluted 1:33 with 1 U / mL thrombin. When diluted chylomicrons from mice treated with oral PPACK NPs were tested, the thrombin inhibition IC50 at 1 U / mL was shown to be 0.4 μL of chylomicrons (FIG. 10C), while for pre-injected PPACK NPs the IC50 was 0.03 μL (FIG. 10D). The data indicate that each μL of chylomicrons was found to contain an amount equivalent to approximately 0.075 μL (0.03 / 0.4) of PPACK NPs at their original concentration. An initial dose containing 250 μL of PPACK NPs (concentrated to 50 μL and added to 400 μL of olive oil) was force-fed as 1 μL containing an amount equivalent to 0.56 μL of chylomicrons (250 / 450). This corresponded to 13.4% of the activity of the force-fed dose in the recovered chylomicrons.

[0105] Fluorescent nanoparticle tracking analysis (F-NTA) was used to evaluate the transport from oral gavage of fluorescent lipids formulated in PPACK NPs to chylomicrons (Figure 11A). In chylomicron samples from mice treated with oral PPACK NPs, F-NTA determined the size and concentration of nanoparticles containing fluorescent lipids derived from PPACK NPs. In this example, it has been shown that the size of the nanoparticles in chylomicrons containing fluorescent lipids did not match the size of the pre-injected nanoparticles, demonstrating that the lipids from PPACK NPs were incorporated into chylomicrons / endogenous vesicles rather than being transported as part of intact nanoparticles. The results show a shift in the size of nanoscale lipid vesicles / aggregates found in chylomicrons from mice treated with oral PPACK NPs compared to sham-treated mice, as shown in Figures 11B - 11D.

[0106] To evaluate the distribution of PPACK NPs in vivo, the radioactive tracer (111)In was incorporated into the NPs (as shown in Figure 12). The movement of the radioactive tracer along the gastrointestinal tract of mice was tracked for 24 hours after administration. The results revealed that the tracer molecules accumulated in various parts of the gastrointestinal tract, including the stomach, duodenum, jejunum, ileum, cecum, colon, and the mesentery of the pancreas. However, after 24 hours (Figures 13A and 13C), the tracer had been removed from these tissues. The tracer was found to penetrate into multiple organs such as the heart, lungs, liver, spleen, kidneys, and brain at much lower concentrations than in the tissues of the gastrointestinal tract. The levels of the tracer in all tissues gradually decreased and returned to normal within 24 hours (Figure 13B). Only trace amounts of the tracer were detected in the blood, and urine analysis revealed its excretion at the 24-hour time point, but the detection of the tracer persisted in chylomicrons.

[0107] Thrombin in plasma was found not to be inhibited by the treatment with oral gavage of PPACK NPs. Figure 14A shows thrombin inhibition (0.0085 μL IC50) by PPACK-NP before gavage. Figure 14B demonstrates that thrombin was inhibited in the chyle of mice treated with oral gavage of PPACK NPs, but not in plasma. Thrombin inhibition in chyle increased over a noisy time course from 0 to 4 hours after gavage, and a significant inhibitory effect was demonstrated 24 hours after gavage (Figure 14C).

[0108] Blood tests were performed at various time points within 24 hours after gavage of PPACK NPs. Analysis of the total white blood cell count, including white blood cells (WBC), lymphocytes (LYM), monocytes (MON), and neutrophils (NEU), showed mild lymphopenia at the early time points after gavage (Figures 15A and 15B). The blood tests further revealed an increase in red blood cell size (Figures 15C and 15D) and a tendency for a decrease in platelet count (Figures 15E and 15F) 24 hours after gavage of PPACK NPs.

[0109] To demonstrate that various antithrombotic drugs can be used in LTAT, a method was devised to modify apixaban, a factor Xa inhibitor, for conjugation to lipids (Figure 16). The modified apixaban was shown to inhibit factor Xa activity similar to unmodified apixaban (Figure 17).

[0110] Discussion An approach for local anticoagulant effects has been demonstrated by formulating anticoagulant molecules on lipids for oral delivery. The anticoagulant molecules bound to lipids can be specifically delivered to lymph and maintain their activity there. At the same time, the anticoagulant molecules bound to lipids are constructed to be removed from unwanted tissues and the bloodstream. The anticoagulant molecules first delivered as liposomes are degraded after administration, and the lipids are redistributed among the modified chylomicrons. The anticoagulant molecules can be formulated as free lipids in liposomes, micelles or oil emulsions. This example demonstrated the conjugation and formulation process and functionality of PPACK complex lipids formulated in liposomes, but formulations containing anticoagulant molecules with higher specificity for thrombin or factor Xa have been developed. Anticoagulant molecules with low oral availability and short half-lives in the bloodstream when not bound to lipids may offer more specific lymph targeting effects. Preferred molecules include those with free acids or free amines that allow conjugation of the non-modified molecule to the lipid head group by covalent bonds to prevent dissociation.

[0111] Method The PPACK liposomes were prepared by: 1) synthesizing liposomes having a bioorthogonal conjugation handle (in this case an azide-terminated lipid); and 2) conjugating PPACK to a linker molecule using a bioorthogonal conjugation handle compatible with the liposomes. In this case, PPACK was combined with dibenzocyclooctyne (DBCO) having an N-hydroxysuccinimide-terminated poly(ethylene) glycol (PEG) linker. N-Hydroxysuccinimide reacted with the primary amine terminus of PPACK to form PPACK-DBCO. The liposomes were prepared using a formulation that has previously been demonstrated to form very stable liposomes using dipalmitoylphosphatidylcholine (DPPC) and cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) having an azide-terminated PEG linker. The liposomes having azide groups were reacted with PPACK-DBCO overnight, and PPACK-DBCO that did not bind to the liposomes was removed from the formulation by centrifugal filtration or size-exclusion chromatography to confirm that the formulation did not contain PPACK that did not bind to the lipids.

[0112] To modify apixaban for conjugation to lipids, a derivative of apixaban having a carboxylic acid group was obtained, and a PEG-(bis)amine homobifunctional linker was attached to the apixaban derivative by carbodiimide coupling with one amine terminus protected by a fluorenylmethyloxycarbonyl (FMOC) group. After deprotection with trifluoroacetic acid, the modified apixaban was purified by high performance liquid chromatography using a reverse-phase column, and the final molecular structure was verified by mass spectrometry and nuclear magnetic resonance.

[0113] The inhibition of thrombin by LTAT was evaluated by a chromogenic substrate assay in either chylous or blood samples from in vitro or LTAT-treated mice. In the chromogenic substrate assay, a chromogenic substrate (e.g., chromozym TH) is cleaved by thrombin or factor Xa, generating an absorbance signature in which the p-nitroaniline group is released and quenched by a tosyl group prior to cleavage. In all chromogenic substrate assays described, a fixed amount of substrate and active enzyme (either thrombin or factor Xa) was added to the assay with different concentrations of inhibitor (either unbound molecule or LTAT), or with samples from mice administered LTAT containing an unknown concentration of inhibitor. The absorbance signature indicates the extent of substrate cleavage by the set concentration of enzyme, and a decrease in the absorbance signature indicates the amount of inhibitor added to the assay.

[0114] Although the subject matter of the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the present disclosure. Further, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufactures, and compositions of matter, methods, and processes described herein. One of ordinary skill in the art will readily appreciate from the present disclosure that processes, machines, manufactures, compositions of matter, or methods currently existing or later developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the present disclosure. Accordingly, the appended claims are intended to include such processes, machines, manufactures, compositions of matter, or methods within their scope.

Claims

**Claim 1** A lipid complex comprising: a. One or more treatment active molecules for lymphatic states, diseases or disorders; and b. A lipid or lipid-like molecule The lipid complex. **Claim 2** The lipid complex according to claim 1, further comprising a linker region. **Claim 3** The lipid complex according to claim 1, further comprising a lipid head group. **Claim 4** The lipid complex according to claim 1, wherein the one or more treatment active molecules for lymphatic states, diseases or disorders are anticoagulant molecules. **Claim 5** The lipid complex according to claim 4, wherein the anticoagulant molecule is an antithrombin, an anti-Xa molecule, or a thrombolytic agent. **Claim 6** The lipid complex according to claim 4, wherein the anticoagulant molecule blocks thrombin or Xa enzyme activity. **Claim 7** The lipid complex according to claim 1, wherein the antithrombin molecule is hirudin, bivalirudin, ximelagatran, a derivative of dabigatran, or a derivative of a tripeptide-type thrombin inhibitor. **Claim 8** The lipid complex according to claim 1, wherein the anti-Xa molecule is selected from the group consisting of edoxaban, rivaroxaban, apixaban, or a combination thereof. **Claim 9** The lipid complex according to claim 1, wherein the lipid comprises a long-chain fatty acid or a monoglyceride. **Claim 10** The active molecule for the lymphatic state, disease or disorder is sepsis, necrotizing enterocolitis, autoimmune disease, Crohn's disease, celiac disease, ulcerative colitis, rheumatoid arthritis, cardiovascular disease, bacterial infection, viral infection, viral hepatitis (including hepatitis C), alcoholic hepatitis, insulin resistance in adipocytes, pancreatitis, metabolic syndrome, trauma-induced inflammation, acute respiratory distress syndrome (ARDS), COVID-19-induced systemic inflammation, organ rejection after transplantation, amyloidosis, lymphangitis, obesity, primary or secondary lymphedema, congenital lymphatic insufficiency, lymphangiogenesis insufficiency, inflammatory bowel disease, chronic granulomatous disease (CGD), malignant lymphoma (including, but not limited to, Hodgkin's disease, non-Hodgkin lymphoma and Castleman disease), Milroy disease, Meige disease, elephantiasis, lymphatic disorders secondary to tissue damage (e.g., infarction, surgical injury, organ or tissue transplantation, radiotherapy, chemotherapy and occlusion or blockage (total or partial) of lymphatic vessels), non-lymphatic malignancies, colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, sepsis, necrotizing enterocolitis, autoimmune disease, and Castleman disease), Milroy disease, Meige disease, elephantiasis, lymphatic disorders secondary to tissue damage (e.g., infarction, surgical injury, organ or tissue transplantation, radiotherapy, chemotherapy, and occlusion or blockage (total or partial) of lymphatic vessels), the lipid complex according to claim 1, selected from active agents capable of treating the lymphatic state, disease or disorder selected from the group consisting of.

11. A method for treating intestinal lymphatic thrombosis, comprising administering to a subject in need of such treatment an effective amount of the lipid complex according to claims 1 to 9, wherein by administering an effective amount of the lipid-based complex, hemostasis in the subject is maintained.

12. The method according to claim 10, wherein the lymphatic thrombosis is associated with at least infection or inflammation in the intestine.

13. The method according to claim 10, wherein the lipid complex is selectively packaged in chylomicrons and transported from the intestine in lymph fluid.

14. The method according to claim 10, wherein the lipid complex is not easily absorbed into the bloodstream.

15. The method according to claim 10, wherein the lipid complex is in the form of tablets, capsules, sachets, suppositories, liquids, oils, or combinations thereof.

16. The method according to claim 10, wherein the lipid complex is administered orally. **Claim 17** The method according to claim 10, wherein the lipid complex is a free lipid in an oil solution, micelle, liposome, or solid lipid nanoparticle. **Claim 18** A method for preventing lymph clot formation using the lipid complex according to any one of claims 1 to 9. **Claim 19** A method for reducing intestinal inflammation using the lipid complex according to any one of claims 1 to 9. **Claim 20** A method for treating intestinal infections using the lipid complex according to any one of claims 1 to 9. **Claim 21** A pharmaceutical composition comprising the lipid complex according to any one of claims 1 to 9. **Claim 22** A kit comprising the lipid complex according to any one of claims 1 to 9.

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