阅读说明：本技术 合成肽，包含该合成肽的药学组合物，以及其在治疗与栓塞相关的疾病中的用途 (Synthetic peptides, pharmaceutical compositions comprising the same, and their use in treating diseases associated with embolism ) 是由 吴汉忠 郭源松 于 2020-03-19 设计创作，主要内容包括：本揭示内容是关于一种生物分子,该生物分子包含一用以标的血栓的合成肽,一种包含该生物分子的药学组合物,以及其在治疗与栓塞相关的疾病中的用途。依据本揭示内容的实施方式,该合成肽具有SEQ ID NO:1、2或3的氨基酸序列,且该合成肽会展现出对血栓的结合亲和力及专一性。因此,该合成肽可作为一种标的元件,用以递送一治疗剂(例如,一抗凝血剂或一溶血栓剂)至血栓处,据以改善该治疗剂的治疗安全性及疗效。(The present disclosure relates to a biomolecule comprising a synthetic peptide for targeting thrombi, a pharmaceutical composition comprising the biomolecule, and its use in treating embolism-related diseases. According to embodiments of the present disclosure, the synthetic peptide has the amino acid sequence of SEQ ID NO 1, 2 or 3, and exhibits binding affinity and specificity for thrombi. Thus, the synthetic peptide can be used as a target element for delivering a therapeutic agent (e.g., an anticoagulant or a thrombolytic agent) to the thrombus site, thereby improving the therapeutic safety and efficacy of the therapeutic agent.)

1. A biomolecule comprising a synthetic peptide for targeting a thrombus, wherein the synthetic peptide has an amino acid sequence of FQNEWFHNFLHD (SEQ ID NO:1), TEANLSSWVFAR (SEQ ID NO:2), or LQKNPFDLVQIL (SEQ ID NO: 3).

2. The biomolecule of claim 1, further comprising a therapeutic agent, wherein the therapeutic agent is complexed with the synthetic peptide, and the therapeutic agent is an anticoagulant or a thrombolytic agent.

3. The biomolecule of claim 2, further comprising a linker, wherein the linker comprises glycine and serine residues, and the therapeutic agent is complexed with the synthetic peptide via the linker.

4. The biomolecule of claim 2, wherein the anticoagulant is coumarin, warfarin, vinpocetine, hydrocinnamatoxin, atromanycin, phenindione, rodenticine, bisrodenticine, heparin, fondaparinux, idoxuridin, biotinylated idoxuridin, rivaroxaban, apixaban, edoxaban, betrixaban, darashban, ridababan, illibasxaban, hirudin, lepirudin, bivalirudin, argatroban, dabigatran, himigraxin, batroxobin, hirudin, cimetidine, aspirin, tacapidine, clopidogrel, or prasugrel.

5. The biomolecule of claim 2, wherein the thrombolytic agent is tissue plasma pro-protein activator, staphylokinase, streptokinase, or urokinase.

6. The biomolecule of claim 5, wherein the tissue plasma pro-protein activator is ralcepase, alteplase, tenecteplase, or lanoproplase.

7. The biomolecule of claim 2, further comprising a reporter molecule, wherein the reporter molecule is complexed with the synthetic peptide or the therapeutic agent, and the reporter molecule is a tag molecule, a radioactive molecule, a fluorescent molecule, a phosphorescent molecule, or a chemiluminescent molecule.

8. The biomolecule of claim 1, further comprising a signal peptide, wherein the signal peptide is located upstream of the synthetic peptide and linked to the synthetic peptide upstream thereof.

9. A pharmaceutical composition for treating an embolism-associated disease comprising the biomolecule of claim 2 and a pharmaceutically acceptable excipient.

10. Use of the biomolecule of claim 2, or the pharmaceutical composition of claim 9 for the preparation of a medicament for the prevention and/or treatment of an embolism-related disorder in a subject in need thereof.

11. The use of claim 10, wherein the anticoagulant is coumarin, warfarin, vinpocetine, hydrocinnamatoxin, coumarine, atonmycin, phenindione, rodenticine, bisrodenticine, heparin, fondaparine, idoxuridine, biotinylated idoxuridine, rivaroxaban, apixaban, edoxaban, betrixaban, darashban, ridababan, irubaban, hirudin, lepirudin, bivalirudin, argatroban, dabigatran, himigraxin, batroxobin, gelucin, cimetidine, tiagabine, clopidogrel, or prasugrel.

12. The use of claim 10, wherein the thrombolytic agent is tissue plasma pro-protein activator, staphylokinase, streptokinase, or urokinase.

13. The use of claim 12, wherein the tissue plasma pro-protein activator is ralcepase, alteplase, tenecteplase, or lanopropride.

14. The use of claim 10, wherein the subject is a human.

Technical Field

The present disclosure generally relates to the technical field of disease treatment. More specifically, the present disclosure relates to three synthetic peptides, and their use in treating embolism-associated diseases.

Background

Thrombosis (thrombosis) refers to the formation of a thrombus (or clot) in a blood vessel or heart chamber. It is a common pathological sign resulting from different embolism-related diseases, including ischemic heart diseases (e.g. angina pectoris (angina), myocardial infarction (myocardiac arrest), and sudden cardiac death (sudden cardiac death)), ischemic stroke, and venous embolism (VTE). According to the World Health Organization (WHO) report, myocardial infarction and stroke are the first two causes of death worldwide, contributing to a total of twenty-five percent of the worldwide mortality. Therefore, it is important to study the molecular mechanism of the disease associated with embolism and provide treatment.

Mainstream therapies for treating embolism-related diseases include surgery, anticoagulation therapy, and thrombolytic therapy. However, these therapies often have deleterious side effects in the treatment of embolism-related diseases and thus the efficacy of these therapies is unsatisfactory. For example, surgery may cause bleeding, wound hematoma, infection, and/or nerve damage; anticoagulation and thrombolytic therapy have been reported to induce off-target effects that can lead to fatal bleeding.

In view of the above, there is a need in the art for a safer and more effective novel method for treating embolism-related diseases.

Disclosure of Invention

The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure, and is intended to neither identify key/critical elements of the disclosure, nor delineate the scope of the disclosure. Its sole purpose is to present some concepts of the disclosure in a simplified form as a prelude to the more detailed description that is presented later.

As embodied and broadly described herein, one aspect of the present disclosure relates to a biomolecule comprising a synthetic peptide for targeting a thrombus. According to an embodiment of the present disclosure, the synthetic peptide has an amino acid sequence of FQNEWFHNFLHD (SEQ ID NO:1), TEANLSSWVFAR (SEQ ID NO:2), or LQKNPFDLVQIL (SEQ ID NO: 3).

Optionally, the biomolecule of the present disclosure further comprises a signal peptide, wherein the signal peptide is located upstream of the synthetic peptide and linked to the synthetic peptide upstream thereof.

According to a preferred embodiment, the disclosed biomolecule further comprises a therapeutic agent, wherein the therapeutic agent is complexed with the synthetic peptide, and the therapeutic agent is an anticoagulant or a thrombolytic agent. Examples of the anticoagulant include, but are not limited to, coumarin (columbin), warfarin (warfarin), acemetacoumarol (acenocoumarol), hydrocumarol (phenoprocouumon), atropine (atromentin), phenindione (phenindione), brodifacoum (brodifacoum), bisfenamic (difacoum), heparin (heparin), fondaparinux (fondaparinux), idraparinux (idraparinux), biotinized idoxaparin (idrabiaparinux), rivaroxaban (rivaroxaban), apixaban (apixaban), edoxaban (edoxaban), betrixaban (ritoxaban), trexaban (ritoxaban), daroxaban (ritoxaban), trexaban (taraxadin), argatropizin (bixaban), lignin (bixaban), lignocandin (bixaban), peganin (bixadin (bixadine), peganin (bixaban), peganin (bixadine), picatin (bixaban), picatin (bixazone), picatin (bixaban), picatin, Clopidogrel (clopidogrel), and prasugrel (prasugrel). Exemplary thrombolytic agents include tissue plasma pro-activator (tPA), e.g., reteplase (reteplase), alteplase (alteplase), tenecteplase (tenecteplase), or lanoproplase (lanoteplase), staphylokinase (staphylokinase), streptokinase (streptokinase), or urokinase (urokinase).

According to certain embodiments of the present disclosure, the biomolecule further comprises a linker comprising glycine (G) and serine (S) residues. In these embodiments, the therapeutic agent is complexed with the synthetic peptide via the linker. According to a working example, the linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 4).

Optionally, the biomolecule further comprises a reporter molecule, wherein the reporter molecule is complexed with the synthetic peptide or the therapeutic agent. Depending on the purpose, the reporter molecule may be a label molecule, a radioactive molecule, a fluorescent molecule, a phosphorescent molecule, or a chemiluminescent molecule.

The present disclosure also encompasses a pharmaceutical composition or medicament for treating a thrombus or embolism-associated disease. The pharmaceutical composition or medicament of the present disclosure comprises a biomolecule of the present disclosure, and a pharmaceutically acceptable excipient, wherein the biomolecule comprises a synthetic peptide and a therapeutic agent, and the therapeutic agent is complexed with the synthetic peptide.

Another aspect of the present disclosure relates to a method for preventing and/or treating a thrombus or embolism-associated disease in a subject in need thereof. The method comprises administering to the subject an effective amount of a biomolecule, pharmaceutical composition, or drug of any aspect or embodiment of the present disclosure.

The subject treatable with a biomolecule, pharmaceutical composition, or drug of the present disclosure is a mammal; preferably, the subject is a human.

The present disclosure also encompasses the use of a biomolecule or pharmaceutical composition of the present disclosure for the preparation of a medicament for the prevention and/or treatment of an embolism-associated disease in a subject in need thereof.

The many features and advantages that are attendant to this disclosure will become more readily apparent upon review of the following detailed description and accompanying drawings.

Drawings

The present disclosure will become more fully understood from the detailed description and the accompanying drawings, wherein:

FIGS. 1A-1B show the results of screening a target human thrombus-derived phage clone, according to one embodiment of the present disclosure. FIG. 1A is a histogram illustrating the titer of eluted phage obtained from different rounds of biopanning (biopanning). FIG. 1B is experimental data of enzyme-linked immunosorbent assay (ELISA) showing the binding affinity of specific phage to human thrombus. Y-axis, absorbance at wavelength 450 nm.

FIGS. 1C-1D illustrate the binding affinity of selected phage clones for a particular protein, according to one embodiment of the present disclosure. FIG. 1C is experimental data of enzyme-linked immunosorbent assay illustrating that specific phage bind to thrombus in a dose-dependent manner. FIG. 1D is the data of ELISA experiments illustrating the binding affinity of specific phage to Bovine Serum Albumin (BSA), which is used as negative control group in this experiment.

FIGS. 2A-2C illustrate the targeting ability of a particular phage clone to thrombus in vivo, according to another embodiment of the present disclosure. Fig. 2A shows photographs before (top photograph) and after (bottom photograph) the mouse right jugular vein induced thrombus. FIG. 2B shows the results of ELISA showing the distribution of specific phage in the tissue. Y-axis, absorbance at wavelength 450 nm. FIG. 2C is the results of a phage titer assay, illustrating the distribution of specific phage in a tissue.

Fig. 3 is a photograph of Immunohistochemistry (IHC) according to one embodiment of the present disclosure, in which specific phage bind specifically to thrombus in vivo, as compared to the negative control group.

FIG. 4 is a graph of the results of an assay for a peptide-tPA fusion protein according to another embodiment of the present disclosure. Panel A is a photograph of a gel electrophoresis performed to analyze the expression of a particular fusion protein. Panel B is a photograph of a Western blot to confirm the expression of a particular fusion protein. Wild-type tPA was used as a positive control group in this experiment. Panel C is a broken line graph illustrating the cleavage activity of a particular fusion protein. tPA activity was detected using Z-Gly-Gly-Arg-AMC as the fluorescent substrate. Once specifically cleaved, the substrate fluoresces (excitation wavelength 380 nm (EX380) and emission wavelength 420 nm (EM 420)). The Y axis is EX380/EM420, and the fluorescence emission is in arbitrary units.

FIGS. 5A-5B illustrate the results of infrared fluorescence imaging of mice treated with specific fusion proteins. FIG. 5A is an infrared fluorescence image of photographs taken at 0 min, 5 min, 20 min, 40 min and 1 hr after treatment of a specific fusion protein. The most intense infrared fluorescence signal is detected near the neck in the right external jugular vein. The arrows indicate the induced thrombus. FIG. 5B is a histogram illustrating the quantification of a particular fusion protein in an induced thrombus. The fluorescence measurements of the specific fusion protein in the induced thrombus are plotted against the time after treatment of the specific fusion protein.

FIG. 6 illustrates the therapeutic effect of a particular fusion protein in the Midle Cerebral Artery Occlusion (MCAO) mouse model. The A-panel is an image of T2-weighted (T2-weighted) MCAO mouse Magnetic Resonance Imaging (MRI), in which vector (PBS), tPA, SP30-tPA or SP66-tPA was injected into the MCAO mouse. The white areas in the T2-weighted MRI represent infarcted areas. Panel B is a histogram illustrating the results of an analysis of infarct zone in the brain of MCAO mice that received a particular treatment.

Detailed Description

To make the disclosure more complete and complete, the following description is given of embodiments and examples of the invention, but this is not intended to be the only form in which the embodiments of the invention may be practiced or utilized. The embodiments are intended to cover the features of the various embodiments as well as the method steps and sequences for constructing and operating the embodiments. However, other embodiments may be utilized to achieve the same or equivalent functions and step sequences.

I. Definition of

For convenience, certain terminology is used in the description, examples, and claims to follow. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, as used herein, the singular form of a noun includes plural forms of that noun and plural forms of that noun may be used without conflicting the context. In particular, in the specification and claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. In addition, in the present specification and claims, the terms "at least one" and "one or more" are used synonymously, and both are meant to include one, two, three or more.

The term "peptide" refers to a polymer of amino acids, regardless of the length of the polymer. Chemical or post-expression modifications of the peptides of the invention are not specified or excluded by the present terminology, although chemical or post-expression modifications of the peptides may be included or excluded in a particular embodiment. Thus, modifications of peptides, for example, covalent linkages comprising glycated groups (glycosylgroups), acetyl groups (acetyl groups), phosphate groups (phosphate groups), lipid groups (lipidgroups), and the like, are expressly contemplated within the scope of the term peptide. In addition, peptides having such modifications may be specifically designated as species individually included or excluded by the present invention. In the present disclosure, the position of any given amino acid residue in a peptide is counted from the N-terminus of the peptide. When an amino acid is not specified as a D-or L-amino acid, the amino acid can be an L-amino acid or a D-or L-amino acid, unless a particular isomer is intended depending on the context. In addition, the notation of peptide amino acid residues used in the present disclosure is a commonly used abbreviation notation in the art.

The term "synthetic peptide" as used herein refers to a peptide that does not include all molecules that are naturally occurring proteins. The peptide is "synthetic" in the sense that the peptide can be prepared by human intervention, for example using the following techniques: phage display (phase-display) technology, chemical synthesis, recombinant genetics technology, or whole antigen fragmentation (fragmentation of whole antigen) technology.

As discussed herein, minor variations in the amino acid sequence of a peptide are considered to be encompassed within the inventive concepts disclosed and claimed herein, provided that the conditions are such that the variations in their amino acid sequence maintain at least 70% sequence similarity, e.g., at least 70%, 71%, 72%, 73%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence similarity. The peptides of the present disclosure may be specifically modified to alter the characteristics of the peptide without affecting its physiological activity. For example, certain amino acids, when altered and/or deleted, do not affect the physiological activity of the peptides of the disclosure (i.e., their ability to be used in target thrombi). In particular, conservative substitutions of amino acids are contemplated. Conservative substitutions are those substitutions that occur within the same amino acid family (amino acid side chains with associations). The commonly encoded amino acids can be classified into the following families: (1) acidic amino acids aspartic acid (aspartate), glutamine (glutamate); (2) basic amino acids, lysine (lysine), arginine (arginine), histidine (histine); (3) non-polar amino acids alanine (alanine), valine (valine), leucine (leucine), isoleucine (isoleucine), proline (proline), phenylalanine (phenylalanine), methionine (methionine), tryptophan (tryptophan); and (4) amino acids with no electric polarity, such as glycine (glycine), asparagine (asparagine), glutamine (glutamine), cysteine (cysteine), serine (serine), threonine (threonine), and tyrosine (tyrosine). Preferred family classifications are: serine (S) and threonine (T) are aliphatic-hydroxy family (aliphatic-hydroxy family); asparagine (N) and glutamine (Q) are amide-containing family (amide-containing family); alanine (a), valine (V), leucine (L), and isoleucine (I) are aliphatic (aliphatic family); and phenylalanine (F), tryptophan (W), and tyrosine (Y) are aromatic family. For example, it is reasonable to expect that a substitution of leucine (L) with isoleucine (I) or valine (V), an substitution of aspartic acid (D) with glutamic acid (E), a substitution of threonine (T) with serine (S), or a substitution of an amino acid with an amino acid of related structure, will not have a significant effect on the binding of the amino acid molecule or on its properties, particularly if the substitution does not include the framework region (framework site) of the amino acid. Whether an amino acid change affects a functional peptide can be confirmed by analyzing the specific activity of the peptide derivative. Fragments or analogs of the proteins/peptides can be prepared by one of ordinary skill in the art. The amine or carboxyl end of preferred fragments or derivatives will be present in the vicinity of functional domains. In one embodiment, a conservative substitution (e.g., to leucine (L)) occurs at one amino acid residue (e.g., valine (V)) in a synthetic peptide of the disclosure. In other embodiments, two amino acid residues in the synthetic peptides of the present disclosure are conservatively substituted with other suitable amino acid residues, for example, valine (V) and arginine (R) are substituted with a pair of amino acids including, but not limited to, methionine (M) and lysine (K), lysine (K) and proline (P), tryptophan (W) and isoleucine (I), isoleucine (I) and proline (P), asparagine (N) and valine (V), and glutamine (G) and lysine (K), respectively.

The term "percent (%) sequence identity" refers to the percent identity of amino acid residues of a candidate peptide sequence to amino acid residues of a particular peptide sequence; in the alignment, a plurality of sequences are aligned, gaps (gaps) are introduced as needed to achieve the maximum percentage of sequence similarity, and any conservative substitutions (conservative substitutions) are not considered as part of the sequence similarity. Alignment and percent sequence similarity can be determined by a variety of methods known to those skilled in the art, for example, by using computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to maximize alignment of the full-length sequences being aligned. To this end, the sequence alignment between two amino acid sequences was analyzed using the computer software Blastp (protein-protein BLAST) provided on-line by the National Center for Biotechnology Information (NCBI). The percent amino acid sequence similarity between a particular amino acid sequence A and a particular amino acid sequence B (or a particular amino acid sequence A having a certain% amino acid sequence identity to a particular amino acid sequence B) is calculated using the following formula:

wherein X is the number of amino acid residues that result in a consistent match after alignment of A and B by the sequence alignment program BLAST, and Y is the total number of amino acid residues in the A or B sequence (whichever is shorter).

The term "administering" or "administration" refers to the administration of a biomolecule, pharmaceutical composition or drug comprising a peptide of the present disclosure in any mode of delivery, including, but not limited to, intravenous, intramuscular, intraperitoneal, intraarterial, intracranial, or subcutaneous. In certain embodiments, the biomolecule comprising a peptide of the present disclosure is mixed with a suitable excipient (e.g., buffer solution) for intravenous injection prior to use.

In the present disclosure, the terms "treating", "treating" and "treatment" encompass the partial or complete prevention, amelioration, alleviation and/or treatment of a condition (symptom), secondary disorder or symptom (condition) associated with thrombosis. The term "treating" also refers to the administration or administration of one or more biomolecules of the present disclosure to a subject suffering from a condition, secondary condition, or symptom associated with thrombosis to partially or completely alleviate, slow, cure, delay onset, inhibit progression of, reduce severity of, and/or reduce the occurrence of one or more of the condition, secondary condition, or symptom associated with thrombosis. Signs, secondary conditions and/or symptoms associated with thrombosis include, but are not limited to, angina pectoris, myocardial infarction, sudden cardiac death, ischemic stroke, and venous embolism. As used herein, "treating" may also refer to administering to a subject with the signs or symptoms at an early stage to reduce the risk of the subject developing signs, secondary disorders and/or symptoms associated with thrombosis. A treatment is "effective" when it reduces one or more symptoms or clinical markers. Alternatively, a treatment is "effective" when it reduces, slows, or stops the progression of the disease course, sign, or symptom.

The term "effective amount" as used herein refers to an amount of an ingredient sufficient to produce a desired therapeutic response. An effective amount for therapeutic purposes may also refer to an amount of an ingredient that provides a therapeutically beneficial effect over the toxic or deleterious effects of the ingredient. An effective amount of an agent is not required to cure the disease or condition, but will provide treatment for the disease or condition, to delay, block, prevent the onset of, or alleviate the symptoms of the disease or condition. An effective amount may be divided into one, two or more doses and formulated appropriately for administration once, twice or more over a specified period. The specific effective amount or sufficient amount will depend on a variety of factors, such as the particular condition to be treated, the physiological condition of the patient (e.g., the weight, age, or sex of the patient), the type of mammal or animal being treated, the duration of the treatment, the nature of the current therapy (if any), and the specific dosage form employed, as well as the structure of the compound or derivative thereof. For example, an effective amount may be expressed as grams, milligrams or micrograms or a few milligrams per kilogram of body weight (mg/kg). Alternatively, the effective amount may be expressed as the concentration of the active ingredient (e.g., a biomolecule of the present disclosure), such as molar concentration (molarity), weight concentration (mass concentration), volume concentration (volume concentration), molarity (molality), mole fraction (mole fraction), weight fraction (mass fraction), and mixing ratio (mixing ratio). One of ordinary skill in the art can convert the dose into a Human Equivalent Dose (HED) of the drug (e.g., the biomolecule of the present disclosure) based on the dose obtained from the experimental animal model. For example, one of ordinary skill in the art can estimate the highest Safe Dose for human use based on the "estimate of Maximum Safe Starting Dose for an initial Clinical treatment regimen for Adult Healthy Volunteers" as announced by the U.S. Food and Drug Administration, FDA.

The term "pharmaceutically acceptable" refers to a "generally regarded as safe" molecular entity and composition, e.g., which are physiologically tolerable and do not typically produce allergic or similar untoward reactions when administered to a human. Preferably, the term "pharmaceutically acceptable" as used herein means that the molecular entities and compositions have been certified by a regulatory agency of the federal government or a regulatory agency of a state government or are listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "excipient", as used herein, refers to any inert substance (e.g., powder or liquid) that forms a carrier or carrier to encapsulate an active agent. Excipients are generally safe and non-toxic and in a broad sense may be ingredients commonly used in the pharmaceutical industry to prepare pharmaceutical compositions, for example, fillers, diluents, agglutinating agents, binding agents, lubricants, glidants, stabilizers, coloring agents, wetting agents, disintegrants and the like.

The term "subject" refers to a mammal, including a human, that can be treated with the biomolecules, pharmaceutical compositions, medicaments and/or methods of the present disclosure. Unless a particular gender is specified, the term "individual" refers to both males and females.

Detailed description of the invention

The present disclosure is based, at least in part, on the inventors' discovery that three synthetic peptides, comprising the amino acid sequences of FQNEWFHNFLHD (SEQ ID NO:1), TEANLSSWVFAR (SEQ ID NO:2), and LQKNPFDLVQIL (SEQ ID NO:3), respectively, exhibit binding affinity and specificity for thrombi. It is therefore an object of the present disclosure to provide a biomolecule comprising the synthetic peptide, and its use in the treatment of thrombosis.

Accordingly, a first aspect of the present disclosure relates to a biomolecule comprising a synthetic peptide having an amino acid sequence that is at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95%, 99%, or 100%) similar to SEQ ID No. 1, 2, or 3. According to a working example of the present disclosure, the synthetic peptide comprises an amino acid sequence having 100% similarity to SEQ ID NO 1, 2 or 3.

The synthetic peptides of the invention may be prepared by any of the usual methods, for example, phage display techniques (i.e., by performing biopanning of phage-displayed peptides to select a desired peptide that exhibits binding affinity for the target molecule), or t-BOC or FMOC protection of the alpha amino group (i.e., stepwise synthesis, starting at the C-terminus of the peptide, with the addition of a single amino acid each time). The peptides of the invention may also be synthesized by solid phase peptide synthesis methods well known in the art. Alternatively, the synthetic peptides of the invention can be prepared by a host cell (e.g., a HEK293 cell) into which a nucleic acid encoding the peptide is transfected.

For therapeutic purposes, the disclosed biomolecules may further comprise a therapeutic agent complexed with the synthetic peptide. According to some embodiments of the present disclosure, the therapeutic agent is an anticoagulant or a thrombolytic agent. Examples of anticoagulants suitable for use in biomolecules of the present disclosure include, but are not limited to, coumarin, warfarin, vinpocoumarin, hydrocinnamatoxin, atromanycin, phenindione, rodenticine, bisrodenticine, heparin, fondaparinux, idoxuridine, biotinylated idoxuridine, rivaroxaban, apixaban, edoxaban, batroxiban, daraxababan, illibasxaban, hirudin, lepirudin, bivalirudin, argatroban, dabigatran, himigraxin, batroxobin, gelucin, cimetidine, aspirin, tacapidine, clopidogrel, and prasugrel. With respect to the portion of the thrombolytic agent, it may be any of tPA (e.g., raltitrase, alteplase, tenecteplase, or lanoproplase), staphylokinase, streptokinase, or urokinase. It is to be understood that the therapeutic agent may be other alternative molecules so long as it exhibits prophylactic and/or therapeutic efficacy on the thrombus (e.g., inhibiting thrombus formation, or dissolving occlusive thrombus), for example, fibrinolytic agents (fibrinolytic agents) or antiplatelet agents (antiplatelet agents). According to a particular operating embodiment, the therapeutic agent is tPA.

Optionally, the synthetic peptide and the therapeutic agent are complexed via a linker. According to some embodiments of the present disclosure, the amino acid sequence of the linker comprises a plurality of glycine (G) and/or serine (S) residues. In a specific embodiment, the linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 4).

Optionally, the biomolecule of the present disclosure may further comprise a reporter molecule complexed with the synthetic peptide or the therapeutic agent. The reporter molecule may be a tag molecule (e.g., Chitin Binding Protein (CBP), Maltose Binding Protein (MBP), glutathione-S-transferase (GST), Thioredoxin (TRX), streptag tag (STREPT), FLAG, etc., depending on the purpose of use^TMA tag, a Myc tag, a human influenza virus Hemagglutinin (HA) tag, or a polyhistidine (His) tag); a radioactive molecule (e.g., gallium-67, molybdenum-99, indium-111, or thallium-201); a fluorescent molecule (e.g., Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), or enhanced variants of these fluorescent proteins); a phosphorescent molecule (e.g., europium-doped (europeium), dysprosium-doped (dysprosium), or terbium-doped (terbi)um), strontium aluminate, or strontium magnesium silicate; copper-activated zinc sulfide (copper-activated zinc sulfide); silver-activated zinc sulfide (silver-activated zinc sulfide); copper-activated cadmium zinc sulfide (copper-activated zinc-cadmium sulfide); or bismuth-activated calcium-strontium sulfide (bismuth-activated calcium-sulfide)); or a chemiluminescent molecule (e.g., coelenterazine, lucigen, luciferin, luciferase, or an oxalate-containing dye). According to one embodiment of the present disclosure, the biomolecule of the present disclosure includes a Myc tag complexed with the therapeutic agent. According to another embodiment of the present disclosure, the biomolecule of the present disclosure includes a His-tag complexed with the therapeutic agent. According to yet another embodiment of the present disclosure, the biomolecule of the present disclosure includes a Myc tag and a His tag, wherein the Myc tag is complexed with the therapeutic agent and the His tag is complexed with the Myc tag.

According to some embodiments, the biomolecule is a fusion protein. In these embodiments, a signal peptide is placed upstream of the synthetic peptide of the present disclosure and linked to the synthetic peptide upstream to directly regulate or enhance expression or secretion of the synthetic peptide. The signal peptide may be a natural signal peptide or a synthetic signal peptide. According to a particular embodiment, the signal peptide is derived from a mouse Immunoglobulin (IG) kappa chain. The fusion proteins of the present disclosure comprise an order, i.e., from N-terminus to C-terminus: signal peptides, synthetic peptides, linkers, therapeutic agents, Myc tags, and His tags.

The present disclosure also encompasses the use of the disclosed biomolecules for the preparation of a medicament or a pharmaceutical composition for the treatment of thrombosis or embolism associated disease. The drug or pharmaceutical composition comprises a biomolecule of the present disclosure, and a pharmaceutically acceptable excipient, wherein the biomolecule comprises a synthetic peptide, and a therapeutic agent, wherein the therapeutic agent is complexed with the synthetic peptide. Preferably, the therapeutic agent is an anticoagulant or a thrombolytic agent.

Generally, the weight of the biomolecule of the present disclosure is from about 0.01% to about 99.9% of the total weight of the pharmaceutical or pharmaceutical composition of the present disclosure. In certain embodiments, the weight of the biomolecule of the present disclosure is at least about 0.1% of the total weight of the pharmaceutical or pharmaceutical composition of the present disclosure. In certain embodiments, the weight of the biomolecule of the present disclosure is at least about 5% of the total weight of the drug or pharmaceutical composition of the present disclosure. In yet other embodiments, the weight of the biomolecule of the present disclosure is at least about 10% of the total weight of the drug or pharmaceutical composition of the present disclosure. In yet other embodiments, the weight of the biomolecule of the present disclosure is at least about 25% of the total weight of the drug or pharmaceutical composition of the present disclosure.

Depending on the route of administration, the drug or pharmaceutical composition may comprise different types of excipients or carriers. The pharmaceutical or pharmaceutical compositions of the present disclosure may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally (intramural), intracranially (intracraranially), intranasally, intrapleurally (intrapleurally), intratracheally (intratracheally), intrarectally (intrarectalyl), topically, intramuscularly, subcutaneously, intravascularly (intrapericardially), intrapericardially (intrapericardially), intraocularly (intraocularly), orally (orally), topically (locally), by injection, inhalation, infusion, topically (locally), by injection, by inhalation, by infusion, by liquid, by aerosol, and the like, and may be formulated as powders, creams, liquids, and sprays, and the like.

In certain embodiments, the pharmaceutical or pharmaceutical compositions of the present invention are formulated in solid dosage forms suitable for oral administration. Such solid dosage forms may be capsules, sachets, tablets, pills, dragees, powders or granules. In such dosage forms, the active ingredient (e.g., any of the biomolecules described above) is mixed with at least one pharmaceutically acceptable excipient. Optionally, any of the solid dosage forms described may contain coatings (coatings) and shells (shells), e.g., enteric coatings, as well as coatings to modify the release rate of any ingredient. Examples of such coatings are well known in the art. In one embodiment, the pharmaceutical or pharmaceutical composition of the invention is a tablet, for example, an immediate release (quick-release) tablet. In another embodiment, the pharmaceutical or pharmaceutical composition of the invention is formulated as a sustained release dosage form. In yet another embodiment, the pharmaceutical or pharmaceutical composition of the invention is a powder and is encapsulated in soft and hard gelatin capsules.

In certain embodiments, the pharmaceutical or pharmaceutical compositions of the present disclosure are formulated in liquid dosage forms suitable for oral administration. The liquid dosage form may further comprise a buffer to facilitate maintenance of the appropriate pH. The liquid dosage form may also be filled into soft gelatin capsules. For example, the liquid dosage form may comprise a solution, suspension, emulsifier, microemulsion, precipitate, or any desired liquid medium to carry any of the above biomolecules, or a pharmaceutically acceptable derivative, salt or solvate thereof, or a combination thereof. The liquid dosage form may be designed to improve the solubility of the active biomolecules described above, for example, to form a drug-containing emulsion or a dispersed phase upon release of the drug.

In certain embodiments, the medicaments or pharmaceutical compositions of the present disclosure may be formulated in a dosage form suitable for non-oral administration, such as administration by injection, including, but not limited to, subcutaneous, bolus injection (bolus injection), intramuscular, intraperitoneal, and intravenous injection. The pharmaceutical or pharmaceutical composition may be formulated as an oily or aqueous isotonic suspension, solution or emulsion, and may contain a prescribed agent, for example, a suspending, stabilizing or dispersing agent. Alternatively, the pharmaceutical or pharmaceutical composition may be in a dry form, e.g., as a powder, crystals or as a freeze-dried solid, supplemented with sterile and pyrogen-free (pyrogen-free) water or isotonic saline solution prior to use. The composition may also be placed in sterile ampoules or vials.

When the biomolecules of the present disclosure are formulated for intravenous, transdermal, or subcutaneous injection, the peptides can be prepared as pyrogen-free non-orally acceptable liquid solutions. One skilled in the art would understand how to prepare the non-orally acceptable liquid solution, taking into account pH, isotonicity, and stability. In addition to the biomolecules of the present disclosure, preferred pharmaceutical or pharmaceutical compositions for intravenous, transdermal or subcutaneous injection should comprise an isotonic excipient, such as sodium chloride injection, ringer's injection, dextrose and sodium chloride injection, lactated ringer's injection, or other known excipients. The pharmaceutical or pharmaceutical compositions of the present disclosure may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. The time interval between intravenous administrations of the pharmaceutical or pharmaceutical compositions of the present disclosure will vary with the severity of the disease and the condition and potential idiosyncrasies of each individual. In continuous intravenous administration, the time interval between each administration of a biomolecule of the present disclosure may be 12 to 24 hours. The attending physician will ultimately decide on the course of intravenous therapy.

The biomolecules of the present disclosure may be formulated into physiologically acceptable dosage forms for local, systemic, or regional administration. For example, the biomolecules of the present disclosure may be applied to the surface of an implant (Impplant) or device. Further, the composition may be coated or injected into an adhesive dosage form as needed to facilitate delivery to the thrombus. As noted above, other useful agents may be included in the medicaments or pharmaceutical compositions of the present disclosure, or the medicaments or pharmaceutical compositions of the present disclosure may be administered simultaneously or sequentially.

Another aspect of the present disclosure relates to a method for preventing and/or treating a thrombosis or embolism associated disorder in an individual in need thereof using a biomolecule of the present disclosure, wherein the biomolecule comprises a synthetic peptide and a therapeutic agent, and the therapeutic agent is complexed with the synthetic peptide. The method comprises administering to the subject an effective amount of the biomolecule, or a pharmaceutical or pharmaceutical composition comprising the biomolecule.

The effective dosage of the biomolecule, drug or pharmaceutical composition of the present disclosure will vary depending on a number of factors, such as the particular condition to be treated, the severity of the condition, individual parameters of the patient including age, physiological condition, size, sex and weight of the individual, the duration of treatment, the nature of concurrent therapy (if any), the particular route of administration, etc., as determined by the medical practitioner with the expertise and experience of the practitioner.

In one embodiment, the individual is a mouse. To elicit therapeutic efficacy in a mouse, a biomolecule of the disclosure can be administered to the mouse at about 0.1 to 100 mg/kg per dose (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 80, 91, 80, 84, 85, 93, 85, 93, 85, 83, 82, 83, 82, 96. 97, 98, 99, or 100 mg/kg) body weight; preferably, a dosage of about 0.5 to 50 mg/kg body weight per dose of a biomolecule of the present disclosure is administered; more preferably, the biomolecule of the present disclosure is administered in a dose of about 1 to 20 mg/kg body weight per dose. According to one example of practice, administration of a dose of about 2 to 10 mg/kg body weight of a biomolecule of the present disclosure per dose is sufficient to elicit therapeutic efficacy (e.g., reduction in thrombus volume, or restoration of blood flow) in a subject.

One skilled in the art can convert the Human Equivalent Dose (HED) of the disclosed biomolecules based on the dose obtained for the animal model. Accordingly, an effective dose of a biomolecule of the present disclosure for use in a human can be a dose between 10 micrograms/kg and 10 milligrams/kg (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 micrograms/kg, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 milligrams/kg) of body weight per dose; preferably, a dose of 50 microgram/kg to 5 mg/kg body weight per dose; more preferably, the effective HED is about 100 to 1,000 micrograms/kg per dose.

When it is understood that the methods of the present disclosure are administered to an individual, either alone or in combination with other therapies that have some beneficial effect for preventing or treating thrombosis or embolism-related disorders. Depending on the purpose of use, the methods of the present disclosure may be administered to the subject before, during, or after the administration of the other therapy.

The embolism-associated disease may be ischemic heart disease (e.g., myocardial infarction), limb ischemia, venous embolism (VTE, including deep venous embolism (DVT) and Pulmonary Embolism (PE)), arterial embolism (ATE), tumor, hemolytic anemia (haemolytic anemia), inflammation, sepsis, hypercortisolism (hyperadrenocorticism), cerebrovascular disease (e.g., stroke), or other disease or disorder associated with or caused by thrombosis.

Further aspects of the present disclosure relate to a method of using a biomolecule of the present disclosure to identify the presence or location of a thrombus or embolism in an individual in need thereof (e.g., an individual having or suspected of having a thrombus or embolism), wherein the biomolecule comprises a synthetic peptide complexed with a reporter molecule. The method comprises administering an effective amount of the biomolecule to the subject and detecting the signal from the reporter molecule to confirm the presence or location of the thrombus or embolism.

According to a specific example of practice of the present disclosure, the subject is a mouse, wherein a dose of the biomolecule of the present disclosure is administered to the mouse of about 1 to 20 mg/kg body weight per dose to confirm the presence or location of the thrombus. One skilled in the art can readily adapt the HED of the biomolecules of the present disclosure based on the dose obtained from the animal model provided in the working examples of this specification.

In addition to humans and mice, the subject may be other alternative mammals, for example, rats, hamsters, guinea pigs, rabbits, dogs, cats, cows, goats, sheep, monkeys, and horses.

Various embodiments are set forth below to illustrate certain aspects of the present disclosure to facilitate the practice of the invention by those of ordinary skill in the art to which the invention pertains. These examples should not be construed as limiting the scope of the invention. It is believed that one skilled in the art, having the benefit of the description set forth herein, can utilize and practice the present disclosure to its fullest extent without undue interpretation. All publications cited herein are incorporated by reference in their entirety.