Nucleic acid molecules having anti-inflammatory and anti-coagulant and organ protective properties

Abstract

The present invention relates to a nucleic acid molecule or a composition comprising said molecule for use as an anti-inflammatory and / or anticoagulant and / or organ protective drug. The present invention also relates to the use of said nucleic acid molecule or said composition for the manufacture of a medicament for the prevention, treatment, regression, cure and / or delay of a disease or condition in which inflammation and / or coagulation and / or organ impairment or failure occurs in an individual. Finally, the present invention further relates to a method for alleviating one or more symptoms and / or parameters and / or improving a disease or condition in which inflammation and / or coagulation and / or organ impairment or failure occurs in an individual, the method comprising administering to said individual said nucleic acid molecule or said composition.

Description

Technical Field

[0001] The present invention relates to nucleic acid molecules or compositions comprising said molecules suitable for use as anti-inflammatory and / or anticoagulant and / or organ protective drugs. The present invention also relates to the use of said nucleic acid molecules or said compositions for the preparation of a medicament for preventing, treating, resolving, curing and / or delaying a disease or condition in which inflammation and / or coagulation and / or organ damage or failure occurs in an individual. Finally, the present invention further relates to a method for alleviating one or more symptoms and / or characteristics and / or ameliorating parameters of a disease or condition in which inflammation and / or coagulation and / or organ damage or failure occurs in an individual, the method comprising administering said nucleic acid molecules or said compositions to said individual. Background Art

[0002] Disseminated intravascular coagulation (DIC) is characterized by systemic activation of coagulation caused by various underlying diseases, among which sepsis is the main cause (Gando S et al., 2008).

[0003] Sepsis and DIC are common, severe, and acute diseases that occur when infectious organisms, cancer, surgery, or trauma trigger an excessive host immune response and are the leading cause of death in hospitalized patients (Clowes GH et al 1970, Parrillo JE et al, 1990, and Lei MG et al, 2003). As a defense mechanism, the immune system triggers a rapid increase in proinflammatory and thrombotic mediators, leading to fatal systemic tissue damage and multiple organ failure or multiple organ dysfunction syndrome.

[0004] Today, health care systems lack effective treatments to reduce the risk of organ damage or failure until infections are controlled, and clinical needs are high.

[0005] Currently, tremendous efforts are devoted to uncovering the molecular mechanisms leading to inflammation and coagulation alterations.

[0006] There are currently no known effective drugs that can be used to specifically prevent, treat, resolve, cure and / or delay the onset of diseases or conditions involving inflammation and / or blood coagulation and / or organ damage or failure.

[0007] Therefore, there remains a need for new treatments for diseases or conditions associated with inflammation and / or coagulation and / or organ damage or failure. Summary of the Invention

[0008] Surprisingly, it has been found that the nucleic acid molecules defined herein exhibit unexpected anti-inflammatory, anticoagulant and organ protective properties.

[0009] In a first aspect of the present invention, a nucleic acid molecule for use as an anti-inflammatory and / or anticoagulant and / or organ protective drug is provided, wherein the nucleic acid molecule is represented by a nucleotide sequence having 95% sequence identity with a nucleotide sequence comprising SEQ ID NO: 1 or SEQ ID NO: 2.

[0010] SEQ ID NO: 1 is the following nucleic acid sequence:

[0011]

[0012] SEQ ID NO: 2 is the following nucleic acid sequence:

[0013]

[0014] Each C and U of SEQ ID NO: 2 is fluorinated (ie, U F , C F ) and increases the stability of the molecule in plasma.

[0015] In the context of the present invention, a nucleic acid molecule may be a synthetic or natural or recombinant or modified molecule in the sense that compared to SEQ ID NO: 1 defined above:

[0016] - 1, 2, 3, 4 or 5 or at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90% or 100% of its nucleotides have been deleted or added compared to the corresponding nucleotides present in the native molecule, and / or

[0017] - One, two, three, four or five or at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90% or 100% of the nucleotides, nucleosides, nucleobases, nucleobase linker moieties and / or backbone moieties have been replaced or substituted by corresponding nucleotides, nucleosides, nucleobases, nucleobase linker moieties and / or backbone moieties not found in the native molecule.

[0018] In the context of the present invention, the nucleic acid molecule may be a modified molecule compared to SEQ ID NO: 2 identified above, in the sense that:

[0019] - 1, 2, 3, 4 or 5 or at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90% or 100% of its nucleotides have been deleted or added compared to the corresponding nucleotides present in the native molecule, and / or

[0020] - One, two, three, four or five or at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90% or 100% of the nucleotides, nucleosides, nucleobases, nucleobase linker moieties and / or backbone moieties have been replaced or substituted by corresponding nucleotides, nucleosides, nucleobases, nucleobase linker moieties and / or backbone moieties not found in the native molecule.

[0021] Preferably, the nucleic acid molecule is represented by a sequence comprising or consisting of SEQ ID NO: 1 or 2. Preferred nucleic acid molecules are nucleic acid molecules comprising SEQ ID NO: 1 or 2 and having a length that is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides longer than SEQ ID NO: 1 or 2.

[0022] Compared with the structure of RNA molecule, the chemical structure of the nucleotide of nucleic acid molecule can be modified, replaced, substituted to increase stability, binding affinity and / or specificity.Described nucleic acid molecule can comprise RNA molecule or preferably modified RNA molecule, or be composed of it.Preferably modified RNA molecule comprises modified sugar.An example of this modification is to introduce 2'-O-methyl or 2'-O-methoxyethyl group or halogen (that is, fluoro, chloro, bromo or iodo) group on this nucleic acid to improve nuclease resistance and RNA binding affinity.Preferred halogen group in this context is fluoro, as shown in SEQ ID NO:2.Another example of this type of modification is to introduce 2'-O atom and 4'-C atom connecting this nucleic acid to lock the methylene bridge (locked nucleic acid (LNA)) of conformation, to improve the affinity to complementary single-stranded RNA.A third example is to introduce phosphorothioate group as the linker between nucleic acid in RNA chain to improve the stability of anti-nuclease attack. The binding affinity of nucleic acid molecules can be further modified to improve their binding properties as described in Hasegawa H et al., which is incorporated herein by reference. More detailed information on other chemical modifications is provided in the general section dedicated to definitions.

[0023] Therefore, preferred nucleic acid molecules for use as defined herein are single-stranded and / or nucleic acid molecules wherein the nucleotides of the nucleic acid molecule are modified compared to the nucleotides present in the RNA.

[0024] According to a more preferred embodiment, the nucleic acid molecule for use is such that one of its pyrimidines is fluorinated and preferably all of its pyrimidines are fluorinated, as shown in SEQ ID NO:2.

[0025] Identity can be at least 95%, 96%, 97%, 98%, 99% or 100%. Identity is preferably assessed over the entire SEQ ID NO identified herein. However, identity can also be assessed over a portion of a given SEQ ID NO. 1. A portion can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the length of the SEQ ID NO. More detailed information on identity assessment is provided in the general section dedicated to the definitions.

[0026] The nucleic acid molecules of the invention are preferably used as anti-inflammatory and / or anticoagulant and / or organ protective drugs, since they exhibit anti-inflammatory and / or anticoagulant and / or organ protective properties or characteristics as explained below and shown in the experimental part.

[0027] In one embodiment, the nucleic acid molecules of the invention are useful as anti-inflammatory and / or anticoagulant and / or organ protective drugs, since they exhibit anti-inflammatory and / or anticoagulant and / or organ protective properties or characteristics as explained below and shown in the experimental part.

[0028] In one embodiment, the preferred activity of the nucleic acid molecules of the present invention is to induce detectable anti-inflammatory activity (i.e., inflammatory activity is reduced and / or anti-inflammatory activity is increased). Any compound or any parameter known to be associated with increased or decreased inflammation can be used as a readout value for evaluating the activity of the nucleic acid molecules of the present invention.

[0029] Anti-inflammatory activity can be assessed by detecting an increase in the expression level of anti-inflammatory cytokines and / or a decrease in the expression level of pro-inflammatory cytokines. Examples of pro-inflammatory cytokines are IL1β, IL-6, TNFα, and HMGB1. An example of an anti-inflammatory cytokine is IL-10.

[0030] Another parameter related to inflammation is the detection of inflammatory infiltration of leukocytes.

[0031] Another parameter related to inflammation is the detection of the expression of the pro-inflammatory receptors MAC-1 (CD11b / CD18) or P-selectin or CD36 or EPCR (endothelial protein C receptor).

[0032] Another parameter associated with inflammation is the detection of the expression of L-selectin, CD35, CD88, ICAM-1 (intercellular adhesion molecule 1) or PECAM-1 (platelet endothelial cell adhesion molecule 1).

[0033] Another parameter related to inflammation is the measurement of fibrinogen, a positive acute phase protein.

[0034] In the context of the present invention, the parameters associated with inflammation are selected from the group consisting of: increased expression levels of anti-inflammatory cytokines, decreased expression levels of pro-inflammatory cytokines, IL1β, IL-6, TNFα, HMGB1, IL10, leukocyte inflammatory infiltration, MAC-1, P-selectin, CD36, EPCR, CD35, CD88, ICAM-1, PECAM-1 and fibrinogen.

[0035] In another embodiment, another preferred activity of the nucleic acid molecules of the present invention is the induction of detectable anticoagulant activity.Any compound or any parameter known to be associated with increased or decreased coagulation can be used as a readout to assess the activity of the nucleic acid molecules of the present invention.

[0036] Preferred parameters related to coagulation and used in this context are APTT (activated partial thromboplastin time), fibrin clot density, thrombin formation, bleeding time, D-dimer, fibrinolytic activity (tPA (tissue plasminogen activator), PAI-1 (plasminogen activator inhibitor-1) and the complex between tPA, anticoagulant protein-S and / or expression of EPCR. Another parameter related to coagulation and used in this context is expression of fibrinogen (factor I), heparan sulfate or thrombomodulin, PECAM-1 or protein C. Low levels of fibrinogen are often associated with fibrinogen depletion, for example, as seen in disseminated intravascular coagulation (DIC) and abnormal fibrinolysis.

[0037] In the context of the present invention, the parameters related to coagulation are selected from: APTT, fibrin clot density, thrombin formation, bleeding time, D-dimer, fibrinolytic activity (tPA, complex between PAI-1 and tPA), expression of anticoagulant protein-S, expression of EPCR, fibrinogen, heparan sulfate, expression of thrombomodulin, expression of PECAM-1 and expression of protein C.

[0038] In another embodiment, another preferred activity of the nucleic acid molecules of the present invention is the induction of detectable organ protective activity, preferably against kidney and / or liver damage or failure. Any compound or any parameter known to be associated with an increase or decrease in organ damage or failure, preferably associated with kidney and / or liver damage or failure, can be used as a readout for evaluating the activity of the nucleic acid molecules of the present invention.

[0039] Preferred parameters that are associated with organ damage or failure and used in this context are serum levels of AST (aspartate aminotransferase), ALT (alanine aminotransferase), GGT (gamma-glutamyl transpeptidase), ALP (alkaline phosphatase), LDH (lactate dehydrogenase), creatine kinase, BUN (blood urea nitrogen), albumin, creatinine, glucose, bile acid, total bilirubin, conjugated bilirubin, phosphorus, total protein, LDL cholesterol, HDL cholesterol, cholesterol and / or triglycerides.

[0040] Preferred parameters associated with liver damage or failure are AST, ALT, GGT, ALP, LDH, albumin, bile acid, total bilirubin, conjugated bilirubin and / or phosphorus.

[0041] Preferred parameters associated with renal damage or failure are creatinine, BUN and / or phosphorus.

[0042] This activity, parameter defined herein, can be evaluated in an individual, an animal model, a cell, preferably an animal model or an individual's cell. The activity can be evaluated using techniques known to the skilled person. Examples of analytical tests are provided in the experimental section.

[0043] At the mRNA level, preferably using RT-qPCR, the expression of pro-inflammatory or anti-inflammatory cytokines can be evaluated. Evaluation of cytokine expression can be performed at the protein level, preferably using tests that detect protein expression, such as Western blot analysis, ELISA, flow cytometry, immunohistochemistry or immunofluorescence analysis of cross sections and / or using the test defined later in this article. Detection of inflammatory infiltration of leukocytes or pro-inflammatory receptors MAC-1 or P-selectin or CD36 or EPCR can be performed using immunofluorescence. Detection of L-selectin, CD35, CD88, ICAM-1 or PECAM-1 can be performed using immunofluorescence.

[0044] APTT, fibrin clot density, thrombin formation, bleeding time, fibrinolytic activity (tPA, complex between PAI-1 and tPA), expression of anticoagulant protein-S and / or EPCR are evaluated using techniques known to the skilled person, preferably as described in the experimental section. Expression of fibrinogen, heparan sulfate, thrombomodulin, PECAM-1 and / or protein C are evaluated using techniques known to the skilled person, preferably as described in the experimental section.

[0045] Serum levels of AST, ALT, GGT, ALP, LDH, creatine kinase, BUN, albumin, creatinine, glucose, bile acids, total bilirubin, conjugated bilirubin, phosphorus, total protein, LDL cholesterol, HDL cholesterol, cholesterol and / or triglycerides are assessed using techniques known to those skilled in the art, preferably as described in the experimental section.

[0046] The evaluation is preferably performed at several time points for a given subject or at one or several time points for a given subject and healthy controls. Such evaluation can be performed weekly or monthly.

[0047] The detection of the presence of nucleic acid molecules of the present invention can be performed using any technique known to the skilled person. Preferably, the expression level or presence of the molecule is evaluated using classical molecular biology techniques such as (real-time) qPCR, microarrays, bead arrays, RNAse protection assays or Northern analysis. It will be appreciated by the skilled person that, alternatively or in conjunction with the quantitative determination of the molecule, the quantitative determination of the function or activity of the molecule using a specific assay for a substrate or any compound known to be associated with the function of the molecule is encompassed within the scope of the present invention.

[0048] The nucleic acid molecules defined herein can be used as naked molecules (with or without chemical modification), or can be present in or formulated into compositions or used in conjunction with moieties. Thus, in a further aspect, compositions comprising nucleic acid molecules as defined herein for use as defined herein are provided, wherein the compositions are preferably pharmaceutical compositions comprising pharmaceutical carriers, adjuvants, salts, diluents and / or excipients. Preferred compositions comprise the molecules encapsulated into particles, preferably nanoparticles or liposome structures. Other compositions are disclosed in the general section dedicated to the definitions.

[0049] In a preferred embodiment, the nucleic acid molecule or composition for use as defined herein can be used for intravenous, oral, subcutaneous or intramuscular administration. If inflammation is to be eliminated, oral or intramuscular injection is preferred. If coagulation is to be prevented, intravenous administration is preferred. Excellent anticoagulant effects are obtained using intravenous administration. If both are to be antagonized, oral, intramuscular and / or intravenous administration is preferred.

[0050] In the context of the present invention, any disease or condition in which inflammation and / or coagulation and / or organ damage or failure is involved or associated or occurs can be prevented, delayed, cured, resolved and / or treated using the nucleic acid molecules defined herein or compositions comprising the molecules. In a disease or condition as defined herein, inflammation and / or coagulation and / or organ damage or failure can be detected during the development of the disease or condition, i.e., after symptoms of the disease or condition appear. Alternatively, inflammation and / or coagulation and / or organ damage or failure can be detectable before the disease or condition develops.

[0051] In one embodiment, the disease or condition in which inflammation occurs is selected from rheumatoid arthritis (RA), juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease including Crohn's disease or ulcerative colitis, hepatitis, sepsis, alcoholic liver disease, non-alcoholic steatosis, sarcoidosis, autoimmune diabetes, diabetes, uveitis, multiple sclerosis, controlling allograft rejection after organ transplantation, graft-versus-host disease (GVHD), inflammatory lung diseases including asthma and chronic obstructive pulmonary disease (COPD), systemic lupus erythematosus (SLE), sarcoidosis, atopic dermatitis and cancer, or the effects of complications or progression associated with one of these diseases or conditions.

[0052] In another embodiment, the disease or condition in which coagulation occurs is selected from acute coronary syndrome (ACS), thrombosis, peripheral vascular occlusion, obstructive arteriosclerosis, vasculitis, dysfunction occurring after cardiac surgery, complications arising from organ transplantation, angina pectoris, transient ischemic attack, pregnancy toxemia (preeclampsia, eclampsia), diabetes, hepatic veno-occlusive disease (VOD), deep vein thrombosis (DVT), sepsis, septic shock, severe sepsis, trauma, acute respiratory distress syndrome (ARDS) and disseminated intravascular coagulation (DIC), or the effects of complications or progression associated with one of these diseases or conditions.

[0053] In another embodiment, the disease or condition in which the organ damage or failure occurs is selected from infection, poisoning, aspiration syndrome, hypoperfusion or shock, heat-induced illness, ischemia, ischemia-reperfusion injury (IRI), autoimmune disease, trauma, hemorrhage, pancreatitis, bacteremia, burns, sepsis, septic shock, severe sepsis, acute respiratory distress syndrome (ARDS), pregnancy toxemia (preeclampsia), eclampsia, systemic inflammatory response syndrome (SIRS), disseminated intravascular coagulation (DIC) and multiple organ failure or multiple organ dysfunction syndrome, or the effects of a complication or progression associated with one of these diseases or conditions. The organ damage or failure is preferably liver damage or failure, or renal damage or failure.

[0054] There are currently no effective known drugs that can be used specifically to prevent, treat, resolve, cure and / or delay diseases or conditions associated with inflammation and / or coagulation and / or organ damage or failure.

[0055] The present invention includes increasing the activity or steady-state level or expression level or amount of a nucleic acid molecule as defined herein or a composition comprising said molecule. The activity or steady-state level of said nucleic acid molecule is increased in a subject, in a cell of said subject, in a tissue of said subject or in a body fluid of said subject.

[0056] The activity or steady-state level or expression level or amount of the at least one nucleic acid molecule is increased to induce detectable anti-inflammatory and / or anticoagulant and / or organ protective activity in a subject, preferably in cells from the subject.

[0057] The expression level of the nucleic acid molecule as defined herein is preferably evaluated in a biopsy or biopsy section at several time points in a given subject or at one or several time points in a given subject and healthy controls. Such evaluation can be performed at regular intervals, for example, every week, every month. Thus, an increase / decrease can be evaluated at regular intervals, for example, every week, every month. Preferably, as explained later herein, an evaluation is performed for a decrease in detectable inflammation and / or a decrease in detectable coagulation and / or a decrease in detectable organ damage or failure to define an anti-inflammatory effect and / or an anticoagulant effect and / or an organ protective effect.

[0058] The activity or steady-state level or expression level of the nucleic acid molecule can be increased by, for example, providing the molecule to a subject, preferably to a cell of the subject, or to a tissue of the subject, or to an organ of the subject, or providing the molecule to the subject from an exogenous source, with the level of the molecule itself. In order to provide the molecule from an exogenous source, the molecule can be conveniently produced by expressing a nucleic acid encoding the molecule or a source thereof encoding the molecule in a suitable host cell as described below, or as a fully synthetic molecule by chemical synthesis.

[0059] The activity of the nucleic acid molecule or the steady-state level or the expression level are increased by regulating the expression level of the nucleotide sequence encoding the molecule. Preferably, the expression level of the nucleic acid molecule is regulated in the cell of the experimenter or in the tissue of the experimenter or in the experimenter. Can be by introducing the molecule or expression construct (or vector) into the cell, tissue, organ or body fluid of the experimenter, or in the experimenter, thus the expression vector comprises the nucleotide sequence containing the molecule, and thus the nucleotide sequence is under the control of the promoter that can drive the nucleotide sequence to express in the cell, tissue, organ, experimenter and increase the expression level of the nucleic acid molecule. Can also be by introducing the expression construct into the cell, tissue, organ, experimenter, thus the construct comprises the nucleotide sequence of the factor that coding can reversely activate the endogenous nucleotide sequence encoding the molecule and increase the expression level of the molecule.

[0060] The activity or steady-state level or amount of the nucleic acid molecule can be increased in a subject, in the cells of the subject, in the tissue of the subject or in a body fluid of the subject by increasing the concentration of the nucleic acid molecule within the cell, tissue or body fluid.

[0061] In the present invention, cells, tissues, organs or body fluids are preferably from subjects suspected of having, for example, a high risk of developing a disease or condition related to inflammation and / or coagulation and / or organ damage or failure due to their age or genetic background or their dietary habits. Alternatively, in another preferred embodiment, the present invention is applied to subjects diagnosed with a high risk of developing a disease or condition related to inflammation and / or coagulation and / or organ damage or failure from cells, tissues, organs or body fluids. Alternatively, cells, tissues or organs to be treated can be selected based on the risk of progression of the disease or condition related to inflammation and / or coagulation and / or organ damage or failure. This risk of progression can be evaluated using classical clinical pathology standards known to the skilled person or based on the prognosis of biomarkers. As explained in detail in the background of the present invention, an example of a situation in which coagulation may occur is DIC. The potential cause of DIC may be infectious organisms, cancer, surgery or trauma or other.

[0062] In the present invention, the cells, tissues, organs or body fluids are preferably derived from subjects who are suspected of having a high risk of developing a disease or condition associated with inflammation and / or coagulation and / or organ damage or failure and / or in which a parameter associated with inflammation, coagulation or organ damage or failure is increased / upregulated, due to their age or genetic background or their diet or the country in which they live or DIC. Alternatively, in another preferred embodiment, the present invention is applied to cells, tissues, organs or body fluids from subjects who are diagnosed as having a predicted risk of later developing a disease or condition associated with inflammation and / or coagulation and / or organ damage or failure and / or in which a parameter associated with inflammation, coagulation or organ damage or failure is increased / upregulated, e.g., due to their age or genetic background or their diet or the country in which they live or DIC.

[0063] The nucleic acid molecules determined herein preferably have an acceptable level of anti-inflammatory and / or anticoagulant and / or organ protective activity. An acceptable level of anti-inflammatory and / or anticoagulant and / or organ protective activity refers to at least 50%, 60%, 70%, 80%, 90%, 100% or more of the activity of the molecule represented by SEQ ID NO: 1 or 2 measured under the same conditions. An acceptable level of anticoagulant activity can also be equal to at least 50%, 60%, 70%, 80%, 90%, 100% or more of the activity of rivaroxaban measured under the same conditions.

[0064] The nucleic acid molecules or compositions of the invention may be combined with standard treatments for diseases or conditions in which inflammation and / or coagulation and / or organ damage or failure occurs, such as rivaroxaban or warfarin as anticoagulants.

[0065] In the context of the present invention, preventing, treating, resolving, curing and / or delaying a disease or condition associated with inflammation and / or coagulation and / or organ damage or failure may refer to:

[0066] - At least one symptom of the disease or condition has improved, and / or

[0067] - At least one parameter associated with the disease or condition has improved.

[0068] - Such improvement can be determined over a treatment period of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days, 1 week, 1 month, 6 months or more. Preferred parameters related to inflammation and / or coagulation and / or organ damage or failure are defined herein.

[0069] In the context of the present invention, preventing, treating, resolving, curing and / or delaying a disease or condition associated with inflammation and / or coagulation and / or organ damage or failure may be replaced by achieving an anti-inflammatory and / or anti-coagulation and / or organ protective effect. Unless otherwise indicated, the anti-inflammatory and / or anticoagulant and / or organ protective effects are preferably evaluated or detected in the treated subject before and at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months or more after treatment.

[0070] The anti-inflammatory effect is preferably determined in a subject as:

[0071] - a detectable decrease in the expression levels of proinflammatory cytokines, preferably IL1β, IL-6, TNFα and / or HMGB1, and / or

[0072] - A detectable decrease in the expression levels of anti-inflammatory cytokines such as IL-10 and / or

[0073] - A decrease in the number of inflammatory leukocytes detected, and / or

[0074] - a detectable decrease in the expression of proinflammatory receptors such as MAC-1, P-selectin, EPCR and / or CD36, and / or

[0075] - a detectable decrease in the expression levels of proinflammatory receptors such as L-selectin, CD35, CD88, ICAM-1 and / or PECAM-1, and / or

[0076] - a detectable decrease in serum levels of fibrinogen, which is a positive acute phase protein, and / or

[0077] - Patient survival is prolonged by at least one month, several months or longer (compared to those patients who were not treated or treated with a control, or compared to the subject at the start of treatment).

[0078] In the context of the present invention, the patient may be alive and may be considered disease-free. Alternatively, the disease or condition may have been arrested, delayed or resolved.

[0079] A detectable decrease in the expression level of IL1β, IL-6, HMGB1, and / or TNFα can be a decrease of at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, or 75% or more. This decrease in expression level can be assessed using known techniques, preferably as described in the experimental section. The expression can be assessed at the RNA or protein level. This decrease can be assessed at least 3, 4, 5, 6, or 7 days after transfection with a given nucleic acid molecule.

[0080] A detectable increase in IL-10 expression levels can be an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, or 75% or more. This increase in expression levels can be assessed using known techniques, preferably as described in the experimental section. This expression can be assessed at the RNA or protein level. This increase can be assessed at least 3, 4, 5, 6, or 7 days after transfection with a given nucleic acid molecule.

[0081] The reduction in the number of inflammatory infiltrates of leukocytes detected can be a reduction of at least 1%, 5%, 10%, 15%, 20%, 25% or more. Such an increase can be assessed at least 3, 4, 5, 6, or 7 days after transfection with a given nucleic acid molecule. Detection of inflammatory infiltrates of leukocytes can be performed using techniques known to those skilled in the art, preferably using immunofluorescence analysis by FACS.

[0082] The reduction in expression of proinflammatory receptors such as MAC-1, P-selectin, EPCR, and / or CD36 can be a reduction of at least 1%, 5%, 10%, 15%, 20%, 25%, or more. Detection of such receptors can be performed using techniques known to those skilled in the art, preferably by immunofluorescence analysis via FACS analysis. Such an increase can be assessed at least 3, 4, 5, 6, or 7 days after transfection with a given nucleic acid molecule.

[0083] The reduction in expression of proinflammatory receptors such as L-selectin, CD35, CD88, ICAM-1 and / or PECAM-1 can be a reduction of at least 1%, 5%, 10%, 15%, 20%, 25% or more. Such receptors can be detected using techniques known to the skilled artisan, preferably evaluated by immunofluorescence analysis using FACS analysis. Such an increase can be evaluated at least 3, 4, 5, 6, or 7 days after transfection with a given nucleic acid molecule.

[0084] The reduction in serum levels of fibrinogen (which is a positive acute phase protein) can be a reduction of at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more. Detection of fibrinogen can be assessed using techniques known to those skilled in the art, preferably as described in the experimental section. This reduction can be assessed after at least 3, 4, 5, 6, or 7 days following transfection with a given nucleic acid molecule.

[0085] The anticoagulant effect is preferably determined in a subject as:

[0086] - a detectable increase in APTT and / or a detectable increase in anticoagulant protein-S and / or a detectable increase in EPCR is observed, and / or

[0087] - a detectable increase in thrombomodulin and / or a detectable increase in protein C and / or a detectable increase in PECAM-1 is observed, and / or

[0088] - A detectable decrease in fibrin clot density and / or bleeding time is observed, and / or

[0089] - Reduction of thrombin formation, and / or

[0090] - a decrease in D-dimer, and / or

[0091] - Decreased heparan sulfate, and / or

[0092] - Patient survival is prolonged by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days, one month, several months or longer compared to those who are not treated or treated with a control, or compared to the subject at the start of treatment, and / or

[0093] - a detectable increase in fibrinolytic activity (increase in tPA, decrease in the complex between PAI-1 and tPA), and / or

[0094] - Detectable reduction in fibrinogen.

[0095] In the context of the present invention, the patient may be alive and may be considered disease-free. Alternatively, the disease or condition may be arrested, delayed or regressed.

[0096] The detectable increase of APTT and / or anticoagulant protein-S and / or EPCR can be an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more. APTT, anticoagulant protein-S and EPCR can be evaluated using known techniques, preferably as evaluated in the experimental part. Such an increase can be assessed at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, or 10 days after transfection with a given nucleic acid molecule.

[0097] The detectable increase in thrombomodulin and / or protein C and / or PECAM-1 may be an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more. Thrombomodulin, protein C and PECAM-1 can be assessed using known techniques, preferably as described in the experimental section. Such an increase can be assessed at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, or 10 days after transfection with a given nucleic acid molecule.

[0098] The detectable reduction in fibrin clot density and / or bleeding time can be at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more reduction. Fibrin clot density and bleeding time can be evaluated using known techniques, preferably as evaluated in the experimental section. This reduction can be evaluated after at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days after transfection with a given nucleic acid molecule.

[0099] The detectable reduction that thrombin forms can be at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more reduction.The formation of thrombin can use known technology to evaluate, preferably as experimental part evaluation.This increase can be evaluated after at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days with given nucleic acid molecule transfection.

[0100] In some embodiments, the present invention provides the method for the reduction of D-dimer and / or heparan sulfate that detects.D-dimer and / or heparan sulfate can be at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more reduction.D-dimer and / or heparan sulfate can use known technology to evaluate, preferably as experimental part evaluation.This increase can be evaluated after at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days with given nucleic acid molecule transfection.

[0101] The detectable increase in fibrinolytic activity (increase in tPA) can be an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more. Detection of free active tPA can be assessed using known techniques, preferably as described in the experimental section. Such an increase can be assessed at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days after transfection with a given nucleic acid molecule.

[0102] The detectable increase in fibrinolytic activity can also be assessed by a reduction in the complex between PAI-1 and tPA. The reduction can be a reduction of at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more. The detection of PAI-1 and tPA can be assessed using known techniques, preferably as described in the experimental section. Such reduction can be assessed at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, or 10 days after transfection with a given nucleic acid molecule.

[0103] In some embodiments, the present invention provides the method for the minimizing evaluation of fibrinolytic activity.The detectable increase of fibrinolytic activity can also be evaluated by the minimizing of fibrinogen.Described minimizing can be at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more minimizing.The detection of fibrinogen can use known technology to evaluate, preferably as experimental part evaluation.This minimizing can be evaluated after at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days with given nucleic acid molecule transfection.

[0104] Organ protection is preferably determined in a subject as:

[0105] - Detectable improvement in parameters associated with organ damage or failure, such as AST, ALT, GGT, ALP, LDH, creatine kinase, BUN, albumin, creatinine, glucose, bile acids, total bilirubin, conjugated bilirubin, phosphorus, total protein, LDL cholesterol, HDL cholesterol, cholesterol, and / or triglycerides. Improvement is defined as an increase or decrease in the parameter value, bringing it closer to the normal range for healthy subjects.

[0106] - Patient survival is prolonged by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days, one month, several months, or more compared to those who are untreated or treated with a control, or compared to the subject at the start of treatment.

[0107] In the context of the present invention, the patient may be alive and may be considered disease-free. Alternatively, the disease or condition may have been arrested, delayed or resolved.

[0108] A detectable improvement in AST and / or ALT and / or GGT and / or ALP and / or LDH and / or total bilirubin and / or conjugated bilirubin and / or BUN and / or creatine kinase and / or creatinine and / or bile acids and / or phosphorus and / or triglycerides and / or LDL cholesterol and / or HDL cholesterol and / or cholesterol may be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold or more reduction. AST, ALT, GGT, ALP, LDH, total bilirubin, conjugated bilirubin, BUN, creatine kinase, creatinine, bile acid, phosphorus, triglycerides, LDL cholesterol, HDL cholesterol and cholesterol can be evaluated using techniques known to those skilled in the art, preferably as described in the experimental section. Such reduction can be evaluated at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days after transfection with a given nucleic acid molecule.

[0109] The detectable improvement of albumin and / or total protein can be at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more increase.Albumin and total protein can be evaluated using techniques known to those skilled in the art, preferably as evaluated in the experimental section.This increase can be evaluated after at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, 10 days with a given nucleic acid molecule transfection.

[0110] The detectable improvement of glucose can be an increase or decrease of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 10 times or more. Glucose can be evaluated using technology known to those skilled in the art. Such an increase or decrease can be assessed at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, 78 hours, 82 hours, 86 hours, 90 hours, 94 hours, 4 days, 5 days, 6 days, 7 days, or 10 days after transfection with a given nucleic acid molecule.

[0111] Thus, in a preferred embodiment, the nucleic acid molecule as defined herein before is used as a medicament as an anti-inflammatory and / or anticoagulant and / or organ protective drug,

[0112] (a) wherein parameters associated with inflammation such as pro-inflammatory cytokine levels, pro-inflammatory integrin levels and / or the number of pro-inflammatory infiltrates have been reduced, and / or

[0113] (b) wherein parameters related to coagulation such as fibrin clot formation and / or thrombin concentration have been reduced, and / or

[0114] (c) wherein parameters associated with organ damage or failure such as AST, ALT, GGT, ALP, LDH, creatine kinase, BUN, albumin, creatinine, glucose, bile acids, total bilirubin, conjugated bilirubin, phosphorus, total protein, LDL cholesterol, HDL cholesterol, cholesterol and / or triglycerides have improved.

[0115] In a further preferred embodiment, the nucleic acid molecule as defined herein before is used as a medicament for use as an anti-inflammatory and / or anticoagulant and / or organ protective drug,

[0116] (a) wherein parameters associated with inflammation such as pro-inflammatory cytokine levels, pro-inflammatory integrin levels and / or the number of pro-inflammatory infiltrates have been reduced, and / or

[0117] (b) wherein parameters related to coagulation such as fibrin clot formation and / or thrombin concentration have been reduced, and / or

[0118] (c) wherein parameters associated with organ damage or failure such as AST, ALT, GGT, ALP, LDH, creatine kinase, BUN, albumin, creatinine, glucose, bile acids, total bilirubin, conjugated bilirubin, phosphorus, total protein, LDL cholesterol, HDL cholesterol, cholesterol and / or triglycerides have improved, and / or

[0119] (d) wherein parameters related to immune stimulation have been added.

[0120] Parameters related to immune stimulation are known to the skilled person and include the activation of immune cells, preferably T, B and / or NK cells.

[0121] In a further aspect, there is provided a nucleic acid molecule for use, preferably as previously defined herein, wherein the use is as a medicament for preventing, treating, resolving, curing and / or delaying a disease or condition in which inflammation and / or coagulation and / or organ damage or failure occurs, wherein the nucleic acid molecule is represented by a nucleotide sequence having at least 95% sequence identity to a nucleotide sequence comprising SEQ ID NO: 1 or 2. Each feature of this further aspect has been defined herein.

[0122] In a further aspect, there is provided the use of a nucleic acid or a composition comprising said nucleic acid molecule for the preparation of a medicament for preventing, treating, resolving, curing and / or delaying a disease or condition associated with inflammation and / or coagulation and / or organ damage or failure. Each of the features of this further aspect has been defined herein.

[0123] In a further aspect, a method is provided for preventing, treating, resolving, curing and / or delaying a condition or disease associated with inflammation and / or coagulation and / or organ damage or failure in a subject in need thereof. Each of the features of this further aspect has been defined herein.

[0124] In a preferred embodiment, the nucleic acid molecules for use, the compositions for use, the uses or the methods as defined herein are suitable for use in a disease or condition in which inflammation and / or coagulation and / or organ damage or failure occurs and are preferably selected from the group consisting of rheumatoid arthritis (RA), juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease including Crohn's disease or ulcerative colitis, hepatitis, sepsis, alcoholic liver disease, non-alcoholic steatosis, sarcoidosis, autoimmune diabetes, diabetes mellitus, uveitis, multiple sclerosis, controlling allograft rejection after organ transplantation, graft versus host disease (GVHD), including asthma and chronic obstructive pulmonary disease (COPD). Inflammatory lung diseases such as COPD, systemic lupus erythematosus (SLE), sarcoidosis, atopic dermatitis and cancer, acute coronary syndrome (ACS), thrombosis, peripheral vascular occlusion, obstructive atherosclerosis, vasculitis, functional impairment following cardiac surgery, complications from organ transplantation, angina pectoris, transient ischemic attack, pregnancy toxemia (preeclampsia, eclampsia), diabetes mellitus, hepatic veno-occlusive disease (VOD), deep vein thrombosis (DVT), septic shock, severe sepsis, trauma, acute respiratory distress syndrome (ARDS) and disseminated intravascular coagulation (DIC), or the effects of complications or progression associated with any of these diseases or conditions.

[0125] In another preferred embodiment, the nucleic acid molecule for use, the composition for use, the use or the method as defined herein is applicable to a disease or condition in which inflammation and / or coagulation and / or organ damage or failure occurs and is preferably selected from the group consisting of infection, poisoning, aspiration syndrome, hypoperfusion or shock, heat-induced illness, ischemia, ischemia-reperfusion injury (IRI), autoimmune diseases, hemorrhage, pancreatitis, bacteremia, burns, systemic inflammatory response syndrome (SIRS) and multiple organ failure or multiple organ dysfunction syndrome or the effects of a complication or progression associated with one of these diseases or conditions.

[0126] The preferred condition is DIC. The organ damage or failure is preferably liver damage or failure, kidney damage or failure.

[0127] Common definitions and common techniques mentioned in this article

[0128] Nucleic Acids

[0129] The term "nucleic acid" is well known in the art. "Nucleic acid" as used herein generally refers to a molecule (one or more chains) of DNA, RNA, or its derivatives or analogs comprising a nucleobase. Nucleobases include, for example, naturally occurring purine or pyrimidine bases in DNA (e.g., adenine "A," guanine "G," thymine "T," or cytosine "C") or in RNA (e.g., A, G, uracil "U," or C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide," each of which is a subgenus of the term "nucleic acid."

[0130] As used herein, "hybridization," "hybridizes," or "capable of hybridizing" should be understood to refer to the formation of double-stranded or triple-stranded molecules or molecules with partially double-stranded or triple-stranded properties using techniques known to those skilled in the art, such as southern blotting procedures. As used herein, the term "annealing" is synonymous with "hybridization." The terms "hybridization," "hybridize(s)," or "capable of hybridizing" may refer to "low," "medium," or "high" hybridization conditions as defined below.

[0131] Low to medium to high stringency conditions refer to prehybridization and hybridization in 5X SSPE, 0.3% SDS, 200 pg / mL sheared and denatured salmon sperm DNA, and 25%, 35%, or 50% formamide at 42°C for low to medium to high stringency, respectively. Subsequently, the hybridization reactions were washed three times using 2X SSC, 0.2% SDS, and 55°C, 65°C, or 75°C for low to medium to high stringency for up to 30 minutes each.

[0132] In some embodiments, the nucleic acid molecules of the present invention will comprise any of the nucleic acid molecules described in SEQ ID NO: 1 or 2. It is contemplated that the nucleic acid sequences of the present invention derived from SEQ ID NO: 1 or 2 may have, at least, or at most 85, 86, 87, 88, 89, 90, 91 consecutive nucleotides (or any range derivable therein) of SEQ ID NO: 1 or 2. In other embodiments, the nucleic acid is, at least, or at most 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% homologous to the nucleic acid sequence of SEQ ID NOs: 1 or 2.

[0133] Nucleobase (or base)

[0134] As used herein, "nucleobase" refers to a heterocyclic base, e.g., a natural nucleobase (i.e., A, T, G, C, or U) found in at least one naturally occurring nucleic acid (i.e., DNA and RNA), as well as naturally occurring or non-naturally occurring derivatives and analogs of such nucleobases. A nucleobase can typically form one or more hydrogen bonds ("anneal" or "hybridize") with at least one naturally occurring nucleobase in a manner that may replace naturally occurring nucleobase pairing (e.g., hydrogen bonds between A and T, G and C, and A and U).

[0135] "Purine" and / or "pyrimidine" nucleobases encompass naturally occurring purine and / or pyrimidine nucleobases and derivatives and analogs thereof, including, but not limited to, those of purines or pyrimidines substituted with one or more of an alkyl, carboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), sulfhydryl, or alkylthiol moiety. Preferred alkyl (e.g., alkyl, carboxyalkyl, etc.) moieties contain about 1, about 2, about 3, about 4, about 5, to about 6 carbon atoms. Other non-limiting examples of purines or pyrimidines include deazapurines, 2,6-diaminopurine, 5-fluorouracil, xanthine, hypoxanthine, 8-bromoguanine, 8-chloroguanine, bromothymine, 8-aminoguanine, 8-hydroxyguanine, 8-methylguanine, 8-thioguanine, azaguanine, 2-aminopurine, 5-ethylcytosine, 5-methylcytosine, 5-bromouracil, 5-ethyluracil, 5-iodouracil, 5-chlorouracil, 5-propyluracil, thiouracil, 2-methyladenine, methylthioadenine, N,N-dimethyladenine, azaadenine, 8-bromoadenine, 8-hydroxyadenine, 6-hydroxyaminopurine, 6-thiopurine, 4-(6-aminohexyl / cytosine), etc. Other examples are well known to those skilled in the art.

[0136] Nucleobases can be included in nucleosides or nucleotides using any chemical or natural synthesis method described herein or known to those of ordinary skill in the art.Such nucleobases can be labeled, or it can be part of a molecule that is labeled and includes the nucleobase.

[0137] Nucleosides

[0138] As used herein, "nucleoside" refers to a single chemical unit comprising a nucleobase covalently linked to a nucleobase linker moiety. Non-limiting examples of "nucleobase linker moieties" are sugars comprising 5-carbon atoms (i.e., "5-carbon sugars"), including but not limited to, deoxyribose, ribose, arabinose, or derivatives or analogs of 5-carbon sugars. Non-limiting examples of derivatives or analogs of 5-carbon sugars include 2'-fluoro-2'-deoxyribose or carbon-substituted carbocyclic sugars having an oxygen atom in the sugar ring.

[0139] Different types of covalent attachment of nucleobases to nucleobase linker moieties are known in the art. By way of non-limiting example, nucleosides comprising a purine (i.e., A or G) or a 7-deazapurine nucleobase typically covalently attach the 9-position of the purine or 7-deazapurine to the 1'-position of a 5-carbon sugar. In another non-limiting example, nucleosides comprising a pyrimidine nucleobase (i.e., C, T or U) typically covalently attach the 1-position of the pyrimidine to the 1'-position of a 5-carbon sugar (Kornberg and Baker, 1992).

[0140] Nucleotides

[0141] As used herein, "nucleotide" refers to a nucleoside that also includes a "backbone portion". The backbone portion typically covalently links the nucleotide to another molecule comprising a nucleotide or another nucleotide to form a nucleic acid. The "backbone portion" in naturally occurring nucleotides typically includes a phosphorus portion that is covalently linked to a 5-carbon sugar. The connection of the backbone portion typically occurs at the 3'- or 5'-position of the 5-carbon sugar. However, other types of connections are known in the art, particularly when the nucleotide includes derivatives or analogs of naturally occurring 5-carbon sugars or phosphorus portions.

[0142] Nucleic acid analogs

[0143] Nucleic acid can comprise a derivative or analog of a naturally occurring nucleic acid that may exist, a nuclear base, a nuclear base linker moiety and / or a backbone moiety or be composed entirely of it. RNA with nucleic acid analogs can also be labeled according to the method of the present invention. As used herein, "derivative" refers to a chemically modified or altered form of a naturally occurring molecule, and the term "simulation" or "analog" refers to a molecule that may or may not be similar to a naturally occurring molecule or part in structure and have a similar function. As used herein, "part" generally refers to the smaller chemical or molecular assembly of a larger chemical or molecular structure. Nuclear bases, nucleosides and nucleotide analogs or derivatives are well known in the art and have been described (see, e.g., Scheit, 1980, incorporated herein by reference).

[0144] Nucleic acid molecules can be further modified to obtain optimized / enough binding affinity. The primary sequence can also be modified. One / multiple hydrophobic moieties can also be added to the nucleic acid molecules to expand the diversity of its interaction with the target. All of these possibilities are described in detail in the document Hasegawa H et al., which is incorporated herein by reference.

[0145] The term "recombinant" may be used, and this generally refers to a molecule that has been manipulated in vitro, or is the replication or expression product of that molecule.

[0146] Recombination methods

[0147] Recombinant methods for producing nucleic acids in cells are well known to those skilled in the art. These include using vectors, plasmids, cosmids and other vectors that deliver nucleic acids to cells that may be target cells or simply host cells (to produce a large amount of desired RNA molecules). Alternatively, as long as there is a reagent for producing RNA molecules, such vectors may be used in the case of a cell-free system. Such methods include those described in Sambrook, 2003, Sambrook, 2001 and Sambrook, 1989, which are incorporated herein by reference. In certain embodiments, the present invention relates to non-synthetic nucleic acid molecules. In some embodiments, nucleic acid molecules have the chemical structure of naturally occurring nucleic acids and the sequence of naturally occurring nucleic acids. Except using recombinant techniques, such non-synthetic nucleic acids can be produced by chemical means, such as by employing the technology for producing oligonucleotides.

[0148] Host cells and target cells

[0149] The cell into which the nucleic acid molecule of the present invention is introduced or in which the presence of the molecule is assessed can be derived from or contained in any organism. Preferably, the cell is a vertebrate cell. More preferably, the cell is a mammalian cell. Even more preferably, the cell is a human cell.

[0150] Mammalian cells can be from the germline or somatic, totipotent or pluripotent, dividing or non-dividing epithelial, immortalized or transformed, etc. The cell can be an undifferentiated cell, such as a stem cell, or a differentiated cell, such as a cell from an organ or tissue. Alternatively, the cell can be characterized as an epithelial cell, brain, breast, cervix, colon, gastrointestinal tract, heart, kidney, large intestine, liver, lung, ovary, pancreas, heart, prostate, bladder, small intestine, stomach, testicle, or uterus.

[0151] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably. All of these terms also include their progeny, which are any offspring formed by cell division. It should be understood that all offspring may not be identical due to deliberate or unintentional mutations. Host cells can be "transfected" or "transformed," which refers to the process by which exogenous nucleic acids are transferred or introduced into host cells. Transformed cells include primary subject cells and their progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells are intended to refer to cells into which exogenous nucleic acid sequences, such as, for example, nucleic acid molecules or template constructs encoding reporter genes, have been introduced. Thus, recombinant cells may be distinguished from naturally occurring cells that do not contain recombinantly introduced nucleic acids.

[0152] Tissues can include host cells or cells to be transformed or contacted with nucleic acid delivery compositions and / or additional agents. Tissues can be part of an organism or separated from an organism. In certain embodiments, tissues and their constituent cells can include, but are not limited to, brain, stem cells, liver, lung, bone, breast, cervix, colon, endometrium, epithelium, esophagus, goblet cells, kidney, ovary, pancreas, prostate, bladder, skin, small intestine, stomach, testicles, heart.

[0153] In some embodiments, the host cell or tissue can be contained in at least one organism. In some embodiments, the organism can be a mammal, a human, a primate, or a murine. Those skilled in the art will further understand the conditions for cultivating all of the above-mentioned host cells to maintain them and allow them to divide and form offspring.

[0154] Delivery Method

[0155] In some embodiments, the present invention involves delivering nucleic acids to cells. This can be done as part of a therapeutic application.

[0156] RNA molecules can be encoded by nucleic acid molecules contained in vectors. The term "vector" is used to refer to vector nucleic acid molecules into which a nucleic acid sequence can be inserted and introduced into cells into which it can replicate. The nucleic acid sequence can be "exogenous," meaning that it is exogenous to the cell into which the vector is introduced, or the sequence is homologous to the sequence in the cell, but is in a position in the host cell nucleic acid where the sequence is not usually present. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, slow viruses, and plant viruses) and artificial chromosomes (e.g., YACs). By standard recombinant techniques, those skilled in the art will be well prepared to construct vectors, which are described in Sambrook et al., 1989 and Ausubel et al., 1996, both of which are incorporated herein by reference. In addition to encoding modified polypeptides such as modified gelatin, vectors can also encode unmodified polypeptide sequences such as markers or targeting molecules. Targeting molecules are molecules that direct desired nucleic acids to specific organs, tissues, cells, or other locations in the subject's body.

[0157] The term "expression vector" refers to a vector that contains a nucleic acid sequence encoding at least a portion of a gene product capable of being transcribed. Expression vectors may contain various "control sequences," which are nucleic acid sequences necessary for the transcription and possible translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that have other functions, which are described herein.

[0158] Administration and formulation

[0159] The nucleic acid molecules of the present invention may be present in a composition, preferably a pharmaceutical composition. The nucleic acid molecules of the present invention may be administered to a subject alone or in the form of a composition or pharmaceutical composition for the treatment of a condition or disease as defined herein. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that assist in processing the protein into a pharmaceutically acceptable preparation. Suitable formulations depend on the selected route of administration. For topical administration, the protein of the present invention may be formulated into solutions, gels, ointments, creams, suspensions, etc., as are well known in the art. Systemic formulations include those designed for administration by injection, for example, subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, and those designed for transdermal, transmucosal, inhalation, oral or pulmonary administration. For injection, the nucleic acid of the present invention may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, for example, Hanks' solution, Ringer's solution or a physiological saline buffer. The solution may include preparatons such as suspending agents, stabilizers and / or dispersants. Alternatively, the nucleic acid molecule can be in powder form for constitution with a suitable carrier, such as sterile pyrogen-free water, before use. For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are well known in the art. For oral administration, the nucleic acid can be easily formulated by combining the molecule with pharmaceutical carriers well known in the art. Such carriers enable the nucleic acid of the present invention to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral administration to the patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, for example, lactose, sucrose, mannitol and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP); granulating agents; and binders. If necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or its salt such as sodium alginate can be added. If necessary, solid dosage forms can be coated with sugar or enteric coating using standard techniques. For oral liquid preparations such as suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. In addition, flavorings, preservatives, colorants, etc. can be added. For oral administration, the molecule can be in the form of tablets, lozenges, etc. formulated in a conventional manner. In order to be administered by inhalation, molecules suitable for use according to the present invention use suitable propellants, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases, conveniently delivered in the form of an aerosol spray by a pressurized package or nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.Gelatin capsules and cartridges suitable for use in an inhaler or insufflator can be formulated containing a powder mix of the nucleic acid and a suitable powder base such as lactose or starch. The nucleic acid molecules can also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0160] In addition to the preparations described previously, the molecule can also be formulated into long-lasting preparations. Such long-acting preparations can be given by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Therefore, for example, the molecule can be formulated with suitable polymerization or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as a poorly soluble derivative, for example, as a poorly soluble salt. Alternatively, other drug delivery systems can be used.

[0161] Liposomes and emulsions are well-known examples of delivery vehicles that can be used to deliver the nucleic acids of the present invention.

[0162] The nucleic acids of the present invention can be administered in combination with a carrier or lipid to increase cellular uptake. For example, the nucleic acid molecules can be administered in combination with a cationic lipid. Examples of cationic lipids include, but are not limited to, lipofectin, DOTMA, DOPE, and DOTAP. The disclosure of WO0071096, specifically incorporated by reference, describes different formulations that can be effectively used for gene therapy, such as DOTAP; cholesterol or cholesterol derivative formulations. Other disclosures also discuss different lipid or liposome formulations, including nanoparticles and methods of administration. These include, but are not limited to, U.S. Patent Publications 20030203865, 20020150626, 20030032615, and 20040048787, which are specifically incorporated herein by reference to the extent they disclose formulations of nucleic acids and other related aspects of administration and delivery. Methods for forming particles are also disclosed in US Patent Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900, which are incorporated herein by reference. Nucleic acids can also be administered in combination with cationic amines such as poly-L-lysine.

[0163] Nucleic acids can also be conjugated to chemical moieties such as transferrin and cholesterol groups. In addition, oligonucleotides can be targeted to certain organelles by attaching specific chemical groups to the oligonucleotide. For example, attaching the oligonucleotide to an appropriate array of mannose residues will target the oligonucleotide to the liver. Other targeting ligands are described in Liu B., Brief Funct. Genomic Proteomic 6: 112-119, 2007. Additional examples are carbohydrate sugars such as galactose, N-acetylgalactosamine, mannose; vitamins such as folic acid; small molecules, including naproxen, ibuprofen or other known protein binding molecules, cyclodextrins, which target the transferrin receptor (Hu-Lieskovan et al., 2005), PEI (RGD-targeted PEG-PEI, Schifeleers et al., 2004), anisidine, RGD-peptide or RGD mimetic, polyarginine, anti-TfR single chain antibody fragment / TfRscFv, annexin A5 (targeting phosphatidylserine exposed membranes, Garnier et al., 2009, 11: 2114-22), WO 2009 / 126933 describing compositions and methods for site-directed delivery of nucleic acids by combining nucleic acids with targeting ligands and endolytic enzyme components. Targeting of nucleic acids can also be achieved by using aptamer technology as described in WO 2005 / 111238. Furthermore, other lipid moieties of the above-mentioned delivery vehicles, such as PEG-lipids, cholesterol, endolytic helper sugars or peptides (WO2009 / 046220) or the overall morphology of the resulting nanoparticles (characterized by charge and size) can confer targeting specificity.

[0164] In addition, this molecule can use sustained release system, as the semipermeable matrix of solid polymer containing therapeutic agent is delivered.Various slow release materials are established, and they are well known to those skilled in the art.Depending on its chemical property, slow release capsule can discharge this molecule and reach several weeks, until exceed 100 days.Depend on the chemical property and biological stability of chimeric molecule, can adopt other strategies for molecule stabilization.

[0165] Alternatively, the molecule may be delivered using a delivery system incorporating coordination chemistry as described in WO2007011217, which is expressly incorporated herein by reference.

[0166] Nucleic acids can be included in any of the above formulations as free acids or bases or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts that substantially retain the biological activity of the free bases and are prepared by reaction with inorganic acids. Pharmaceutically acceptable salts tend to be more soluble in water and other protic solvents than the corresponding free base forms.

[0167] The pharmaceutical composition of the present invention comprises an effective amount of nucleic acid molecules dissolved or dispersed in a pharmaceutical carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce or produce acceptable adverse, allergic or other adverse reactions when appropriately administered to animals, such as humans. Whether a specific adverse reaction is acceptable is determined based on the severity of the disease. According to the present disclosure, the preparation of a pharmaceutical composition comprising at least one nucleic acid molecule is known to those skilled in the art, see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, incorporated herein by reference. In addition, for administration to animals (e.g., humans), it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Bureau of Biological Standards.

[0168] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes and the like materials and combinations thereof, as known to those of ordinary skill in the art (e.g., see Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical composition is contemplated.

[0169] The nucleic acid molecule can comprise different types of carriers, depending on whether it is to be administered in solid, liquid, or aerosol form, and whether sterility is required for routes of administration such as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intraalveolarly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, topically, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion, by direct bathing of target cells, by catheter, by lavage, in cream, in lipid compositions (e.g., liposomes), or by other methods known to those of ordinary skill in the art or any combination thereof (e.g., see Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, incorporated herein by reference).

[0170] The actual dosage of the composition of the present invention administered to an animal or patient can be determined by physiological and physiological factors such as body weight, severity of the disease, type of disease being treated, previous or concurrent therapeutic interventions, patient's own disease state and route of administration. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.

[0171] In certain embodiments, the pharmaceutical composition can include, for example, at least about 0.1% of the active compound. In other embodiments, the active compound can account for, for example, about 2% to about 75% of the weight of the unit, or about 25% to about 60%, and any range derivable therein. In other non-limiting examples, dosage can also include each administration of less than 1 microgram / kg body weight, or 5 micrograms / kg body weight, 10 micrograms / kg body weight, 50 micrograms / kg body weight, 100 micrograms / kg body weight, 200 micrograms / kg body weight, 350 micrograms / kg body weight, 500 micrograms / kg body weight, 1 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, 50 mg / kg body weight, 100 mg / kg body weight, 200 mg / kg body weight, 350 mg / kg body weight, or 500 mg / kg body weight, to 1000 mg / kg body weight or more, and any range derivable therein. In non-limiting examples of ranges derivable from the numbers listed herein, ranges of 5 mg / kg to 100 mg / kg, 5 micrograms / kg to 500 mg / kg, etc. can be administered based on the numbers described above.

[0172] In any case, the composition may contain various antioxidants to prevent oxidation of one or more components. Additionally, prevention of the action of microorganisms may be achieved by preservatives such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0173] The compounds can be formulated into compositions in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, for example, those formed with free amino groups of the protein composition, or with inorganic acids such as hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxides; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

[0174] In embodiments where the composition is in liquid form, the carrier can be a solvent or dispersion medium, including, but not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin; by maintaining the desired particle size by dispersing in a carrier such as a liquid polyol or lipid; by using a surfactant such as hydroxypropylcellulose; or a combination of such methods. In many cases, it is preferred to include an isotonic agent, such as a sugar, sodium chloride, or a combination thereof.

[0175] In other embodiments, eye drops, nasal drops or sprays, aerosols or inhalers can be used. Such compositions are generally designed to be compatible with the target tissue type. In non-limiting examples, nasal solutions are generally aqueous solutions designed to be administered to the nasal passages in the form of drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, thereby maintaining normal ciliary action. Therefore, in preferred embodiments, nasal aqueous solutions are generally isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, if desired, antimicrobial preservatives similar to those used in ophthalmic preparations, drugs or suitable drug stabilizers may be included in the formulation. For example, various commercially available nasal preparations are known and include drugs such as antibiotics or antihistamines. In certain embodiments, the molecules are prepared for administration by routes such as oral ingestion. In these embodiments, solid compositions can include, for example, solutions, suspensions, emulsions, tablets, pills, capsules (for example, hard or soft shell gelatin capsules), sustained release formulations, buccal compositions, lozenges, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions can be directly mixed with the food in the diet. Preferred carriers for oral administration include inert diluents, absorbable edible carriers, or combinations thereof. In other aspects of the present invention, oral compositions can be made into syrups or elixirs. Syrups or elixirs can include, for example, at least one active agent, sweetener, preservative, flavoring, dye, preservative, or combinations thereof.

[0176] In certain preferred embodiments, oral compositions may include one or more binders, excipients, disintegrants, lubricants, flavorings, and combinations thereof. In certain embodiments, the composition may include one or more of the following: binders, such as gum tragacanth, gum arabic, corn starch, gelatin, or a combination thereof; excipients, such as dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or a combination thereof; disintegrants, such as corn starch, potato starch, alginic acid, or a combination thereof; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose, saccharin, or a combination thereof; flavorings, such as peppermint, oil of wintergreen, cherry flavoring, orange flavoring, or the like, or a combination thereof. When the dosage unit form is a capsule, in addition to the above-mentioned types of materials, it may also include a carrier, such as a liquid carrier. Various other materials may be present as a coating or otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both.

[0177] The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be minimized to maintain safe levels, for example, less than 0.5 ng / mg protein.

[0178] In a specific embodiment, prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate, gelatin, or a combination thereof.

[0179] Any of the embodiments discussed above with respect to delivery or trafficking to cells can also be employed with respect to practicing the delivery of pharmaceutical compounds discussed in this section.

[0180] Effective dose

[0181] The molecules of the present invention will generally be used in an amount effective to achieve the intended purpose. For use in treating or preventing a disease condition, the molecules of the present invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. A therapeutically effective amount is an amount effective to ameliorate or prevent a symptom or parameter associated with inflammation or coagulation, as previously defined herein, or to prolong the survival of the patient being treated. Determination of a therapeutically effective amount, especially in light of the detailed disclosure provided herein, is well within the capabilities of those skilled in the art.

[0182] For systemic administration, the therapeutically effective dose can be initially estimated from in vitro assays. For example, a dose can be formulated in an animal model to achieve a circulating concentration range that includes the EC50 determined in cell culture. Such information can be used to more accurately determine a dose useful in humans.

[0183] Initial doses can also be estimated based on in vivo data, for example, animal models, using techniques well known in the art. One of ordinary skill in the art can readily optimize administration to humans based on animal data.

[0184] Dosage and interval can be adjusted individually to provide plasma levels of the molecule sufficient to maintain therapeutic effect. Typical patient dosages administered by injection range from 0.01 to 0.1 mg / kg / day, or 0.1 to 5 mg / kg / day, preferably 0.5 to 1 mg / kg / day or higher. Therapeutically effective serum levels can be achieved by multiple daily doses.

[0185] In cases of local administration or selective uptake, the effective local concentration of the protein may not be related to plasma concentration. One skilled in the art will be able to optimize the therapeutically effective local dose without undue experimentation.

[0186] The amount of the molecule administered will, of course, be dependent on the subject being treated, the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

[0187] Treatment may be repeated intermittently when symptoms are detectable or even when no symptoms are detectable. Treatment may be given alone or in combination with other drugs or treatments, including surgery.

[0188] toxicity

[0189] Preferably, a therapeutically effective dose of the molecules described herein will provide therapeutic benefit without causing substantial toxicity. The toxicity of the molecules described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxicity and therapeutic efficacy is the therapeutic index. Proteins exhibiting high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is nontoxic to humans. The dosage of the proteins described herein is preferably within a circulating concentration range that includes an effective dose with minimal or no toxicity. The dosage can vary within this range, depending on the dosage form employed and the route of administration used. The exact formulation, route of administration, and dosage can be selected by the individual physician based on the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p1).

[0190] Pendant groups

[0191] "Pendant groups" can be connected or conjugated to nucleic acids. Pendant groups can increase the uptake of nucleic acids by cells. Pendant groups can be attached to any part of the nucleic acid, but are typically attached to the end of the oligonucleotide chain. Examples of pendant groups include, but are not limited to: acridine derivatives (i.e., 2-methoxy-6-chloro-9-aminoacridine); cross-linking agents such as psoralen derivatives, azidobenzoyl, proflavine and azidoproflavine; artificial endonucleases; metal complexes such as EDTA-Fe(II), o-phenanthroline-Cu(I) and porphyrin-Fe(II); alkylating moieties; nucleases such as amino-1-hexanol staphylococcal nuclease and alkaline phosphatase; terminal transferases; antibody enzymes; cholesterol moieties; lipophilic carriers; peptide conjugates; long-chain alcohols; phosphates; amino groups; sulfhydryl groups; radiolabels; non-radioactive labels such as dyes; and polylysine or other polyamines. In one example, the nucleic acid is conjugated to a carbohydrate, a sulfated carbohydrate or a polysaccharide.

[0192] Sequence identity

[0193] "Sequence identity" is defined herein as the relationship between two or more nucleic acid (nucleotide, polynucleotide, RNA, DNA) sequences as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM Those in J. Applied Math., 48: 1073 (1988).

[0194] Preferred methods for determining identity are designed to provide the largest match between the sequences tested. Methods for determining identity and similarity are incorporated into publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Mol. Biol. 215: 403-410 (1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0195] Preferred parameters for nucleic acid alignment include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison Matrix: Number of Matches = +10, Number of Mismatches = 0; Gap Penalty: 50; Gap Length Penalty: 3. The GAP program available from Genetics Computer Group in Madison, Wisconsin, is the default parameter for nucleic acid alignment.

[0196] In this document and in its claims, the verb "to comprise" and its conjugations are used in their non-limiting sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb "consisting of" can be replaced by "consisting essentially of" to indicate that the nucleic acid molecules defined herein or the compositions comprising the nucleic acid molecules may contain one or more other components in addition to those specifically identified, and the one or more other components do not alter the unique characteristics of the invention. In addition, the verb "consisting of" can be replaced by "consisting essentially of" to indicate that the methods defined herein may include one or more other steps in addition to the steps specifically identified, and the one or more other steps do not alter the unique characteristics of the invention. In addition, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element be present. Thus, the indefinite article "a" or "an" generally means "at least one".

[0197] All patent and literature references cited in this specification are hereby incorporated by reference in their entirety.

[0198] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0199] Figure 1 : Anticoagulant activity of SEQ ID NO:2 in the tail bleeding model. Rats were intravenously injected with SEQ ID NO:2 at dose levels of 5, 20, and 40 mg / kg saline solution. Rivaroxaban and warfarin were used as positive controls. Rivaroxaban was given by oral gavage 1 hour before the bleeding time test. Bleeding time was measured in male (A) and female (B) rats 5 minutes after injection or 1 hour after gavage. Each bar represents mean ± SE (one-way ANOVA, followed by post-test Dunnett's multiple comparisons; *p≤0.05, **p<0.01, and ***p<0.001).

[0200] Figure 2 :The anticoagulant activity of SEQ ID NO:2 in the arteriovenous shunt model. According to their body weight, male rats were intravenously injected with saline (vehicle) or SEQ ID NO:2 solution. Rivaroxaban was orally administered to the positive control group. 5-90 minutes after administration, the rats were anesthetized with pentobarbital sodium (pentobarbital-na) (50 mg / kg). The left jugular vein and right carotid artery of the anesthetized rats were connected with two catheters respectively. The weight of the thrombus formed on the line was calculated by subtracting the average weight of the thick nylon line (A). Prothrombin time (B) was measured, and activated partial thromboplastin time (APTT) (C) was analyzed. Each bar represents mean ± SEM (for vehicle and rivaroxaban groups, n = 8, for SEQ ID NO:2 group, n = 3), relative to vehicle, *p < 0.05, **p < 0.01, ***p < 0.001.

[0201] Figure 3 Figure 2: Anticoagulant activity of SEQ ID NO:2 in an arterial thrombosis model induced by ferric chloride. Before ferric chloride induced arterial thrombosis, mice were treated with 3 doses of SEQ ID NO:2. Five minutes before the ferric chloride test, mice were intravenously injected with NO:2 at a dosage level of 5, 20, and 40 mg / kg. Arterial thrombosis was performed to measure thrombosis weight (A) and size (B). Each bar represents mean ± SE (two-way ANOVA statistical analysis followed by Bonferroni post hoc comparisons; *p<0.05; **p<0.01; ***p<0.001).

[0202] Figure 4: Anti-inflammatory cytokine production induced by LPS injection in C57BL / 6 mice. Mice were given vehicle (negative control), dexamethasone (positive control) or customer compound (customer compound) (one of 3 doses) and then injected with LPS 5 minutes later. Mice were bled two (2) hours after LPS injection and serum was separated. The concentrations of IL-10, TNF, IL-6 and IL-1β in serum were measured using eBioscience's Luminex kit. Each bar represents mean ± SEM (n = 8 for vehicle and rivaroxaban groups, n = 3 for SEQ ID NO: 2 group), *p < 0.05, **p < 0.01, ***p < 0.001 relative to vehicle.

[0203] Figure 5 :The effect of SEQ ID NO:2 on the cellular immune response induced by LPS injection in C57BL / 6 mice. SEQ ID NO:2 was injected into C57BL / 6 mice at doses of 5, 25 and 50 mg / kg 5 minutes before intravenous LPS injection. Two hours after LPS injection, blood samples were collected. Blood samples were prepared for flow cytometry analysis to determine the expression of CD11b and CD14. The amount of CD11b+CD14+ and CD11b+CD14- leukocytes was evaluated. Each bar represents the mean ± SEM (for vehicle and rivaroxaban groups, n = 8, for SEQ ID NO:2 group, n = 3), relative to vehicle, *p < 0.05, **p < 0.01, ***p < 0.001.

[0204] Figure 6 : Effect of post-LPS treatment on survival in C57BL / 6 mice. Female C57BL / 6 mice were injected intravenously twice with SEQ ID NO: 2 at 10, 25, 50, and 100 mg / kg. The first injection was given 15 minutes after LPS injection, and the second was given 1 hour after LPS injection. A sixth group received two injections, 60 minutes and 2 hours after LPS administration. Survival was determined 72 hours after LPS injection (A). Percent survival is plotted every 4 hours in (B) (n=20 mice / group). Survival was analyzed using the standard Mantel-Cox log-rank test.

[0205] Figure 7: Effect of SEQ ID NO:2 treatment on serum LPS-induced mortality in C57BL / 6 mice: Extended toxicology analysis. Female C57BL / 6 mice were injected intravenously twice with SEQ ID NO:2 at 10, 25, 50, and 100 mg / kg. The first injection was given 15 minutes after LPS injection, and the second was given 1 hour after LPS injection. Group 7 received two injections 60 minutes and 2 hours after LPS administration. (A) Serum AST, ALT, creatine kinase, BUN, and phosphorus concentrations in each group. Blood was collected 28 hours later. (B) Serum albumin, creatinine, total protein, cholesterol, and triglyceride concentrations in each group. Blood was collected 28 hours later. P values ​​were calculated by t-test. P values ​​for Group 2 (vehicle) are given relative to Group 1 (vehicle, no LPS). P values ​​for all other groups were determined relative to Group 2 (vehicle).

[0206] Figure 8 : Effect of treatment with SEQ ID NO: 2 on serum fibrinogen and D-dimer analysis of LPS-induced death in C57BL / 6 mice. Female C57BL / 6 mice were injected intravenously twice with SEQ ID NO: 2 at 10, 25, 50, and 100 mg / kg. The first injection was given 15 minutes after LPS injection, and the second was given 1 hour after LPS injection. Group 7 received two injections 60 minutes and 2 hours after LPS administration. Serum fibrinogen and D-dimer levels are shown. Blood was collected 28 hours later. P values ​​were calculated by t-test. The P value of Group 2 (vehicle) is given relative to Group 1 (vehicle, no LPS). The P values ​​of all other groups were determined relative to Group 2 (vehicle). DETAILED DESCRIPTION

[0207] Example

[0208] Study 1: Anticoagulant Effect of SEQ ID NO: 2

[0209] Example 1: Anticoagulant activity of SEQ ID NO: 2

[0210] Tail bleeding model

[0211] Materials and methods

[0212] rats

[0213] A total of 36 Sprague-Dawley male and female rats were used and divided into 6 groups of 6 animals each. Animal handling was performed according to the guidelines of the National Institute of Health and the Association for Assessment and Accreditation of Laboratory Animal Care. Animals had free access to a commercially available rodent diet (Teklad certified global 18% protein diet catalog number: 2018SC). Animals had free access to autoclaved and acidified drinking water (pH 2.5-3.5) obtained from a municipal supply. Animals were raised under standard laboratory conditions, and the air was conditioned and filtered with an adequate source of fresh air. Animals were kept in a climate-controlled environment.

[0214] Compounds and preparations

[0215] SEQ ID NO: 2 (Biospring) (100 mg) was dissolved in 5 mL of saline to form a 20 mg / mL stock solution. The vehicle (saline) was provided by Pharmaseed for IV administration. Rivaroxaban (Bayer, Pharmaseed Ltd) dosing solution was diluted to 10 mg / kg (Group 2M). Warfarin (Ceriliant, Pharmaseed Ltd) was diluted to 0.5 mg / kg (Group 3M). Both rivaroxaban and warfarin are clinical standards with anticoagulant activity. To obtain a 5 mg / kg dose level and 2 mg / kg dose volume of SEQ ID NO: 2 dosing solution (2.5 mg / mL) for IV injection, 0.375 mL of the above 20 mg / mL stock solution was diluted with 2.625 mL of saline (Group 4M). To obtain a 20 mg / kg dose level and a 2 mL / kg dose volume of SEQ ID NO: 2 dosing solution (10 mg / mL) for iv injection, 1.5 mL of the above 20 mg / mL stock solution was diluted with 1.5 mL of saline (Group 5M). To obtain a 40 mg / kg dose level and a 2 mL / kg dose volume of SEQ ID NO: 2 dosing solution (20 mg / mL) for iv injection, the above 20 mg / mL stock solution was used (Group 6).

[0216] Compound administration

[0217] Five minutes before the bleeding time test, rats were injected intravenously. The number of rats in each group is shown in Table 1. Three treatment groups received SEQ ID NO:2 at dosage levels of 5, 20, and 40 mg / kg saline solution. In each gender, the control group received saline vehicle. The positive control group received rivaroxaban (10 mg / kg). Rivaroxaban was given by oral gavage 1 hour before the bleeding time test. The second positive control group received warfarin (0.5 mg / kg).

[0218]

[0219] Table 1 – Treatment options

[0220] Tail bleeding time measurement

[0221] Male and female rats were anesthetized with an intraperitoneal injection of ketamine / xylazine. Bleeding time was monitored as described by Stupnisek et al. (2012) until blood flow stopped for a complete 30-second interval or until the end of the 30-minute period. In deeply anesthetized rats (placed in the ventral position), the tail was cut with a scalpel 2 mm from the tip (and vertically immersed in a tube filled with 40 mL of saline at room temperature), and the duration and amount of bleeding were measured to evaluate the hemostatic effect of drug or saline administration. After the completion of the bleeding time evaluation, all rats were immediately killed by CO2 asphyxiation.

[0222] Statistical analysis

[0223] Numerical results are given as mean ± standard error. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison post hoc test, where applicable. A probability of 5% (p ≤ 0.05) was considered significant. In the figures, the degree of statistically significant differences between the groups is shown as *p ≤ 0.05, **p < 0.01, and ***p < 0.001.

[0224] Results and discussion

[0225] The antithrombotic activity of SEQ ID NO: 2 administered intravenously at three different doses was evaluated in rats. Figure 1 As shown, compared with the matched vehicle group (1M), in males ( Figure 1 A) and female ( Figure 1 B) In all treatment groups, the mean bleeding time was significantly increased. In the positive control group (rivaroban and warfarin) and in male rats ( Figure 1 A) and female rats ( Figure 1Comparable bleeding times were measured across all doses of SEQ ID NO: 2 tested in B). In matched treatment groups, bleeding times were similar for male and female rats. A dose-response effect of SEQ ID NO: 2 was observed in male rats but not in female rats.

[0226] SEQ ID NO: 2 showed a significant anticoagulant effect on bleeding time in both male and female rats. However, a dose-responsive anticoagulant effect of SEQ ID NO: 2 was observed only in male rats. The anticoagulant activity of SEQ ID NO: 2 at all doses was equivalent to that of the clinical standard of care (rivaroban and warfarin).

[0227] Example 2: Anticoagulant activity of SEQ ID NO: 2.

[0228] Arteriovenous shunt model

[0229] Materials and methods

[0230] rats

[0231] This study was conducted at WuXi AppTec Co., Ltd. in Shanghai according to WuXi IACUC standard animal procedures and in compliance with the Animal Welfare Act, the IACUC guidelines of the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare (OLAW). Sprague-Dawley male rats (Vital River; Beijing, PR China) weighing 150-200 g were fed a rodent diet ad libitum. The animals had free access to autoclaved and acidified drinking water. The animals were housed under standard laboratory conditions, with air conditioning and filtration using an adequate fresh air source. The animals were housed in a climate-controlled environment.

[0232] Compound

[0233] Rivaroxaban (10 mg / kg) was dissolved in water and stored at 4° C. 20 mg of SEQ ID NO: 2 (Biospring) was dissolved in 3 mL of saline, vortexed for 5 minutes, and sonicated in a water bath for 1 minute to achieve 6.66 mg / mL of SEQ ID NO: 2.

[0234] SEQ ID NO: 2 gives

[0235] Animals were divided into three dose groups according to body weight (see Table 2). 90 minutes before the shunt test, animals receiving vehicle treatment were intravenously injected with saline. 90 minutes before the shunt test, animals in the positive control group were orally administered rivaroxaban 10 mg / kg. 5 minutes before the shunt test, SEQ ID NO: 2 was injected intravenously. The dose volume is shown in Table 2.

[0236]

[0237] Table 2 – Treatment options

[0238] Thrombometry (arteriovenous shunt test)

[0239] 60 minutes before the shunt test, rat was anesthetized with sodium pentobarbital (ip, 50mg / kg, 20mg / mL, 2.5mL / kg).Separate left jugular vein and right common carotid artery, and with two 6cm long PE-60 catheter cannulas that are full of saline.Polyethylene catheter (American Health and Medical Supply International Corp.) is connected with the PE-160 polyethylene tube of 8cm long thick nylon line (size: 3-0, weight 3.5mg) that contains the 6cm long that is folded into two strands.Keep extracorporeal circulation 15 minutes, calculate the thrombus weight that forms on the line by the average weight of deducting thick nylon line.

[0240] Activated partial thromboplastin time (APTT) measurement

[0241] After the thrombus was removed, a blood sample (2 mL) was immediately drawn from the carotid artery catheter and collected into a plastic tube containing 1 / 10 volume of 3.2% trisodium citrate. Subsequently, the sample was centrifuged at 7000 rpm for 10 minutes at 4°C to collect plasma. Samples for APTT analysis were analyzed in duplicate using a BCS-XP coagulation analyzer (Siemens, Marburg, Germany) according to the manufacturer's protocol. The reagents used were the APTT reagent Actin FSL (Siemens, Marburg, Germany) and sterile 0.9% NaCl.

[0242] Statistical analysis

[0243] The significance of the differences between the control and treatment groups was analyzed by analysis of variance (ANOVA) using EXCEL office software. A P value of less than 0.05 was considered statistically significant.

[0244] Results and discussion

[0245] like Figure 2As shown in Figure A, rats treated with SEQ ID NO: 2 showed significantly lower thrombus weight compared to the vehicle group (48.1% less than the vehicle group). The positive control group given a dose of rivaroxaban 90 minutes before extracorporeal circulation also had significantly less thrombus formation (26.5% less than the vehicle group).

[0246] like Figure 2 As shown in B, the positive control rivaroxaban significantly prolonged the prothrombin time, while the SEQ ID NO: 2 group did not show any significant difference compared with the vehicle group. Figure 2 As shown in Figure C, rats treated with SEQ ID NO: 2 showed a significant increase in activated partial thromboplastin time (APTT), while the positive control, rivaroxaban, did not show any difference compared to the vehicle group. These positive effects of SEQ ID NO: 2 on APTT have been confirmed by comparable in vitro studies in mice, dogs, non-human primates, and humans.

[0247] In the rat arteriovenous shunt model, SEQ ID NO: 2 significantly reduced thrombus weight, demonstrating its anti-thrombotic effect. The positive compound rivaroxaban also significantly inhibited thrombus formation, but its effect was not as effective as that of compound SEQ ID NO: 2.

[0248] Although SEQ ID NO: 2 had no effect on prothrombin time, it did prolong the activated partial thromboplastin time (APTT). This may indicate that SEQ ID NO: 2 inhibits thrombosis mainly due to the prolongation of the activated partial thromboplastin time, while rivaroxaban mainly prolongs the prothrombin time.

[0249] Example 3: Anticoagulant activity of SEQ ID NO: 2 in a ferric chloride-induced arterial thrombosis model

[0250] Materials and methods

[0251] rats

[0252] Animal care was performed according to the guidelines of the National Institute of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). A total of 36 Sprague–Dawley rats (Envigo RMS) were divided into 6 groups, each including 6 animals (3 males and 3 females). At the beginning of the study, Sprague–Dawley male rats had an average body weight of 350 g. Female rats had a body weight of 243 g. Both were fed a rodent diet ad libitum. Animals had free access to autoclaved and acidified drinking water. Animals were housed under standard laboratory conditions, with air conditioned and filtered with an adequate source of fresh air. Animals were maintained in a climate-controlled environment.

[0253] Compounds and preparations

[0254] SEQ ID NO: 2 (Biospring) (100 mg) was dissolved in 5 mL of saline to form a 20 mg / mL stock solution. Vehicle (saline, Teva) was prepared for IV administration. Rivaroxaban (Bayer, Pharmaseed Ltd) dosing solution was diluted to 10 mg / kg (Group 2M). Warfarin (Ceriliant, Pharmaseed Ltd) was diluted to 0.5 mg / kg (Group 3M). Both rivaroxaban and warfarin are clinical standards with anticoagulant activity. To obtain a 5 mg / kg intravenous dose level and a 2 mL / kg dose volume of SEQ ID NO: 2 dosing solution (2.5 mg / mL) for IV injection, 0.375 mL of the above 20 mg / mL stock solution was diluted with 2.625 mL of saline (Group 4M). To obtain a 20 mg / kg dose level and a 2 mL / kg dose volume of SEQ ID NO: 2 dosing solution (10 mg / mL) for iv injection, 1.5 mL of the above 20 mg / mL stock solution was diluted with 1.5 mL of saline (Group 5M). To obtain a 40 mg / kg dose level and a 2 mL / kg dose volume of SEQ ID NO: 2 dosing solution (20 mg / mL) for iv injection, the above 20 mg / mL stock solution was used (Group 6).

[0255] Compound administration

[0256] The study was divided into six groups, each group including six rats (3 males and 3 females). In each sex, 1 male rat and 1 female rat of the control group were intravenously (iv) injected with vehicle. 1 hour before the iron chloride test, the first positive control group (2 males and 2 females) was orally treated with rivaroxaban (10mg / kg). 5 minutes before the iron chloride test, the second positive control group, including 3 males and 3 female rats, was intravenously injected with warfarin (0.5mg / kg). Finally, 5 minutes before the iron chloride test, SEQ ID NO:2 was intravenously injected to three groups (composed of 4-6M and 4-6F) at a dosage level of 5, 20 and 40mg / kg.

[0257] Ferric chloride-induced arterial thrombosis

[0258] Male and female rats were anesthetized with an intraperitoneal injection of ketamine / xylazine. Arterial thrombosis was performed as described by Lee J. et al., (2009). The right carotid artery was isolated and dissected to remove the vagus nerve and surrounding tissue. Arterial thrombosis was performed by wrapping a 2 mm saturated with 50% ferric chloride (FeCl3; w / v, in distilled water) around the right carotid artery. 2 The time required for occlusion to occur was measured up to 60 minutes, and vessels that occluded within this time were assigned an occlusion time of 60 minutes.

[0259] Clot formation measurement

[0260] After the application of FeCl3, clot formation was allowed to proceed for 60 minutes. Immediately following the completion of the clotting time evaluation, all rats were sacrificed with a Pental overdose. The arterial wall was longitudinally incised. The clot was removed, weighed, and measured with a caliper.

[0261] Statistical analysis

[0262] Numerical results are expressed as mean ± standard error. Statistical analysis was performed using two-way or one-way ANOVA, followed by a Bonferroni post hoc test, if applicable. A probability of 5% (p ≤ 0.05) was considered significant. In the accompanying figures, the degree of statistically significant differences between the groups is shown as *p ≤ 0.05, **p < 0.01, and ***p < 0.001.

[0263] Results and discussion

[0264] FeCl3 was applied for 60 minutes for clot formation. Compared with the control group (vehicle), clot weight ( Figure 3 A) and size ( Figure 3Compared with the rivaroxaban positive control and the high dose of SEQ ID NO: 2, the lowest dose (5 mg / kg) of SEQ ID NO: 2 in male rats treated with clot size ( Figure 3 B) and weight ( Figure 3 A) showed statistically significant differences.

[0265] Study 2. Anti-inflammatory activity of SEQ ID NO: 2.

[0266] Example 4: Effects of post-LPS treatment on cytokine release in C57BL / 6 mice.

[0267] Materials and methods

[0268] rats

[0269] Female C57BL / 6 mice (Taconic Biosciences) were 8 weeks old at the start of the study. Animals had free access to autoclaved, acidified drinking water. Animals were housed under standard laboratory conditions, with air conditioning and filtered air supplied by an adequate fresh air source. Animals were maintained in a climate-controlled environment.

[0270] Compound

[0271] SEQ ID NO: 2 (Biospring) (100 mg) was dissolved in 5 mL of saline to form a 20 mg / mL stock solution. The vehicle was saline solution.

[0272] A dexamethasone stock solution was prepared by mixing dexamethasone and β-cyclodextrin at a ratio of 4:96, dissolving in PBS at 2 mg dexamethasone / mL, and storing at -20°C. The dexamethasone dosing solution was prepared by diluting the stock solution to 1 mg / mL with PBS. Dexamethasone administered ip at 5 to 10 mg / kg was used as a positive control for immune response suppression.

[0273] Compound administration

[0274] Five minutes before the intravenous injection of lipopolysaccharide (LPS) (100 ng / 200 μl), the mice were dosed as shown in Table 3.

[0275] Group #mouse deal with dose way frequency volume Purpose 1 10 carrier - iv once 10 mL / kg Negative control 2 10 Dexamethasone 10 mg / kg ip once 10 mL / kg Positive control 3 10 SEQ ID NO:2 5mg / kg iv once 10 mL / kg test 4 10 SEQ ID NO:2 25 mg / kg iv once 10 mL / kg test 5 10 SEQ ID NO:2 50 mg / kg iv once 10 mL / kg test

[0276] Table 3 – Treatment options

[0277] Cytokine measurement

[0278] Two hours after LPS injection, mice were bled, serum isolated, and added to EDTA in a Gel Clot Activator tube. A minimum of 100 μL was analyzed. Serum concentrations of IL-10, TNF, IL-6, and IL-1β were measured using Luminex kits from eBioscience according to the manufacturer's protocol. A single analysis was performed for each sample.

[0279] result

[0280] To determine the anti-inflammatory activity of SEQ ID NO: 2, mice were treated with SEQ ID NO: 2 prior to LPS stimulation. Serum samples from treated mice were evaluated for various pro-inflammatory cytokines (TNF, IL-6, and IL-1β) and the anti-inflammatory cytokine IL-10.

[0281] The serum concentration of IL-10 is shown in Figure 4 A. Compared with the vehicle group, an increase in IL-10 was observed for SEQ ID NO: 2, and the difference was significant in the groups administered with 25 mg / kg and 50 mg / kg.

[0282] Depend on Figure 4 As can be seen from Figures 4B, 4C, and 4D, the group administered with SEQ ID NO: 2 at 5 mg / kg had a cytokine concentration in serum similar to that of the vehicle group.

[0283] The groups administered SEQ ID NO: 2 at doses of 25 and 50 mg / kg had lower TNF, IL-6, and IL-1β concentrations in serum, but only the reduction in IL-1β concentration in the group administered 50 mg / kg was statistically significant compared to the vehicle group.

[0284] Taken together, these results demonstrate that SEQ ID NO: 2 has anti-inflammatory properties in the LPS-induced cytokine production model.

[0285] Example 5: Effect of SEQ ID NO: 2 treatment on cell-based immune responses after LPS injection in C57BL / 6 mice Influence.

[0286] Materials and methods

[0287] mice

[0288] Female C57BL / 6 mice (Taconic Biosciences) were 8 weeks old at the start of the study. Animals had free access to autoclaved, acidified drinking water. Animals were housed under standard laboratory conditions, with air conditioning and filtered air supplied by an adequate fresh air source. Animals were maintained in a climate-controlled environment.

[0289] Compound

[0290] SEQ ID NO: 2 (Biospring) (100 mg) was dissolved in 5 mL of saline to form a 20 mg / mL stock solution. The vehicle was saline solution.

[0291] A dexamethasone stock solution was prepared by mixing dexamethasone and β-cyclodextrin at a ratio of 4:96, dissolving in PBS at 2 mg dexamethasone / mL, and storing at -20°C. The dexamethasone dosing solution was prepared by diluting the stock solution to 1 mg / mL with PBS. Dexamethasone administered ip at 5-10 mg / kg was used as a positive control for immune response suppression.

[0292] Compound administration

[0293] Five minutes before the intravenous injection of lipopolysaccharide (LPS) (100 ng / 200 μL), the mice were dosed as shown in Table 4.

[0294]

[0295]

[0296] Table 4 – Treatment options

[0297] Flow cytometry

[0298] Two hours after LPS injection, mice were bled and blood samples were collected. Flow cytometry studies were performed on a BD FACSCalibur (lasers: 488 nm; 635 nm and UV). In our study, we measured the expression of the integrin Mac-1 (CD11b / 18) on leukocytes by flow cytometry. CD11b expression was assessed on all leukocytes, including monocytes and granulocytes, using dual fluorescent staining (CD45 / CD11b) by flow cytometry.

[0299] Results and discussion

[0300] Figure 5 The figure shows the ratio of CD11b+CD14+ cells and the expression of CD11b in leukocytes. Compared with the vehicle group, the dexamethasone group had significantly reduced CD11b expression on CD11b+ cells, but had a similar ratio of CD11b+CD14+ cells in white blood cells. Compared with the vehicle group, the group administered 5 mg / kg of SEQ ID NO: 2 had similar CD11b expression in leukocytes and a similar ratio of CD11b+CD14+ cells.

[0301] Compared with the vehicle group, the groups administered with 25 and 50 mg / kg of SEQ ID NO: 2 had significantly lower proportions of CD11b+CD14+ cells in blood leukocytes. Similarly, these groups had lower CD11b expression, and for the group administered with 50 mg / kg, the difference was statistically significant.

[0302] Overall, the results of this study indicate that SEQ ID NO: 2 has anti-inflammatory properties in an LPS-induced cytokine production model.

[0303] Example 6: Effect of SEQ ID NO: 2 treatment on LPS-induced death in C57BL / 6 mice

[0304] Materials and Methods

[0305] mice

[0306] In this study, female C57BL / 6 mice (Taconic Biosciences) aged 10-12 weeks were used. Animals had free access to autoclaved, acidified drinking water. Animals were housed under standard laboratory conditions, with air conditioning and filtered air supplied by an adequate fresh air source. Animals were maintained in a climate-controlled environment.

[0307] Compound

[0308] Lipopolysaccharide (LPS) from Escherichia coli (O111:B4) was diluted in PBS.

[0309] SEQ ID NO: 2 was stored in powder form at 4° C. until use. A stock solution of SEQ ID NO: 2 was prepared by adding the powder to saline and vortexing into solution.

[0310] deal with

[0311] Mice were treated as shown in Table 5. One group of 20 control mice did not receive lipopolysaccharide. All other groups received an intraperitoneal injection of LPS at a dose of 10 mg / kg. SEQ ID NO: 2 was injected intravenously at concentrations of 10, 25, 50, and 100 mg / kg 15 and 60 minutes after LPS administration, with the exception of Group 6, which received an intravenous injection 15 and 120 minutes after LPS administration.

[0312]

[0313] Table 5. Treatment options

[0314] Survival rate trial

[0315] When high doses of LPS are administered, animals will experience LPS-induced shock and can be observed to die. Mice were checked for death several times daily for 72 hours after LPS injection. All surviving mice were euthanized at the end of the study (day 3).

[0316] Results and discussion

[0317] like Figure 6 A and the corresponding survival chart ( Figure 6 As shown in Figure B), administration of SEQ ID NO: 2 at 25, 50, and 100 mg / kg significantly increased survival compared to vehicle-treated mice. These findings indicate that intravenous administration of SEQ ID NO: 2 effectively treated LPS-induced shock in this study.

[0318] Example 7: Extended Toxicological Analysis of SEQ ID NO: 2 Treatment on Serum LPS-Induced Death in C57BL / 6 Mice The impact of analysis.

[0319] Materials and methods

[0320] mice

[0321] In this study, female C57BL / 6 mice (Taconic Biosciences) aged 10-12 weeks were used. Animals had free access to autoclaved, acidified drinking water. Animals were housed under standard laboratory conditions, with air conditioning and filtered air supplied by an adequate fresh air source. Animals were maintained in a climate-controlled environment.

[0322] Compound

[0323] Lipopolysaccharide (LPS) from Escherichia coli (O111:B4) was diluted in PBS. SEQ ID NO: 2 was stored in powder form at 4°C until use. A stock solution of SEQ ID NO: 2 was prepared by adding the powder to saline and vortexing the solution.

[0324] deal with

[0325] Mice were treated as shown in Table 6. One group of 20 control mice did not receive lipopolysaccharide. All other groups received an intraperitoneal injection of LPS at a dose of 10 mg / kg. SEQ ID NO: 2 was injected intravenously at concentrations of 10, 25, 50, and 100 mg / kg 15 and 60 minutes after LPS administration, with the exception of Group 7, which received an intravenous injection at 15 and 120 minutes after LPS administration.

[0326]

[0327]

[0328] Table 6. Treatment options

[0329] Blood collection and analysis

[0330] Blood collected at 28 hours was analyzed for extended toxicology evaluation by IDEXX Laboratories, Inc. (N. Grafton, MA). We attempted to collect enough blood to perform five analyses per group.

[0331] Extended Toxicology Panel

[0332] The extended toxicology analysis measured concentrations of AST, ALT, creatine kinase, albumin, total protein, BUN, cholesterol, glucose, phosphorus, triglycerides, HDL cholesterol, bile acids, LDL cholesterol, total bilirubin, GGT, conjugated bilirubin, and creatinine. Here, the results for AST, ALT, creatine kinase, albumin, total protein, BUN, cholesterol, phosphorus, triglycerides, cholesterol, and creatinine are shown.

[0333] Results and discussion

[0334] Figure 7 A shows the concentrations of AST, ALT, creatine kinase, BUN, and phosphorus. These analytes showed significant improvement in the previous Hooke study 20170510-2 (Aptahem study APT1-17-PPD018) and also showed improvement in this study, but the improvements in ALT and creatine kinase did not reach statistical significance.

[0335] Figure 7 B shows the concentrations of albumin, creatinine, total protein, cholesterol, and triglycerides. Administration of 50 mg / kg of SEQ ID NO:2 at 60 and 120 minutes after LPS administration showed significant improvement in triglycerides. Administration of 50 mg / kg of SEQ ID NO:2 at 15 and 60 minutes and at 60 and 120 minutes after LPS administration showed significant improvement in creatinine. Administration of 25 mg / kg of SEQ ID NO:2 and 50 mg / kg of SEQ ID NO:2 at 15 and 60 minutes, respectively, showed significant improvement in cholesterol.

[0336] Overall, serum concentrations of AST, creatinine, BUN, phosphorus, cholesterol, and triglycerides were significantly improved in the group treated intravenously with SEQ ID NO: 2 compared to the vehicle-treated group (levels were more similar to young healthy mice).

[0337] These findings suggest that intravenous administration of SEQ ID NO: 2 can effectively prevent organ damage or failure, particularly liver and / or kidney damage or failure, during LPS-induced shock.

[0338] Example 8: Effect of SEQ ID NO: 2 treatment on serum LPS-induced death in C57BL / 6 mice and fibrinogen Effect of D-dimer analysis.

[0339] Materials and Methods

[0340] mice

[0341] In this study, female C57BL / 6 mice (Taconic Biosciences) aged 10-12 weeks were used. Animals had free access to autoclaved, acidified drinking water. Animals were housed under standard laboratory conditions, with air conditioning and filtration using an adequate fresh air source. Animals were maintained in a climate-controlled environment.

[0342] Compound

[0343] Lipopolysaccharide (LPS) from Escherichia coli (O111:B4) was diluted in PBS. SEQ ID NO: 2 was stored in powder form at 4°C until use. A stock solution of SEQ ID NO: 2 was prepared by adding the powder to saline and vortexing the solution.

[0344] deal with

[0345] Mice were treated as shown in Table 7. One group of 20 control mice did not receive lipopolysaccharide. All other groups received an intraperitoneal injection of LPS at a dose of 10 mg / kg. SEQ ID NO: 2 was injected intravenously at concentrations of 10, 25, 50, and 100 mg / kg 15 and 60 minutes after LPS administration, with the exception of Group 7, which received an intravenous injection at 15 and 120 minutes after LPS administration.

[0346]

[0347]

[0348] Table 7. Treatment options

[0349] Blood collection and analysis

[0350] Blood collected at 28 hours was analyzed for fibrinogen and D-dimer analysis from citrated plasma by IDEXX Laboratories, Inc. (N. Grafton, MA). The Hook laboratory attempted to collect enough blood for each group to perform each analysis five times, but in one case (citrated plasma for D-dimer, Group 4), we were able to collect enough blood for only four analyses.

[0351] Results and discussion

[0352] Figure 8Fibrinogen and D-dimer concentrations are shown. There was no significant difference in plasma fibrinogen concentration between mice treated with SEQ ID NO: 2 and mice treated with vehicle. Plasma D-dimer concentrations were significantly lower at 60 and 120 minutes in the 100 mg / kg SEQ ID NO: 2-treated group and the 50 mg / kg SEQ ID NO: 2-treated group compared to the vehicle-treated group.

[0353] Thus, Apta-1 treatment reduced D-dimer in a dose-dependent manner, with the maximum reduction observed in the plasma of the Apta-1 group treated at 60 and 120 min after LPS administration.

[0354] The above findings indicate that intravenous administration of SEQ ID NO: 2 significantly inhibits D-dimer, a marker associated with blood coagulation and a marker of adverse outcomes under various harsh conditions of LPS-induced shock.

[0355] References

[0356] Clowes GH., Jr. "Survival or death from sepsis." Surgery 67 (1970): 374-382.

[0357] Gando S, Saitoh D, Ogura H, Mayumi T, Koseki K, Ikeda T, Ishikura H, Iba T, Ueyama M, Eguchi Y, Ohtomo Y, Okamoto K, Kushimoto S, Endo S, Shimazaki S, "Japanese Association for Acute Medicine Disseminated Intravascular Coagulation (JAAMDIC) Study Group: Natural history of disseminated intravascular coagulationdiagnosed based on the newly established diagnostic criteria for critically ill patients: results of a multicenter, prospective survey." Crit Care Med 36 (2008): 145-150.

[0358] Hasegawa H et al,Methods for Improving Aptamer Binding Affinity,Molecules 2016,21(4),421.

[0359] Lei MG,Gao JJ,Morrison DC,Qureshi N.″Pathogenesis of sepsis:current concepts and emerging therapies.Mo Med 100(2003):524-529.

[0360] Parrillo JE,Parker MM,Natanson C,et al.″Septic shock in humans:advances in the understanding of pathogenesis,cardiovascular dysfunction,and therapy.″Ann Intern Med 113(1990):227-242.

[0361] Stupnisek M,Franjic S,Drmic D,Hrelec M,Kolenc D,Radic B,et al.(2012)

[0362] Pentadecapeptide BPC 157 reduces bleeding time and thrombocytopenia after amputation in rats treated with heparin,warfarin or aspirin.Thromb Res 129:652-659

[0363] Lee JJ,Jin YR,Yu JY,Munkhtsetseg T,Park ES,Lim Y,Kim TJ,Pyo MY,Hong JT,Yoo HS.et al.Antithrombotic and antiplatelet activities of fenofibrate,a lipid-lowering drug.Atherosclerosis.2009;206(2):375-382. Sequence Listing <110> Aptamer, Inc. <120> Nucleic acid molecules with anti-inflammatory, anticoagulant and organ protective properties <130> P6068978PCT <150> EP17194536.3 <151> 2017-10-03 <150> EP18171219.1 <151> 2018-05-08 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 90 <212> RNA <213> Artificial sequence <220> <223> Artificial sequence 1 <400> 1 gggaauucga gcucgguacc aacaauacga cuacaccauc aaaaguauua ucuugcaucg 60 aagguuggca cguagcaagc ucugcagucg 90 <210> 2 <211> 90 <212> RNA <213> Artificial sequence 2 <220> <223> Artificial sequence 2 <220> <221> misc_feature <222> (6)..(7) <223> Uracil is fluorinated <220> <221> misc_feature <222> (8) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (12)..(12) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (13) <223> Uracil is fluorinated <220> <221> misc_feature <222> (14)..(14) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (17) <223> Uracil is fluorinated <220> <221> misc_feature <222> (19)..(20) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (23)..(23) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (26) <223> Uracil is fluorinated <220> <221> misc_feature <222> (28)..(28) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (31)..(31) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (32)..(32) <223> Uracil is fluorinated <220> <221> misc_feature <222> (34)..(34) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (36)..(37) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (39)..(39) <223> Uracil is fluorinated <220> <221> misc_feature <222> (40)..(40) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (46)..(46) <223> Uracil is fluorinated <220> <221> misc_feature <222> (48)..(49) <223> Uracil is fluorinated <220> <221> misc_feature <222> (51)..(51) <223> Uracil is fluorinated <220> <221> misc_feature <222> (52)..(52) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (53)..(54) <223> Uracil is fluorinated <220> <221> misc_feature <222> (56)..(56) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (58)..(58) <223> Uracil is fluorinated <220> <221> misc_feature <222> (59)..(59) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (65)..(66) <223> Uracil is fluorinated <220> <221> misc_feature <222> (69)..(69) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (71)..(71) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (73)..(73) <223> Uracil is fluorinated <220> <221> misc_feature <222> (76)..(76) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (80)..(80) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (81)..(81) <223> Uracil is fluorinated <220> <221> misc_feature <222> (82)..(82) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (83)..(83) <223> Uracil is fluorinated <220> <221> misc_feature <222> (85)..(85) <223> Cytosine is fluorinated <220> <221> misc_feature <222> (88)..(88) <223> Uracil is fluorinated <220> <221> misc_feature <222> (89)..(89) <223> Cytosine is fluorinated <400> 2 gggaauucga gcucgguacc aacaauacga cuacaccauc aaaaguauua ucuugcaucg 60 aagguuggca cguagcaagc ucugcagucg 90

Claims

1. Use of a nucleic acid molecule for producing an anti-inflammatory and / or anti-coagulant and / or anti-organ damage or failure drug, wherein the nucleic acid molecule is represented by SEQ ID NO:

2.

2. The use according to claim 1, (a) wherein parameters associated with inflammation have been reduced, and / or (b) wherein parameters related to coagulation have been reduced, and / or (c) wherein a parameter associated with organ damage or failure has been improved.

3. The use according to claim 2, (a) wherein a parameter associated with inflammation selected from the group consisting of pro-inflammatory cytokine levels, pro-inflammatory integrin levels and / or the number of pro-inflammatory infiltrates has been reduced, and / or (b) wherein a parameter related to coagulation selected from fibrin clot formation and / or thrombin concentration has been reduced, and / or (c) wherein a parameter associated with organ damage or failure selected from AST, ALT, GGT, ALP, LDH, creatine kinase, BUN, albumin, creatinine, glucose, bile acids, total bilirubin, conjugated bilirubin, phosphorus, total protein, LDL cholesterol, HDL cholesterol, cholesterol and / or triglycerides has been improved.

4. The use according to claim 1, wherein The medicament is a medicament for preventing, treating, resolving, curing and / or delaying a disease or condition in which inflammation and / or coagulation and / or organ damage or failure occurs. The method according to claim 1 , wherein the nucleic acid molecule is single-stranded.

6. The use according to claim 1, wherein The nucleic acid molecule is contained in a composition.

7. The use according to claim 6, wherein The composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, salt, diluent and / or excipient.

8. The use according to claim 6, wherein The nucleic acid molecule or the composition is for intravenous administration.

9. The use according to claim 4, wherein A disease or condition in which inflammation and / or coagulation and / or organ damage or failure occurs is thrombosis.

Images