Lymphocyte-targeted anticoagulant for prevention of lymphatic thrombosis

Abstract

Provided herein are novel methods, compositions, and kits for targeting antithrombotic (LTAT) molecules using lymph. The LTAT molecule can be used for treating or preventing lymphatic thrombosis caused by infection or inflammation, in particular in the intestinal tract.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 393,409, filed on July 29, 2022, the entire contents of which are incorporated herein by reference.

[0003] Authorization Information

[0004] This disclosure was made with government support under DK123528 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] The disclosed subject matter relates to intestinal lymphatic targeted anti-thrombotic molecules for treating or preventing intestinal lymphatic thrombosis. The disclosed subject matter also provides methods, compositions and kits for treating inflammation, coagulation and cell aggregation that impair intestinal lymphatic function. Background Art

[0006] The lymphatic system is a system of vessels that collects interstitial fluid and returns it to the bloodstream, transports immune cells, and transports fat absorbed from the intestine. Interstitial fluid is produced by fluid leaking from the bloodstream into the tissues and then collected by the lymphatic system for return to the bloodstream. It has long been known that lymphatic fluid contains high levels of molecular components of the coagulation cascade and that lymphatic fluid can form fibrin clots in vitro and in ex vivo models of clot initiation. Lymphatic clots can be associated with infections involving lymphatic vessels, but such lymphatic thrombi are rare, and whether they truly arise independently of blood and blood-derived cells is unclear.

[0007] The intestinal lymphatic network drains interstitial fluid and immune cells from the stomach, small intestine, and colon. The intestinal lymphatics also play a special role in the absorption of fat from the intestine by transporting chylomicrons formed by the intestinal epithelium from dietary lipids. These chylomicrons are delivered from the intestine to the collecting lymphatic system along with lymphatic fluid and to the blood system via lymphatic venous nodes. The intestinal lymphatics play a key role in transporting intestinal immune cells, which are challenged by the constant exposure of the intestinal tissue to endogenous and exogenous bacteria, as well as other challenges. Immune surveillance of the intestine may require patent lymphatics to transport and clear activated immune cells and regulate the duration and intensity of inflammation. Summary of the invention

[0008] The objects and advantages of the disclosed subject matter will be set forth in and will be apparent from the description which follows, and will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and attained by the techniques particularly pointed out in the written description and claims as well as the accompanying drawings.

[0009] To achieve these and other advantages and in accordance with the purposes of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter comprises lymphatic targeted antithrombotic (LTAT) molecules and methods for using the molecules to treat inflammatory conditions and thrombotic conditions affecting the intestinal lymphatic system. In addition to the methods of treatment, the disclosed subject matter also provides pharmaceutical compositions and kits comprising the molecules and suitable pharmaceutical carriers.

[0010] In a first aspect, the present disclosure provides a lipid conjugate, wherein the lipid conjugate comprises: one or more lymphatic pathology, disease or condition therapeutic active molecules; and, a lipid or lipidoid molecule. In certain embodiments, the lipid conjugate further comprises a linker region. In certain embodiments, the lipid conjugate further comprises a lipid head group.

[0011] In certain embodiments, one or more lymphatic conditions, diseases or conditions therapeutically active molecules are anticoagulant molecules. In certain embodiments, the anticoagulant molecules are antithrombin, anti-Xa molecules or thrombolytic agents. In certain embodiments, the anticoagulant molecules are antithrombin, anti-Xa molecules or thrombolytic agents. In certain embodiments, the anticoagulant molecules block thrombin or Xa enzymatic activity.

[0012] In certain embodiments, the antithrombin molecule is hirudin, bivalirudin, ximelagatran, a dabigatran derivative, or a tripeptide thrombin inhibitor derivative. In certain embodiments, the anti-Xa molecule is selected from the group consisting of edoxaban, rivaroxaban, apixaban, or a combination thereof.

[0013] In certain embodiments, the lipids comprise long chain fatty acids or monoglycerides.

[0014] In certain embodiments, the lymphatic condition, disease or disorder therapeutic active molecule is selected from an active agent that can treat a lymphatic condition, disease or disorder selected from the group consisting of sepsis, necrotizing enterocolitis, autoimmune diseases, Crohn's disease, celiac disease, ulcerative colitis, rheumatoid arthritis, cardiovascular disease, bacterial infection, viral infection, viral hepatitis (including hepatitis C viral hepatitis), alcoholic hepatitis, adipocyte insulin resistance, pancreatitis, metabolic syndrome, trauma-induced inflammation, acute respiratory distress syndrome (ARDS), COVID-19-induced systemic inflammation, post-transplant organ rejection, amyloidosis, lymphangitis, obesity, primary or secondary lymphedema, congenital lymphatic insufficiency, lymph Lymphatic dysgenesis, inflammatory bowel disease, chronic granulomatous disease (CGD), lymphoid malignancies (including but not limited to Hodgkin's disease, non-Hodgkin's lymphoma and Castleman's disease), Milo's disease, syphilis, elephantiasis, lymphatic system disorders secondary to tissue damage (e.g., infarction, surgical injury, organ or tissue transplantation, radiation therapy, chemotherapy, lymphatic obstruction or blockage (full or partial)), non-lymphoid malignancies (colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, sepsis, necrotizing enterocolitis, autoimmune diseases and Castleman's disease), Milo's disease, syphilis, elephantiasis, lymphatic system disorders secondary to tissue damage (e.g., infarction, surgical injury, organ or tissue transplantation, radiation therapy, chemotherapy, lymphatic obstruction or blockage (full or partial)).

[0015] In certain embodiments, the present disclosure provides a method of treating intestinal lymphatic thrombosis, the method comprising: administering an effective amount of a lipid conjugate to a subject in need of such treatment, wherein administration of the effective amount of the lipid-based conjugate maintains hemostatic function in the subject.

[0016] In certain embodiments, lymphatic thrombosis is associated with infection or inflammation in at least the intestinal tract.

[0017] In certain embodiments, the lipid conjugate is selectively packaged using chylomicrons and transported from the intestine in the lymph. In certain embodiments, the lipid conjugate is not readily absorbed into the bloodstream.

[0018] In certain embodiments, the lipid conjugate is in the form of a tablet, capsule, sachet, suppository, liquid, oil, or a combination thereof.

[0019] In certain embodiments, the lipid conjugate is administered orally.

[0020] In certain embodiments, the lipid conjugate is a free lipid in an oil solution, micelle, liposome, or solid lipid nanoparticle.

[0021] In certain embodiments, the present disclosure provides a method of preventing lymphatic clot formation using a lipid conjugate.

[0022] In certain embodiments, the present disclosure provides a method for reducing intestinal inflammation using a lipid conjugate.

[0023] In certain embodiments, the present disclosure provides a method for treating intestinal infections using lipid conjugates.

[0024] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a lipid conjugate.

[0025] In certain embodiments, the present disclosure provides a kit comprising a lipid conjugate.

[0026] In a first aspect, the present disclosure provides a method for treating lymphatic thrombosis, the method comprising: administering an effective amount of a lymphatic targeted anti-thrombotic molecule (LTAT) to a subject in need of such treatment. In certain embodiments, lymphatic thrombosis is associated with infection or inflammation in at least the intestinal tract.

[0027] In certain embodiments, LTAT molecules selectively utilize chylomicron packaging, and in lymph (rather than blood), transport from intestinal tract, which is different from non-lipid-based antithrombotic molecules. In certain embodiments, compared with non-lipid-based antithrombotic molecules, LTAT molecules are not easily absorbed into the bloodstream. In certain embodiments, LTAT molecules include lipids or lipidoid molecules conjugated to active agents (such as anticoagulants).

[0028] In certain embodiments, the LTAT molecule further comprises a linker region. In certain embodiments, the LTAT molecule further comprises a lipid head group.

[0029] 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.

[0030] In certain embodiments, the antithrombin molecule is hirudin, bivalirudin, ximelagatran, a dabigatran derivative, or a tripeptide thrombin inhibitor derivative.

[0031] In certain embodiments, the anti-Xa molecule is selected from edoxaban, rivaroxaban, apixaban, or a combination thereof.

[0032] In certain embodiments, the lipids comprise long chain fatty acids or monoglycerides.

[0033] In certain embodiments, LTAT is in the form of a tablet, capsule, sachet, suppository, liquid, oil, or a combination thereof.

[0034] In certain embodiments, the LTAT molecule is administered orally. In certain embodiments, the LTAT molecule is a free lipid in an oil solution, micelle, liposome, or solid lipid nanoparticle.

[0035] In another aspect, the present disclosure provides a method of preventing, reducing or treating lymphatic clot formation 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 infection.

[0036] In another aspect, the present disclosure provides a pharmaceutical composition for practicing the methods described herein.

[0037] In another aspect, the present disclosure provides a kit for performing the methods described herein.

[0038] The present disclosure also provides a bioconjugate, wherein the bioconjugate comprises an active agent, such as an anticoagulant molecule and a lipid or lipidoid molecule. In certain embodiments, the bioconjugate further comprises a linker region. In certain embodiments, the bioconjugate further comprises a lipid head group. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A and Figure 1B The expression of antithrombotic proteins in mouse lymphatic endothelial cells is shown. Figure 1A Representative images of immunohistological staining of mouse tissues for PROX1 expression (green) and EPCR (red) are shown. Figure 1B Representative immunohistological staining of mouse tissues for PROX1 and thrombomodulin (THBD) are shown. Lymphatic vessels (L) and blood vessels (BV) are shown for reference only.

[0040] Figure 2A and Figure 2B Schematic representation of the PAR1-Tango allele that reports thrombin activity in mice. Figure 2A shows the lack of reporter activity when the PAR1 receptor is not exposed to thrombin, whereas Figure 2B Activation of green fluorescent protein (GFP) expression in the presence of thrombin was demonstrated.

[0041] Figure 3A and Figure 3B PAR1-Tango activity in intestinal lymphatic vessels is shown. Figure 3A PAR1-Tango activity was shown not to be in lymphatic vessels of the lung, heart, or skin. Figure 3B PAR1-Tango activity of intestinal lymphatic vessels is shown. Lymphatic vessels were marked by VEGFR3 or LYVE1 staining. Figure 3A The LYVE1-positive vessels in the liver shown are sinusoidal vessels.

[0042] FIG. 4A to FIG. 4E It was shown that PAR1-Tango activity is synergistically produced with intestinal flora and can be reversed by antibiotics. Figure 4A It was demonstrated that the nuclei of cells in LYVE1+ (lymphatic endothelial hyaluronan receptor 1) lymphatic vessels were negative for GFP at P0 and P7, began to show GFP expression at P14, and then showed strong expression of GFP at P21. Figure 4B Quantitative analysis of GFP-positive cells over time is shown. LYVE1-negative cells, GFP-positive cells are likely macrophages in the neonatal intestine. Figure 4C Schematic diagram showing an example design and timeline for accessing the gut microbiota in response to antibiotic treatment. Figure 4D The inhibitory effect of neonatal antibiotic ("ABX") treatment on PAR1-Tango reporter activity is shown and Figure 4E quantified in the literature, confirming that thrombin production is associated with bacterial colonization of the intestine. These data link bacteria in the intestinal lumen to thrombin activity in the intestinal lymphatic compartment.

[0043] FIG. 5A to FIG. 5D Bacterial infection is shown to produce lymphatic clots. Figure 5A The experimental design in which mature mice were orally treated with Salmonella or Shigella by gavage is shown. Figure 5B demonstrated fibrin clots in LYVE1-positive lymphatic vessels of mice infected with Salmonella and Yersinia. Figure 5C It is shown that nearly 20% of intestinal lymphatic vessels become thrombosed after Salmonella infection. Figure 5D In this study, mice were gavaged with GFP-expressing Salmonella and Salmonella were identified within fibrin clots in intestinal lymphatic vessels. P values ​​are indicated by asterisks as follows: **p<0.01.

[0044] FIG. 6A to FIG. 6E Chronic small intestinal inflammation leading to lymphatic clot formation is shown. Fig. 6A A schematic diagram of the dosing schedule for mice using 2.5% DSS to induce intestinal inflammation over 7 days is shown. Figure 6B Representative images of the small intestine of control and DSS-treated mice are shown, stained for fibrin (green) and the lymphatic vessel marker VegFR3 (red). Figure 6C Quantification of the average percentage of fibrin-positive lymphatic vessels is shown, with mean and standard deviation shown. Fig.6D Representative images of human intestinal samples from healthy subjects and Crohn's disease subjects are shown, showing staining for fibrin(ogen) (green) and the lymphatic vessel marker Lyve1 (red). Fig. 6EThe quantitative results of the mean and standard deviation of two groups are shown. P values ​​are indicated by asterisks as follows: ***p<0.001, **p<0.01.

[0045] 7A to 7C Orally delivered warfarin is shown to prevent lymphatic clots in a mouse model of lymphatic thrombosis. Fig. 7A Shown is a schematic outlining the experimental design, in which mice with an inducible knockout allele of thrombomodulin were administered tamoxifen to induce the absence of thrombomodulin expression, while simultaneously given warfarin or vehicle treatment in their water. Figure 7B Representative images of intestinal lymphatic vessels of experimental mice three weeks after the first induction are shown, showing fibrin (green) and Lyve1 (red) as markers of lymphatic vessels. Figure 7C Quantitative data are shown showing the mean and standard deviation of the percentage of fibrin-positive lymphatic vessels after 3 weeks. P values ​​are indicated with asterisks as follows: ***p<0.001.

[0046] FIG. 8A to FIG. 8D The lymphatic-targeted antithrombotic (LTAT) molecule structure and design are shown. Fig. 8A A schematic diagram depicting the general architecture of the LTAT molecule is shown, in which the antithrombotic molecule is attached to the lipid head group via a flexible linker. The LTAT molecule can be delivered as a free lipid or as Figure 8B Delivered as micelles or liposomes with other lipids as shown. Figure 8C Molecular structures of four direct oral anticoagulants are shown. Fig.8D Two sample LTAT molecular designs are shown, showing chemical linkage of dabigatran or rivaroxaban to a lipid.

[0047] 9A to 9E A process for producing anticoagulant lipid-based nanoparticles is exemplified. Fig. 9A Representative lipid formulations used to construct basic liposomes are depicted. Fig. 9B A method for attaching anticoagulant molecules to a lipid matrix to form anticoagulant lipid nanoparticles (NPs) is exemplified. Specifically, the thrombin inhibitor PPACK functionalized with dibenzocyclooctyne is reacted with azide groups present on the liposome surface. Fig. 9C A comparison between the activities of PPACK nanoparticles and self-generated free PPACK is presented to measure thrombin activity using a chromogenic substrate assay. FIG. 9C to FIG. 9D The ability to track PPACKNP in chylomicron was demonstrated. Fig.9D Representative fluorescence images of PPACK NPs after doping in extracted chyle are shown. Fig.9E The size distribution of PPACK NPs doped in chylomicron is shown.

[0048] FIG. 10A to FIG. 10D The inhibitory effect of oral PPACK NP treatment on thrombin in lymph was demonstrated. Fig. 10A It was demonstrated that thrombin activity in the chylomicron of mice treated orally with PPACK NPs was inhibited, as assessed using the thrombin-ChromozymTH reaction assay. Fig. 10B Depicted are the quantification of thrombin activity in lymph of mice treated with oral PPACK NPs, expressed as a percentage of thrombin activity relative to sham-treated chyle. Fig. 10C The dose-response curve of thrombin inhibition in the lymph of mice treated with oral PPACK NPs was plotted to study the effect of different amounts of chyle containing PPACKNPs on thrombin activity. The curve revealed that the IC50 of thrombin inhibition was 1U / mL at 0.4μL of chyle. Fig. 10D The dose-response curve of PPACK NP inhibition on thrombin before injection is shown, which reveals an IC50 of 0.03 μL. Fig. 10D and Fig. 10C Comparison shows the concentration of PPACK lipids in the chylomicron of mice treated with oral PPACK NPs.

[0049] FIG. 11A to FIG. 11C Tracking of coformulated fluorescent lipids in PPACK NPs in chylomicrons was depicted, and it was demonstrated that lipids from orally administered PPACK NPs were transported into chylomicrons and incorporated into chylomicrons. Fig.11A Shown are size and concentration measurements of nanoparticles (including chylomicrons) in chylomicrons containing fluorescently labeled lipids. Fig. 11B Shown is the size distribution of all NPs (including chylomicrons) found in the chyle of mice orally treated with PPACK NPs compared to sham-treated mice. Fig. 11C Shown is the average nanoparticle size of nanoparticles (including chylomicrons) found in the chyle of mice orally treated with PPACK NPs compared to sham-treated mice. Fig.11D Shown is the total concentration of nanoparticles (including chylomicrons) found in the chyle of mice orally treated with PPACK NPs compared to sham-treated mice.

[0050] Fig.12 The process of radiolabeling anticoagulant lipid-based nanoparticles is exemplified.

[0051] FIG. 13A to FIG. 13D Depicted 111 In vivo pharmacokinetics of labeled lipids coformulated in PPACK NPs in mice. Data were acquired within 24 h after gavage. Fig.13A shows the NP from PPACK 111Biodistribution of In labeled lipids along the gastrointestinal (GI) tract. Fig. 13B Non-GI organs (including heart, lung, liver, spleen, kidney and brain) are shown. 111 The concentration of In. Fig. 13C Shows the various tissues found in the gastrointestinal tract 111 The concentration of In. Fig.13D Shows chyle, blood and urine 111 The concentration of In.

[0052] FIG. 14A to FIG. 14C The pharmacokinetics of PPACK lipid-induced thrombin inhibition in chylomicron and plasma are shown. Fig.14A A dose-response curve is depicted showing the inhibition of thrombin by PPACK-NPs at the concentrations prepared prior to gavage, revealing an IC50 of 0.0085 μL. Fig. 14B It was shown that thrombin was inhibited in the chyle of mice treated orally with PPACK NPs, but not in the plasma of mice treated orally with PPACK-NPs, as assessed using the thrombin-chromozym TH reaction assay. Fig. 14C Quantitative analysis of PPACK NP-induced thrombin inhibition in chylomicrons and plasma is shown, expressed as percent inhibition of thrombin activity relative to sham-treated chylomicrons or untreated plasma.

[0053] FIG. 15A to FIG. 15F The effect of PPACK NPs administered via gavage on complete blood counts was demonstrated. Blood tests were performed on mice at different time points over a 24-hour period following gavage of PPACK NPs. Fig.15A Cell counts of total white blood cells (WBC), lymphocytes (LYM), monocytes (MON), and neutrophils (NEU) are presented. Fig. 15B Shown are changes in the size of leukocytes within 24 hours after gavage administration of PPACK NPs. Fig. 15C Blood test results for red blood cells and hemoglobin are shown. Fig.15D Shown are the changes in erythrocyte size after gavage of PPACK NPs. Fig.15E Results of a blood test for platelets are shown. Fig.15F Shown are the changes in platelet size after gavage of PPACK NPs.

[0054] Fig.16A and Fig. 16B An alternative approach to developing anticoagulant lipid conjugates is illustrated. Fig.16A The modification process of the FXa inhibitor apixaban for lipid conjugation is exemplified. Fig. 16B Shown are the Factor Xa inhibitors DX-9065a and YM-60828, which have structures suitable for covalent bonding to lipid head groups.

[0055] Fig.17 It is demonstrated that when the FXa inhibitor apixaban is modified with a linker for conjugation to a lipid, the FXa inhibitor apixaban retains the ability to inhibit FXa. DETAILED DESCRIPTION

[0056] The present disclosure relates to methods, compositions and kits for preventing intestinal lymphatic thrombosis without disrupting hemostasis in the treated individual.

[0057] For the purpose of clarity of disclosure, but not limitation, the detailed description of the disclosed subject matter is divided into the following subsections:

[0058] 5.1. Definitions;

[0059] 5.2 Symptoms;

[0060] 5.3 Lipid conjugates

[0061] 5.4. How to use;

[0062] 5.5. Composition; and

[0063] 5.6. Test kit.

[0064] 5.1. Definitions

[0065] The terms used in this specification generally have their ordinary meanings in the art, within the context of the present disclosure, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners when describing the compositions and methods of the present disclosure and how to make and use them.

[0066] When used in conjunction with the word "comprising" in the claims and / or the specification, the use of the terms "a" or "an" may mean "one", but is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0067] As used herein, the terms "comprise," "include," "having, has," "can," "contain," and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude additional actions or structures. The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of," embodiments or elements presented herein, whether or not explicitly set forth.

[0068] The term "about" or "nearly" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 3 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or even up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and within 2-fold of a value.

[0069] The term "cell" refers to any suitable cell used in the present disclosure, such as a eukaryotic cell. For example, but not limited to, suitable eukaryotic cells include animal cells (e.g., mammalian cells). In certain embodiments, suitable cells are cultured cells. In certain embodiments, suitable cells are host cells, recombinant cells, and recombinant host cells. In certain embodiments, suitable cells are cell lines obtained or derived from mammalian tissues that can grow and survive when placed in a culture medium containing appropriate nutrients and / or growth factors.

[0070] As used herein, the term "expression" (expression or expresses) refers to transcription and translation occurring in a cell (e.g., a mammalian cell). In certain embodiments, the expression level of a gene and / or nucleic acid in a cell can be determined based on 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, mRNA transcribed from a gene and / or nucleic acid is quantified by Northern hybridization in a desired manner. Sambrook et al., Molecular Cloning Laboratory Manual, pp.7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). The protein encoded by a gene and / or nucleic acid can be quantified by measuring the biological activity of the protein or by using an assay independent of the activity, such as a Western blotting method or a radioimmunoassay using an antibody capable of reacting with the protein. Sambrook et al., Molecular Cloning Laboratory Manual, pp.18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0071] The term "thrombosis" herein refers to a thrombus or clot formed in a lymphatic vessel, thereby obstructing lymphatic flow. The term includes that the lymphatic vessel is healthy, but due to the intestinal bacterial environment and the immune cell environment, and the damaged or sick, edema, fibrosis, immune disorders, nutritional failure that may occur in the lymphatic system, and other conditions of lymphatic exposure to high thrombin. Significantly, lymphatic thrombus is mainly composed of a fibrin network, and does not contain prothrombotic cells such as platelets and neutrophils that circulate in the blood but not in the lymph. The term is different from "thrombus" formed in the vascular space. Both are stimulated by the proteolysis of fibrinogen by thrombin, to form cross-linked fibrin, but thrombus is contained and produced by platelets and neutrophils that do not circulate in the lymph.

[0072] The term "disease, disorder or condition" refers to diseases, disorders and conditions that a patient is diagnosed with or suspected of having, particularly diseases, disorders or conditions associated with lymphatic thrombosis. Diseases, disorders or conditions include, but are not limited to, pathogenic infections, inflammation-related conditions, side effects associated with drugs or treatments, and idiopathic conditions characterized by symptoms including inflammation.

[0073] In certain contexts, "lymphatic condition, disease, or disorder" is intended to include all conditions characterized by lymphatic insufficiency or abnormal lymphatic function, including, but not limited to, viral or bacterial infection, trauma, cancer, amyloidosis, sporadic cases, lymphangitis, obesity, primary or secondary lymphedema, congenital lymphadenopathy, lymphatic dysgenesis, cardiovascular disease, heart disease, inflammatory bowel disease, ulcerative colitis, Crohn's disease, chronic granulomatous disease (CGD), lymphatic malignancies (including, but not limited to Crohn's disease, Hodgkin's disease, non-Hodgkin's lymphoma, and Castleman's disease), Milo's disease, syphilis, elephantiasis, lymphatic system disorders secondary to tissue injury (e.g., infarction, surgical injury, organ or tissue transplantation, radiation therapy, chemotherapy, and lymphatic obstruction or blockage (full or partial)).

[0074] In certain contexts, the term "lymphatic endothelial cells" (also referred to as LECs) refers to endothelial cells that line lymphatic vessels, which are related to but distinct from endothelial cells that line blood vessels, known as "blood endothelial cells."

[0075] The term "effective amount" refers to the amount of active ingredients that effectively alleviate or alleviate to a certain extent one or more symptoms of a disease in need of treatment, or delay the onset of clinical markers or symptoms of a disease in need of prevention when a compound is administered. Therefore, an effective amount refers to the amount of active ingredients that exhibit the following effects, such as: (i) reversing the rate of progression of the disease; (ii) inhibiting the further development of the disease to a certain extent; and / or (iii) alleviating (or eliminating) one or more symptoms associated with the disease to a certain extent. The effective amount can be determined empirically by experimenting with related compounds in known in vivo model systems and in vitro model systems of diseases in need of treatment. The context in which the phrase "effective amount" is used may indicate a specific desired effect. For example, "LTAT molecular weight for effectively preventing or treating lymphatic thrombosis" and similar phrases refer to the amount of LTAT protein that will cause a significant improvement in the lymphatic thrombosis state of the subject when applied to the subject. Unlike anticoagulation in the blood system, effective LTAT cannot be measured by laboratory assessment of established coagulation parameters because these parameters are based on blood and plasma. The effective amount may vary according to individual weight, sex, age and medical history, as well as the severity of the patient's condition, the type of disease and the mode of administration. The effective amount can be easily determined using routine experiments (e.g., by titration (increasing the dose until an effective dose is found) and / or by reference to the amount that was effective for previous patients).

[0076] The 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.

[0077] As used herein, the term or "conjugate" refers to two or more components joined by a covalent bond, wherein at least one component is a biomolecule (such as an enzyme, protein or antibody). For example, the presently disclosed conjugates may comprise a lipid or lipidoid molecule covalently joined to an anticoagulant molecule.

[0078] The term "vehicle" refers to a diluent, adjuvant, excipient or carrier used together with a therapeutic drug. Such physiological carriers can be sterile liquids, such as water and oils (comprising oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). When the pharmaceutical composition is administered intravenously, water is a suitable carrier. Saline solutions and liquid glucose and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, dry skim milk, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer.

[0079] The term "agent" as used herein refers to a substance that produces or is capable of producing an effect, and includes, but is not limited to, chemicals, drugs, biologics, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

[0080] 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, reduces, inhibits, eliminates or blocks) the signal transduction function of a protein or pathway. An inhibitor can be any compound or molecule (signal molecule, any molecule related to a named signal molecule or a named related molecule) that changes any activity of a protein (such as RNF167), or interferes with the interaction of a protein (e.g., RNF167) with a signaling partner. Inhibitors also include molecules that indirectly regulate the biological activity of a named protein (e.g., RNF167) by intercepting upstream signaling molecules.

[0081] 5.2 Symptoms

[0082] In certain embodiments, the lipid conjugates, methods, compositions, and kits provided herein can be used to treat any condition or disease / disorder ("disorder") involving intestinal lymphatic thrombosis, including, but not limited to, lymphatic insufficiency or abnormality, including, but not limited to, infection, trauma, cancer, and inflammation.

[0083] In certain embodiments, lymphatic disorders may be associated with any condition that causes intestinal inflammation, including sepsis, necrotizing enterocolitis, autoimmune diseases, Crohn's disease, celiac disease, ulcerative colitis, rheumatoid arthritis, cardiovascular disease, bacterial infections, viral infections, viral hepatitis (including hepatitis C viral hepatitis), alcoholic hepatitis, adipocyte insulin resistance, pancreatitis, metabolic syndrome, trauma-induced inflammation, acute respiratory distress syndrome (ARDS), COVID-19-induced systemic inflammation , post-transplant organ rejection, amyloidosis, lymphangitis, obesity, primary or secondary lymphedema, congenital lymphatic insufficiency, lymphatic dysplasia, inflammatory bowel disease, chronic granulomatous disease (CGD), lymphatic malignancies (including but not limited to Hodgkin's disease, non-Hodgkin's lymphoma and Castleman's disease), Milo's disease, syphilis, elephantiasis, lymphatic system disorders secondary to tissue damage (e.g., infarction, surgical injury, organ or tissue transplantation, radiation therapy, chemotherapy, lymphatic obstruction or blockage (full or partial)). In certain embodiments, lymphatic conditions may also include non-lymphatic malignancies that still affect the lymphatic system, including but not limited to colorectal cancer, liver cancer, gastric cancer, pancreatic cancer. Lymphatic insufficiency or abnormal lymphatic function can be caused, for example, by defects or deficiencies in any component of the lymphatic system, including valves, capillaries, tubes, etc. It is contemplated to use the methods described herein to repair or regulate the expansion of any of these and any other lymphatic system components.

[0084] 5.3 Lipid conjugates

[0085] In certain embodiments, the disclosure relates to lipid conjugates. In certain embodiments, the disclosure relates to lipid components or lipidoid components conjugated to active agents (such as anticoagulant molecules). In certain non-limiting embodiments, anticoagulant molecules are antithrombotic molecules. In certain embodiments, lipid antithrombotic molecules are lymphatic targeted antithrombotic (LTAT) molecules. In certain embodiments, lipid anticoagulant molecules target the intestinal lymphatic system. Lipid anticoagulants can be delivered as free lipids in oil solutions or as micelles, liposomes or solid lipid nanoparticles to enhance the absorbability and availability of anticoagulants in the lymphatic system. The conjugation between lipid components or lipidoid components and anticoagulant molecules prevents the separation of lipids from anticoagulant components during administration, ensuring the integrity and effectiveness of their targeted delivery in the lymphatic system. Lipid anticoagulant molecules can be used for targeted anticoagulant therapy for the lymphatic system.

[0086] Non-limiting examples of lipid molecules or lipid-based molecules include dipalmitoylphosphatidylcholine (DPPC), cholesterol, distearoylphosphatidylethanolamine (DSPE), phosphatidylcholine (PC), and phosphatidylethanolamine (PE).

[0087] In certain embodiments, the lipid comprises a long-chain fatty acid or a monoglyceride. 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 glyceryl monooleate, glyceryl monostearate, monoglyceride, and glyceryl monopalmitate.

[0088] In certain non-limiting embodiments, when the anticoagulant is part of a lipid structure or is present alone, the anticoagulant cannot enter the bloodstream upon oral administration.

[0089] In certain non-limiting embodiments, the anticoagulant is resistant to proteases of the digestive system.

[0090] In certain non-limiting embodiments, the anticoagulant molecule is an antithrombin, an anti-Xa molecule, or a thrombolytic agent. The anticoagulant molecule can block the activity of thrombin or Xa enzymes.

[0091] Non-limiting examples of antithrombin molecules include hirudin, bivalirudin, ximelagatran, derivatives of dabigatran, and derivatives of tripeptide-type thrombin inhibitors.

[0092] Non-limiting examples of anti-Xa molecules include edoxaban, rivaroxaban, apixaban, DX-9065a, and YM-60828.

[0093] In certain non-limiting embodiments, the lipid or lipidoid molecule is conjugated to the anticoagulant molecule by conjugating the lipid or lipidoid molecule to the anticoagulant molecule via click chemistry, esterification, amidation, or another conjugation reaction.

[0094] In certain non-limiting embodiments, the anticoagulant is covalently linked to the lipid head group, wherein the point of attachment is sufficiently far away from the enzyme interface so as not to reduce the inhibitory effect. Non-limiting examples of covalently terminal sulfhydryls, acids, hydroxyls, esters, aldehydes, and amines, among others.

[0095] In certain non-limiting embodiments, lipid molecules or lipid-based molecules include reactive moieties. In certain non-limiting embodiments, anticoagulant molecules include reactive moieties. In certain non-limiting embodiments, reactive moieties are linking moieties, such as but not limited to trans-cyclooctene, tetrazine, cyclooctyne, alkynes or azides, olefins, tetrazole, light-DIBO or cyclopropenone.

[0096] In certain non-limiting embodiments, the conjugation between the lipid or lipidoid molecule and the anticoagulant molecule can be prepared by various reaction schemes known in the art. Examples of suitable reactions include, but are not limited to, Diels-Alder reactions, azide-alkyne-based click reactions (e.g., Cu(I)-catalyzed azide-alkyne cycloadditions and metal-free azide-alkyne cycloadditions), Staudinger ligations, thiol-maleimide additions, oxime ligations, and thiol-ene reactions.

[0097] For purposes of illustration and not limitation, FIG. 8A to FIG. 9B The process of producing lipid conjugates according to the presently disclosed subject matter is schematically illustrated.The lipid formulation serves as a basis for the attachment of anticoagulant molecules, resulting in the formation of anticoagulant lipid nanoparticles (NPs).

[0098] The lipid conjugates of the disclosed subject matter may also include one or more additional components. For example, in certain embodiments, spacers may be provided between the various components of the lipid conjugate. Additionally, spacers may enhance the conjugation between the components of the lipid conjugate; if present, spacers may be used to control the relative position of the lipid or lipidoid molecule and the anticoagulant. In certain embodiments, the spacer may comprise a polymer or a biomolecule. In certain non-limiting examples, the polymer can be polyethylene glycol, polyethylene, polyethylene glycol, dendrimers, polyacrylic acid, hydroxyethyl starch (HES), polylactic acid-glycolic acid, poly-D,L-dioxanone lactic acid-ethylene glycol block copolymer (PLA-DX-PEG), poly(o-)esters, polyglutamates, polyaspartates, polymers of aB-unsaturated monomers (such as (meth)acrylic acid, butenoic acid, maleic acid, maleic anhydride, fumaric acid, itaconic acid / anhydride, etc.), comonomers (containing vinyl ethers, vinyl esters, vinylamine amides, olefins and / or diallyldialkylammonium halides, preferably vinyl ethers, ethylene glycol adipate polyesters, polyethylene imine, polyglycolide, polyureas, polylimonene (=Polylimo), poly(2-methyl-1,3-propylene glycol adipate) as well as graft polymers and graft (block) polymers (e.g., with other polymers).

[0099] For example, but not limited to, polyethylene glycol (PEG) n can be used as a spacer.

[0100] In certain non-limiting embodiments, the size and use of the spacer can be selected to control the overall size of the lipid conjugate. 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 size of the total lipid conjugate can be controlled to induce enhanced absorbability and availability of the drug in the lymphatic system.

[0101] 5.4 Treatment methods

[0102] In certain embodiments, the present disclosure provides a method for treating an infectious, inflammatory, or autoimmune disorder affecting the intestinal lymphatic system, the method comprising administering to a subject in need of such treatment an effective amount of a LTAT molecule that prevents, reduces, or inhibits one or more signs or symptoms of the disorder and does not significantly alter the subject's coagulation dynamics.

[0103] In certain embodiments, the disclosure provides a method for treating or preventing lymphatic thrombosis associated with viral infection or bacterial infection in a subject, the method comprising: administering an effective amount of LTAT molecules to a subject in need of such treatment for blood coagulation in the subject's lymphatic system. In certain embodiments, infection causes inflammation, thereby causing lymphatic clot formation. In certain embodiments, the disclosure provides a method for treating and / or preventing lymphatic thrombosis in a subject.

[0104] In certain embodiments, the present disclosure provides a method for treating lymphatic thrombosis associated with an inflammatory condition in a subject, the method comprising: administering an effective amount of LTAT molecules to a subject in need of such treatment for coagulation 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-related inflammation caused by lymphatic thrombosis.

[0105] In certain embodiments, the present disclosure provides a method of treating or preventing one or more lymph node masses associated with a viral infection or a bacterial infection. In certain embodiments, the present disclosure provides a method of treating or preventing lymph node masses associated with an inflammatory condition, including but not limited to inflammatory bowel disease (such as ulcerative colitis and Crohn's disease).

[0106] 5.5 Pharmaceutical Compositions

[0107] In certain embodiments, pharmaceutical compositions of the present disclosure include lipid conjugates and pharmaceutically acceptable carriers. Suitable carriers that can be used together with the disclosed subject matter have the characteristics of not interfering with the effectiveness of the biological activity of active ingredients (e.g., disclosed inhibitors / anticancer agents), and are nontoxic to patients. 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. Additional non-limiting examples of pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, implant elements containing inhibitors and / or any other suitable vehicles, delivery means or materials or distribution means or materials. Such pharmaceutically acceptable carriers can be prepared by conventional methods, and can be applied to subjects. In certain embodiments, pharmaceutically acceptable carriers may include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as, but not limited to, octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl-p-alkylbenzenes (such as methyl-p-hydroxybenzoate or propyl-p-hydroxyphenylate, 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. Ketones; amino acids such as glycine, glutamine, asparagine, histidine, arginine or 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 may include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol or a combination thereof.

[0108] In certain non-limiting embodiments, the pharmaceutical composition of the present disclosure can be formulated using a pharmaceutically acceptable carrier suitable for oral administration well known in the art. Such carriers enable the pharmaceutical composition to be formulated into tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral or nasal intake of patients to be treated. In certain embodiments, the pharmaceutical composition is formulated as a capsule. In certain embodiments, the pharmaceutical composition can be 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 tablet or a controlled release tablet. In certain embodiments, the solid dose can include an immediate release portion and a sustained release portion or a controlled release portion.

[0109] Of course, the dosage regimen of the compounds of the invention will vary depending on known factors, such as the pharmacodynamic characteristics of the particular formulation and its mode and route of administration; the species, age, sex, health condition, medical condition and weight of the recipient; the nature and extent of symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and hepatic function, and the desired effect. A physician or veterinarian can determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the thromboembolic disorder.

[0110] Dosage forms (pharmaceutical compositions) suitable for administration may contain from about 1 mg to about 1000 mg of active ingredient per dosage unit. In these pharmaceutical compositions, the active ingredient will generally be present in an amount of about 0.1% to 95% by weight based on the total weight of the composition.

[0111] As a general guide, when used to indicate effect, the daily oral dosage of each active ingredient will be between about 0.001 mg per kilogram of body weight to about 1000 mg / kg per kilogram of body weight. The compounds of the present disclosure may be administered in a single daily dose, or the total daily dose may be administered in divided doses twice, three times or four times a day.

[0112] In certain embodiments, the disclosure provides pharmaceutical compositions comprising an LTAT molecule as 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.

[0113] In certain embodiments, the disclosure provides a pharmaceutical composition comprising any one of the LTAT molecules in a therapeutically effective amount, for example but not limited to use with a pharmaceutical carrier such as water or other physiological solvents. The therapeutically effective amount can prevent, reduce or inhibit lymphatic clot formation.

[0114] In certain non-limiting embodiments, the LTAT molecule may be contained in an oil solution, micelle, liposome, or similar structure.

[0115] In certain embodiments, pharmaceutical composition can be liquid, comprising LTAT molecules in a liquid drug carrier, and the liquid drug carrier comprises, for example, water (aqueous carrier) or normal saline. In certain embodiments, the liquid composition can optionally further contain one or more of a buffer or a preservative.

[0116] In certain other embodiments, the pharmaceutical composition of the present disclosure may be in a solid (e.g., tablet, capsule, medicine bag or suppository) form, comprising a dose of LTAT molecules, which, when administered according to a dosing regimen, provide an effective amount of LTAT to a subject in need of such treatment. In certain embodiments, the solid pharmaceutical composition may also include one or more excipients, such as, but not limited to, lactose, sucrose, mannitol, erythritol, carboxymethyl cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, starch, polyvinyl pyrrolidone, etc.

[0117] In certain embodiments, the pharmaceutical composition may include an admixture having antimicrobial activity and / or anti-inflammatory activity. In certain embodiments, such compounds include, but are not limited to, antibiotics, steroids, or nonsteroidal anti-inflammatory agents. In certain other embodiments, the pharmaceutical composition may include an analgesic. In certain further embodiments, the pharmaceutical composition may include an agent that dissolves pre-existing lymphatic clots, such as tissue plasminogen activator (tPA) or active plasminogen.

[0118] Pharmaceutically acceptable salts are well-known and include relatively non-toxic inorganic and organic acid addition salts of the compositions of the disclosed subject matter, including, but not limited to, therapeutic agents, excipients, other materials, and the like. Examples of pharmaceutically acceptable salts include salts derived from inorganic acids such as hydrochloric acid and sulfuric acid, and salts derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of suitable inorganic bases for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed using suitable organic bases, including those that are non-toxic and strong enough to form such salts. For illustrative purposes, the class of such organic bases may include monoalkylamines, dialkylamines, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; monohydroxyalkylamides, dihydroxyalkylamides, or hydroxyalkylamides, such as monoethanolamine, diethanolamine, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N-methylglucamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; (trihydroxymethyl)ethane, and the like; see, for example, J. Pharm. Sci., 66: 1-19 (1977).

[0119] 5.6 Kit

[0120] The subject matter of the present disclosure also provides kits containing materials that can be used to perform the methods and compositions disclosed herein. For example, but not limited to, any combination of materials that can be used in the present disclosure can be packaged together as a kit for use in performing any disclosed method or composition.

[0121] In certain embodiments, the kits of the present disclosure may contain LTAT molecules together with suitable pharmaceutical carriers. In certain embodiments, the kit components may be packaged disposable, suitable for carrying for a single administration.

[0122] In certain embodiments, the kit further comprises a package insert that provides instructions for using the components provided in the kit.For example, a kit of the present disclosure may include a package insert that provides instructions for using the LTAT molecule provided in the kit.

[0123] Alternatively or additionally, the kit may include other materials desirable from a commercial and user perspective, including other buffers, diluents, and filters. In certain embodiments, the kit may include materials for preparing LTAT molecules.

[0124] The kit can provide reagents in pre-measured amounts to simplify the performance of the disclosed method and the administration of the composition. Optionally, the disclosed kit includes instructions for performing the method or administering the composition. Other optional components of the disclosed kit include suitable buffers, formulations, packaging materials, etc. The disclosed kit may also include additional admixtures required for performing the disclosed method and composition use. The reagents of the kit may be in containers in which they are stable, for example, in lyophilized form or in a stable liquid form.

[0125] 6. Examples

[0126] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration and not by way of limitation of the presently disclosed subject matter.

[0127] result

[0128] Antithrombotic properties of the lymphatic endothelium.

[0129] The present disclosure shows that the lymphatic endothelium lining the lymphatic vessels expresses a family of transcription factors, including FOXC2, PROX1, and GATA2, which play a key role in the development of the lymphatic system, particularly the lymphatic vessels, and are also associated with a potent antithrombotic phenotype. The antithrombotic phenotype represented by the expression of FOXC2 and PROX1 was also observed in the venous valve endothelium and plays a role in preventing thrombus formation within the venous valve pocket. The antithrombotic phenotype includes downregulation of several pro-thrombotic and pro-inflammatory endothelial surface proteins (von Willebrand factor and P-selectin), as well as upregulation of the antithrombotic endothelial surface proteins thrombomodulin (THBD) and endothelial protein C receptor (EPCR) ( Figure 1A and Figure 1B). The lymphatic endothelium, especially the lymphatic network in the intestine, shares this antithrombotic expression pattern characterized by high THBD and EPCR expression. The lymphatic endothelium shows a synergistic effect, resulting in a decrease in prothrombotic proteins and an increase in antithrombotic proteins, which in turn produces an additive reaction and a strong thrombotic inhibitory effect on thrombus formation.

[0130] The PAR1-Tango mouse model reports thrombin activity in vivo.

[0131] The present disclosure provides a mouse model that carries the PAR1-Tango allele as a reporter gene for the expression of thrombin, a major procoagulant factor ( Figure 2A and Figure 2B Mice expressing the PAR1-Tango allele showed strong thrombin activity in intestinal lymphatic vessels ( Figure 3B ), whereas when the PAR1 receptor was not exposed to thrombin, a lack of reporter activity was observed in the lung, heart, skin, and liver ( Figure 3A ). The present disclosure shows that the intestinal lymphatic environment is naturally procoagulant.

[0132] Lymphatic clot formation caused by gastrointestinal infection.

[0133] The present disclosure shows that the formation of lymphofibrillar clots is a key part of the intestinal natural immune response, which functions to prevent the spread of pathogenic bacteria introduced into the mouse intestine ( FIG. 4A to FIG. 4E ).like Figure 4A and Figure 4B As shown, PAR1-Tango activity is produced in concert with intestinal flora. It was found that lymphatic vessels (LYVE1 positive) were negative for PAR1-Tango expression at P0 and P7, but showed expression (GFP positive cells) at P14 and stronger expression at P21. The present disclosure demonstrates that PAR1-Tango positive cells are present in the neonatal intestine.

[0134] Clot formation within the intestinal lymphatic vessels blocks the flow of fluid, immune cells, and bacteria, preventing the spread of infection and providing more time for activated immune cells to respond to invading bacteria, thereby preventing the spread of pathogens from the intestine to other parts of the body (i.e., sepsis). Similarly, in viral infections, intestinal lymph clots form and their function is to prevent the spread of pathogens and to focus locally activated immune cells to the area to eliminate the pathogens. In both cases, lymph node clots are beneficial to health. The present disclosure shows the inhibitory effect of neonatal antibiotic ("ABX") treatment on PAR1-Tango reporter activity, confirming that thrombin production is related to intestinal bacterial colonization. The present disclosure establishes a link between bacteria in the intestinal lumen and thrombin activity in the intestinal lymph lumen. The present disclosure further demonstrates that bacterial infections can produce lymphatic clots. As FIG. 5A to FIG. 5D As shown, mature mice orally gavaged with Salmonella or Shigella formed fibrin clots in LYVE1-positive lymphatic vessels. This publication shows that nearly 20% of intestinal lymphatic vessels form thrombi after Salmonella infection ( Figure 5C This was further demonstrated in mice gavaged with GFP-expressing Salmonella, showing that GFP-expressing Salmonella were found at sites of fibrin clot formation within the intestinal lymphatic vessels ( Figure 5D ).

[0135] Enteritis causes lymphatic clots to form in the intestines.

[0136] The present disclosure finds that lymphatic clots may be inappropriately induced in cases of inflammation and autoimmune or inflammatory diseases caused by non-pathogenic sources. In these cases, activated immune cells known to be thrombogenic may activate intraluminal thrombin activity in lymphatic vessels, thereby initiating fibrin clot formation. Both mouse models of intestinal inflammation (induced by dietary intake of DSS) and human samples from patients with chronic inflammatory bowel disease showed extensive lymphatic clot formation relative to healthy controls ( FIG. 6A to FIG. 6E ). In these instances, rather than preventing the spread of pathogens, clots prevent clearance by activated immune cells, thereby exacerbating inflammation and leading to tissue damage and symptoms. Clinical data from patients with chronic inflammation of the gut suggest that they may develop nutritional deficiencies consistent with dysregulated dietary lipid uptake and disruption of intestinal lymphatic flow.

[0137] Natural anticoagulant molecules expressed by intestinal lymphatic vessels prevent intestinal lymph agglomeration.

[0138] The present disclosure demonstrates that genetic deletion of the lymphatic endothelial THBD in mice results in extensive intestinal lymph node mass and obstruction of intestinal lymphatic flow. In this context, oral anticoagulant warfarin can prevent lymph node mass ( 7A to 7C). This demonstrates that antithrombotic drugs prevent the formation of lymphatic clots and lead to restoration of lymphatic function. However, the use of existing antithrombotic drugs produces systemic anticoagulation and carries a significant risk of systemic (including intestinal and brain) bleeding. The risk of systemic anticoagulation (i.e., anticoagulation that affects both the blood and lymphatic vasculature) is significantly higher in patients with chronic inflammation because they have higher rates of clinical bleeding, particularly in the intestine. Therefore, there is a clear need for a functional antithrombotic drug that can be delivered at a therapeutic dose preferentially and act on the intestinal lymphatics. In addition, the volume of lymph in the intestine is much smaller than the volume of blood in the body, meaning that a therapeutic dose of drug delivered directly into the intestinal lymphatics would be substantially diluted to well below therapeutic levels once it reaches the bloodstream, thereby preventing systemic anticoagulation and inappropriate bleeding.

[0139] Lipid conjugates for targeting antithrombotic drugs to intestinal lymphatics.

[0140] The present disclosure demonstrates that lipid conjugate molecules with flexible linkers of varying lengths can be attached to orally active antithrombin molecules to achieve specific lymphatic-targeted antithrombotic drug delivery. Attachment of antithrombotic drugs to lipid head groups results in various LTAT molecules ( FIG. 8A to FIG. 8D ). These molecules can be formulated into isolated lipids, micelles or lipid nanoparticles (liposomes containing solid lipid nanoparticles). These molecules will be used in clinical cases of small intestinal inflammation (such as Crohn's disease) to reduce the intensity and duration of enteritis, and for any viral or bacterial infection where lymph node mass may hinder the drug from entering the cells within the clot. These molecules can be delivered orally in the form of oil capsules, and these molecules will be specifically taken up into the intestinal lymphatic vessels as chylomicrons, during which the antithrombotic drug is exposed to the intraluminal lymph environment, thereby being able to block the thrombin or Xa enzyme activity required for thrombosis. LTAT molecules can be delivered as free lipids, or micelles, or liposomes in oil solutions to enhance the absorbability and availability of drugs in the lymphatic system ( Fig. 8A and Figure 8B LTAT molecules can be the sole lipid used to form micelles or liposomes, or they can be the major component of micelles or liposomes. Unabsorbed drugs can pass through the intestines more easily than non-lipid-based small molecules and are less easily absorbed into the bloodstream, thereby reducing the possibility of excessive blood levels of antithrombotic drugs causing bleeding.

[0141] The formation process of LTAT molecule is as follows Fig. 9A and Fig. 9BAs illustrated. Initially, a 2% molar concentration DSPE-PEG azide formulation was used to prepare the base liposomes. This formulation ensured that the azide groups were located on the outer surface of the liposomes to facilitate 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. The IC50 value of PPACK NPs was nearly 0.8 nM, while the IC50 value of stand-alone PPACK was nearly 0.0002 nM. Therefore, the liposomes proved to be very effective for loading PPACK, with an estimated nearly 400 PPACK molecules per liposome ( Fig. 9C To simulate the oral effect and study the size change of nanoparticles, PPACK NPs were incorporated into mouse-derived chylomicron ( Fig.9D and Fig.9E ).

[0142] like Fig. 10A and Fig. 10B As demonstrated, oral administration of PPACK NPs can inhibit thrombin in chylomicrons. The thrombin activity of chylomicrons diluted 1:33 in 1 U / mL thrombin was evaluated. Chylomicrons from mice treated with oral PPACK NPs demonstrated an IC50 of thrombin inhibition of 1 U / mL at 0.4 μL chylomicrons. Fig. 10C ), while the IC50 of pre-injected PPACK NP was 0.03 μL ( Fig. 10D ). The data indicated that each μL of chyle contained nearly 0.075 μL (0.03 / 0.4) of PPACK NPs at their original concentration. The initial dose contained 250 μL of PPACK NPs (concentrated to 50 μL and added to 400 μL of olive oil), resulting in a 1 μL gavage containing the equivalent of 0.56 μL of chyle (250 / 450). This is equivalent to 13.4% of the gavage dose activity in the chyle at recovery.

[0143] Fluorescence nanoparticle tracking analysis (F-NTA) was used to evaluate the transport of fluorescent lipids formulated in PPACK NPs from oral gavage to chylomicron. Fig.11A ). In chylomicron samples from mice treated with oral PPACK NPs, the size and concentration of nanoparticles containing fluorescent lipids from PPACK NPs were measured by F-NTA. This example shows that the size of nanoparticles in the chylomicron containing fluorescent lipids is inconsistent with the pre-injected nanoparticles, indicating that the lipids from PPACK NPs are incorporated into chylomicrons / endogenous vesicles rather than transported as part of intact nanoparticles. The results indicate that the size of nanoscale lipid vesicles / aggregates found in the chylomicron of mice treated with oral PPACK NPs was altered compared to sham-treated mice, as shown in Figure 2. FIG. 11B to FIG. 11D Proven.

[0144] To evaluate the in vivo distribution of PPACK NPs, the radioactive labeling agent (111)In was incorporated into the NPs (e.g. Fig.12 The movement of the radiolabeled tracer along the gastrointestinal tract of mice was tracked over a 24-hour period 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 pancreatic mesentery. However, after 24 hours ( Fig.13A and Fig. 13C ), the tracer had been cleared from these tissues. The tracer was found to have penetrated into multiple organs (such as the heart, lungs, liver, spleen, kidneys, and brain) at concentrations much lower than those in the gastrointestinal tract. The levels of tracer in all tissues gradually decreased and returned to normal within 24 hours ( Fig. 13B ). Only trace amounts of the tracer were detected in the blood, and urinalysis revealed its excretion after 24 hours, whereas detection of the tracer persisted in the chyle.

[0145] It was found that thrombin in plasma was not inhibited after oral gavage of PPACK NP treatment. Fig.14A Inhibition of thrombin by PPACK-NPs before gavage (IC50 of 0.0085 μL) is shown. Fig. 14B It was demonstrated that thrombin in the chyle, but not in the plasma, of mice treated with oral gavage of PPACK NPs was inhibited. Thrombin inhibition in the chyle showed a fluctuating increase from 0 to 4 hours after gavage, and a significant inhibitory effect was demonstrated at 24 hours after gavage ( Fig. 14C ).

[0146] Blood tests were performed at different time points within 24 hours after PPACK NP gavage. Analysis of complete blood counts (including white blood cells (WBC), lymphocytes (LYM), monocytes (MON), and neutrophils (NEU)) demonstrated mild lymphocytopenia at early time points after gavage ( Fig.15A and Fig. 15B Blood tests further revealed an increase in red blood cell size 24 hours after gavage of PPACK NPs ( Fig. 15C and Fig.15D ), platelet count showed a downward trend ( Fig.15E and Fig.15F ).

[0147] To demonstrate that different antithrombotic drugs can be used for LTAT, a method was designed to modify the factor Xa inhibitor apixaban so that it can be conjugated to a lipid (Figure 16). The modified apixaban was shown to inhibit factor Xa activity as well as the unmodified apixaban ( Fig.17 ).

[0148] discuss

[0149] Anticoagulant molecules are formulated on lipids for oral delivery, demonstrating a method of local anticoagulant effect. Anticoagulant molecules associated with lipids can be specifically delivered to the lymphatic fluid where they remain active. At the same time, anticoagulant molecules connected to lipids are constructed to be removed from non-target (unwanted) tissues and bloodstreams. Anticoagulant molecules initially delivered in liposome form are decomposed after administration, and lipids are redistributed in recombinant chylomicrons. Anticoagulant molecules can be formulated into free lipids in liposomes, micelles or oil emulsions. This example demonstrates the conjugation and formulation process and function of PPACK conjugated lipids prepared in liposomes, but preparations containing anticoagulant molecules with higher specificity for thrombin or factor Xa are being developed. When not combined with lipids and the half-life in the bloodstream is short, anticoagulant molecules with lower oral bioavailability can provide more specific lymphatic targeting. Preferred molecules include those with free acids or amines to allow unmodified molecules to be conjugated to lipid head groups by covalent bonds, thereby preventing dissociation.

[0150] method

[0151] PPACK liposomes were prepared by 1) synthesizing liposomes using a bioorthogonal conjugation handle (in this case, an azide-terminated lipid); and 2) conjugating PPACK to a linker molecule using a bioorthogonal conjugation handle that is compatible with the liposome. In this case, PPACK was conjugated to dibenzocyclooctyne (DBCO) via an N-hydroxysuccinimide-terminated polyethylene glycol (PEG) linker. N-hydroxysuccinimide reacted with the primary amine end of PPACK to form PPACK-DBCO. Liposomes were prepared using dipalmitoylphosphatidylcholine (DPPC) and cholesterol, along with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and an azide-terminated PEG linker, a formulation that has previously been shown to form highly stable liposomes. Liposomes with azide groups were reacted with PPACK-DBCO overnight, and PPACK-DBC not conjugated to lipids was removed from the formulation by centrifugal filtration or size exclusion chromatography to ensure that the formulation did not contain PPACK not conjugated to lipids.

[0152] In order to modify apixaban for conjugation with lipids, a derivative of apixaban with a carboxylic acid group was obtained and conjugated to an amine terminus protected by a fluorenylmethoxycarbonyl (FMOC) group via carbodiimide coupling, and a PEG-(bis)amine high bifunctional linker was conjugated to the apixaban derivative. After deprotection with trifluoroacetic acid, the modified apixaban was purified by high performance liquid chromatography with a reverse phase column, and the structure of the final molecule was verified by mass spectrometry and nuclear magnetic resonance.

[0153] The inhibitory effect of LTAT on thrombin, either in vitro or in chylomicron or blood samples from mice treated with LTAT, was assessed using chromogenic substrate assays in which a chromogenic substrate (such as Chromozym TH) is cleaved by thrombin or factor Xa, thereby releasing the p-nitroanilide group and generating an absorbance signature that is quenched by the tosyl group prior to cleavage. In all chromogenic substrate assays described, a fixed amount of substrate and active enzyme (thrombin or factor Xa) are added to the assay along with varying concentrations of inhibitor, which can be either the unconjugated molecule or LTAT, or samples from mice receiving LTAT containing an unknown concentration of inhibitor. The absorbance signature represents the extent of cleavage of the substrate by a set concentration of enzyme, and a decrease in the absorbance signature represents the amount of inhibitor added to the assay.

[0154] 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 modifications can be made herein without departing from the spirit and scope of the disclosed subject matter. In addition, the scope of the present application is not intended to be limited to the specific embodiments of the process, machine, manufacture and material composition, method and process described in the specification. Those of ordinary skill in the art will be easily understood from the disclosed subject matter of the disclosed subject matter, according to which the disclosed subject matter can be used to perform substantially the same function or achieve substantially the same result with the corresponding embodiment described herein that currently exists or is developed later. Therefore, the appended claims are intended to include these processes, machines, manufacture, material composition or methods within their scope.

Claims

1. A lipid conjugate, comprising: a. one or more lymphatic condition, disease or disorder therapeutically active molecules; and b. Lipid or lipid-like molecule.

2. The lipid conjugate according to claim 1, further comprising a linker region.

3. The lipid conjugate according to claim 1, further comprising a lipid head group.

4. The lipid conjugate of claim 1, wherein the one or more lymphatic condition, disease or disorder therapeutically active molecules are anticoagulant molecules.

5. The lipid conjugate according to claim 4, wherein the anticoagulant molecule is antithrombin, an anti-Xa molecule or a thrombolytic agent.

6. The lipid conjugate of claim 4, wherein the anticoagulant molecule blocks thrombin or Xa enzyme activity.

7. The lipid conjugate of claim 1, wherein the antithrombin molecule is hirudin, bivalirudin, ximelagatran, a dabigatran derivative, or a tripeptide thrombin inhibitor derivative.

8. The lipid conjugate according to claim 1, wherein the anti-Xa molecule is selected from the group consisting of edoxaban, rivaroxaban, apixaban or a combination thereof.

9. The lipid conjugate of claim 1, wherein the lipid comprises a long-chain fatty acid or a monoglyceride.

10. The lipid conjugate of claim 1, wherein the lymphatic condition, disease or disorder therapeutic active molecule is selected from an active agent capable of treating a lymphatic condition, disease or disorder selected from the group consisting of sepsis, necrotizing enterocolitis, autoimmune diseases, Crohn's disease, celiac disease, ulcerative colitis, rheumatoid arthritis, cardiovascular disease, bacterial infection, viral infection, viral hepatitis including hepatitis C viral hepatitis, alcoholic hepatitis, adipocyte insulin resistance, pancreatitis, metabolic syndrome, trauma-induced inflammation, acute respiratory distress syndrome ARDS, COVID-19-induced systemic inflammation, post-transplant organ rejection, amyloidosis, lymphangitis, obesity, primary or secondary lymphedema, Congenital lymphadenopathy, lymphatic dysgenesis, inflammatory bowel disease, chronic granulomatous disease (CGD), lymphatic malignancies - including but not limited to Hodgkin's disease, non-Hodgkin's lymphoma and Castleman's disease, Milo's disease, syphilis, elephantiasis, lymphatic system disorders secondary to tissue damage - such as infarction, surgical injury, organ or tissue transplantation, radiation therapy, chemotherapy, lymphatic obstruction or blockage in whole or in part, non-lymphatic malignancies - colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, sepsis, necrotizing enterocolitis, autoimmune diseases and Castleman's disease, Milo's disease, syphilis, elephantiasis, lymphatic system disorders secondary to tissue damage, such as infarction, surgical injury, organ or tissue transplantation, radiation therapy, chemotherapy, lymphatic obstruction or blockage in whole or in part.

11. A method for treating intestinal lymphatic thrombosis, the method comprising: An effective amount of a lipid conjugate according to claims 1 to 9 is administered to a subject in need of such treatment, wherein administration of the effective amount of the lipid-based conjugate maintains hemostatic function in the subject.

12. The method of claim 10, wherein the lymphatic thrombosis is associated with infection or inflammation at least in the intestinal tract.

13. The method of claim 10, wherein the lipid conjugate is selectively packaged using chylomicrons and transported from the intestine in the lymph.

14. The method of claim 10, wherein the lipid conjugate is not readily absorbed into the bloodstream.

15. The method of claim 10, wherein the lipid conjugate is in the form of a tablet, capsule, sachet, suppository, liquid, oil, or a combination thereof.

16. The method of claim 10, wherein the lipid conjugate is administered orally.

17. The method of claim 10, wherein the lipid conjugate is a free lipid in an oil solution, micelles, liposomes, or solid lipid nanoparticles.

18. A method for preventing lymphatic clot formation using the lipid conjugate according to any one of claims 1 to 9.

19. A method for reducing intestinal inflammation using the lipid conjugate according to any one of claims 1 to 9.

20. A method for treating intestinal infections using the lipid conjugate according to any one of claims 1 to 9.

21. A pharmaceutical composition comprising the lipid conjugate according to any one of claims 1 to 9.

22. A kit comprising the lipid conjugate according to any one of claims 1 to 9.

Images