Composition and delivery method of bioactive substances in cordyceps for the treatment of acute and long-term COVID-19

Abstract

This invention discloses a composition comprising active ingredients found in the genus Cordyceps in defined ratios, which, when administered orally in an appropriate dosage form, is effective in treating COVID and long COVID.

Description

[Background technology]

[0001] Since the WHO declared COVID-19 a pandemic three years ago (March 11, 2020), 680 million cases have been reported, and 1% of those infected (6.8 million people) have died. As of January 2023, the WHO maintains that while the world is in a transitional phase, COVID-19 remains a global emergency.

[0002] Long COVID is the post-COVID crisis. Long COVID will have long-term health and economic impacts. In the United States alone, 70% of Americans have contracted COVID. Of these, 24% have had symptoms for more than three months. More than 16 million working-age people are suffering from long COVID. Annual wage losses exceed $170 billion, and could reach as high as $230 billion.

[0003] There is no consensus on the etiology of long-term COVID, let alone an effective treatment. Several hypotheses exist regarding the pathogenesis of this condition. These hypotheses include, but are not limited to, residual viruses or viral remnants in the body, an overactive immune system that fails to return to normal, microthrombi in the blood and tissues, and organ damage caused by the virus.

[0004] There are over 100 symptoms associated with long-term COVID; the most common are fatigue, exhaustion or lethargy (72.1%), cough (39.3%), shortness of breath (38.5%), brain fog (32.9%), and general weakness (30.9%) (https: / / health-infobase.canada.ca / covid-19 / post-covid-condition / ).

[0005] The effects of COVID infection on the body, particularly on the lungs, brain, digestive tract, immune system, and circulatory system, have been well described.

[0006] Symptoms can fluctuate as the disease progresses, particularly in relation to viral load, immune system imbalance, and bodily damage.

[0007] There is still an unmet need for drugs and treatments for the severe symptoms associated with this disease. Cordyceps species are known to possess antiviral, anti-inflammatory, and immunomodulatory properties (Jedrejko, Lazur et al. 2021, Rabie 2022). Recently, an extract of Cordyceps militaris was tested in patients with COVID-19 (Dubhashi, Sinha et al. 2023). Unfortunately, its efficacy was limited.

[0008] Bioactive compounds in the Cordyceps genus have been suggested to have potential therapeutic effects on COVID-19 (Kaymakci and Guler 2020, Verma 2020, Zivan, Ruiz et al. 2023). However, at the identified and typical doses (3-6 g of fungus per day), none of the compounds showed therapeutic significance when tested individually. These compounds include adenosine and cordycepin (Verma 2020, Zivan, Ruiz et al. 2023). While the clinical efficacy of Cordyceps polysaccharides has not been established, their anti-inflammatory and immunomodulatory activities have been well demonstrated (Miao, Yu et al. 2022).

[0009] For example, while cordycepin is more effective than remdesivir (Rabie 2022), the required daily dose of cordycepin when administered as monotherapy exceeds 450 mg. This means that, assuming a cordycepin content of 1% in the bacterial cells, 450 grams of cordyceps would be needed. This amount is impractical in terms of dietary or medicinal use. [Overview of the project]

[0010] This disclosure describes combination therapies derived from cordyceps. Without being limited by theory, the individual components are thought to have synergistic interactions. As a result, acceptable dosages have been developed, and preliminary human trials have shown that at least one embodiment described herein may be effective in treating COVID-19 and / or long COVID.

[0011] Aspects of the present invention were developed at least in part using data mining techniques to describe the disease networks of COVID and long COVID.

[0012] In one embodiment, a pharmaceutical composition is disclosed comprising a combination of cordycepin, adenosine, and a polysaccharide extracted from a fungus of the genus Cordyceps. Preferably, cordycepin and adenosine are also extracted from a fungus of the genus Cordyceps.

[0013] In another embodiment, an oral dosage form (oral administration formulation) comprising the composition described herein provides an effective treatment for COVID and / or long COVID when administered to a subject in an effective dose. ) will be disclosed.

[0014] In another embodiment, a method for treating COVID and / or long COVID is disclosed, comprising administering to a patient a therapeutically effective dose of a combination of cordycepin, adenosine, and a polysaccharide extracted from the fungus Cordyceps.

[0015] In another embodiment, the use of a composition for treating COVID and / or long COVID is disclosed, wherein the composition comprises a combination of cordycepin, adenosine, and a polysaccharide extracted from the fungus Cordyceps.

[0016] Embodiments of the present invention may include any aspect evident herein and any combination of features or elements described herein. [Brief explanation of the drawing]

[0017] [Figure 1A] This diagram illustrates pathways that regulate cytokine and hypoxia production. In the diagram, the three components are represented by gray ovals labeled with their common names. Potential targets are highlighted in light gray. Target genes associated with pathways potentially regulated by these compounds are indicated with a larger font size. Lines connecting two boxes with an arrow at the end indicate activation. Lines with a T at the end indicate inhibition. Solid lines without a suffix indicate binding / association. Dashed lines with arrows indicate indirect interactions, and dotted lines indicate undetermined interactions. Furthermore, the width of the line between the compound (oval) and the target (rectangular box) represents efficacy, with 1 (thickest) indicating the strongest and 30 (thinnest) indicating the weakest, and the unit of concentration is μM. Finally, ten gray solid and dashed lines are used to highlight downstream signaling pathways of targets likely regulated by the compounds. [Figure 1B] This diagram illustrates pathways that regulate the innate immune system, coagulation, and platelet activity. In the diagram, the three components are represented by gray ovals labeled with their common names. Potential targets are highlighted in light gray. Target genes associated with pathways potentially regulated by these compounds are indicated with a larger font size. Lines connecting two boxes with an arrow at the end indicate activation. Lines with a T at the end indicate inhibition. Solid lines without a suffix indicate binding / association. Dashed lines with arrows indicate indirect interactions, and dotted lines indicate undetermined interactions. Furthermore, the width of the line between the compound (oval) and the target (rectangular box) represents efficacy, with 1 (thickest) indicating the strongest and 30 (thinnest) indicating the weakest, and the concentration is in μM. Finally, ten gray solid and dashed lines are used to highlight downstream signaling pathways of targets likely regulated by the compounds.

[0018] [Figure 2]Shows the simulated 24-hour concentration profiles in human plasma, lung tissue, and muscle after oral administration of 1 mg of cordycepin.

[0019] [Figure 3] Shows the minimum effective dose of cordycepin required to inhibit 50% of the SARS-CoV-2 virus in each organ.

[0020] [Figure 4] Representative HPLC / DAD chromatogram of a sample of SCI-2213. The identity of the compound is confirmed using the overlay of this chromatogram with each respective standard. Red represents the standard, and blue represents the SCI-2213 extract.

[0021] [Figure 5] Shows the HPLC / DAD trace of the hydrolyzed SCI-2213 polysaccharide. As the derivatizing agent, 1-phenyl-3-methyl-5-pyrazolone (PMP) was used. As reported in the literature, glucose is the most abundant monosaccharide in the polysaccharide.

[0022] [Figure 6] Figure showing the polyacrylamide gel electrophoresis separation (PS-PAGE) of polysaccharides derived from Cordyceps militaris.

[0023] [Figure 7] Shows the comparison of Caco-2 cell permeability of pure cordycepin versus cordycepin in SCI-2213. The amount of cordycepin is expressed as a percentage (detected %) relative to the original concentration added to the apical side.

[0024] [Figure 8] Shows the demographic information of the participants included in the SCI-2213 study.

[0025] [Figure 9] Shows the distribution of subjects who participated in the study regarding the number of days after a positive COVID diagnosis.

[0026] [Figure 10] This report shows treatment responses (n=40) to 11 common COVID symptoms and long-term COVID symptoms.

[0027] [Figure 11] This shows the average number of doses required to elicit a significant response to 11 COVID symptoms and long-term COVID symptoms. [Modes for carrying out the invention]

[0028] term The transitional phrases “comprising,” “consisting essentially of,” and “consisting” are intended to imply the meanings generally accepted in patent terminology. That is, “comprising” is synonymous with “including,” “containing,” and “characterized by,” and is an inclusive or non-exclusive term that does not exclude additional elements or methods or processes not described herein. (ii) “consisting of” excludes elements, processes, or components not expressed in the claims. And (iii) “consisting essentially of” limits the scope of the claims or embodiments to the specified materials or processes of the claimed invention or embodiment that “do not substantially affect the basic and novel features.” More specifically, the basic and novel features relate to the ability of a method or use to provide at least one of the benefits described herein, including but not limited to the ability to improve the survivability of a human population compared to the survivability of a comparative human population described elsewhere herein. Embodiments described with respect to the phrase "comprising" (or its equivalent) also provide embodiments described independently with respect to "consisting of" and "consisting essentially of".

[0029] When a value is expressed as an approximation using the descriptor “approximately”, it will be understood that a particular value forms another embodiment. Unless otherwise specified, the term “approximately” means a variation of ±10% of the value in question, but additional embodiments include those where the variation may be ±5%, ±15%, ±20%, ±25%, or ±50%, and in particular the term “approximately” means a variation of ±5% or ±10% of the value in question, more specifically a variation of ±5%.

[0030] Where a list is presented, it should be understood that, unless otherwise specified, each individual element of that list, and all combinations of that list, are distinct embodiments. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments of "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0031] As used herein, the singular forms "a," "an," and "the" include the plural forms.

[0032] As used herein, “patient” is intended to mean any animal, in particular mammals. Therefore, the methods or uses are applicable to humans and non-human animals, but preferably humans. The terms “patient,” “subject,” and “human” may be used interchangeably.

[0033] The terms “to treat” and “to cure” refer to the treatment of a patient suffering from a pathological condition, and include not only effects that alleviate the condition by any mechanism of action, but also effects that inhibit the progression of the condition, and include at least one of the following: slowing the rate of progression, stopping the rate of progression, improving the condition, alleviating symptoms, preventing infection or symptoms, and curing the condition.

[0034] The "therapeutic dose" refers to the amount of medication that is effective in terms of the dose and duration of administration required to achieve the desired therapeutic effect. The therapeutic dose may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the medication or combination of medications to induce the desired response in the individual. Typical indicators of an effective treatment or combination of medications include, for example, improved patient well-being.

[0035] The term "dosage" refers to the amount of medication a subject should take and the frequency of administration. The term "dose" refers to the amount or quantity of medication taken at one time.

[0036] As used herein, the term "COVID" refers to severe acute respiratory syndrome (SARS), caused by the virus known as SARS-CoV-2. The term "long COVID" refers to symptoms that persist beyond the initial infection with the virus, for example, for more than 12 weeks. Long COVID may also be known as post-COVID-19 syndrome.

[0037] As used herein, terms such as “concurrent administration” encompass the administration of selected therapeutic agents to a single patient and are intended to include therapeutic regimens in which the drugs are administered by the same or different routes of administration or at the same or different times.

[0038] As used herein, the term “pharmaceutical combination” means a product resulting from a mixture or combination of two or more active ingredients, and includes both fixed and unfixed combinations of active ingredients. The term “fixed combination” means that any of the active ingredients, e.g., polysaccharides, adenosine, and cordycepin, are administered to the patient simultaneously in the form of a single unit or single dosage form. The term “unfixed combination” means that the active ingredients, e.g., polysaccharides, adenosine, and cordycepin, are administered to the patient simultaneously, in parallel, or sequentially, without specific intervening time limitations, as separate units or separate dosage forms, and such administration provides a safe and effective level of the active ingredients in the human body. The latter also applies to cocktail therapies, e.g., the administration of three or more active ingredients.

[0039] As used herein, “cordyceps polysaccharides” or “polysaccharides” means long-chain polymer carbohydrates composed of glycosidically linked monosaccharide units that can be extracted from fungi of the genus Cordyceps.

[0040] Cordycepin, or 3'-deoxyadenosine, is a derivative of nucleoside adenosine, differing from adenosine in that the hydroxyl group at the 3' position of adenosine is replaced by hydrogen. Cordycepin can be extracted from fungi of the genus Cordyceps, but it may also be produced synthetically.

[0041] Adenosine is a nucleoside and is known as a pharmaceutical ingredient, particularly for the treatment of certain arrhythmias. Adenosine can be extracted from a variety of sources, including the fungi of the genus Cordyceps, but it may also be produced synthetically.

[0042] In the history of Traditional Chinese Medicine (TCM), cordyceps, usually in the form of Cordyceps sinensis, has been used as an adaptogen and medicine. More than 600 species of cordyceps have been described in this genus. The most common species used in TCM are C. sinensis and C. militaris.

[0043] Cordyceps sinensis is typically found at high altitudes. Overharvesting has driven up the price of C. sinensis. Wild C. militaris is even rarer, and its active ingredient profile is considered superior to that of C. sinensis. Because the natural supply of Cordyceps is unsustainable, both species are cultivated, and the chemical profiles of cultivated Cordyceps have been shown to be similar to those found in nature.

[0044] This specification discloses compositions comprising cordycepin, adenosine, and a Cordyceps fungal extract (Cordyceps sinensis extract) containing polysaccharides, preferably in appropriate ratios and dosages as described herein. In preferred embodiments, the cordycepin and adenosine components are also extracted from Cordyceps fungi. Embodiments of this composition were administered to groups of subjects diagnosed with COVID and long COVID. The post-infection period ranged from 1 day to over 800 days.

[0045] The pharmaceutical compositions provided herein include effective amounts of synergistic combinations. The phrase “pharmaceutically or pharmacologically acceptable” means molecular entities and compositions that, where necessary, do not produce adverse reactions, allergic reactions, or other undesirable reactions when administered to animals, such as humans, or that may produce adverse reactions, allergic reactions, or other undesirable reactions, but are tolerable in light of the therapeutic benefits obtained. The preparation of pharmaceutical compositions including synergistic combinations is known to those skilled in the art in light of this disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference. Furthermore, it will be understood that, in the case of administration to animals (e.g., humans), the preparations should meet the standards of sterility, pyrogenicity, general safety, and purity required by regulatory authorities.

[0046] In some embodiments, the composition may be provided in a dosage form, preferably in an oral dosage form, that provides a daily dose of about 200 to about 800 mg of polysaccharides, about 15 to about 50 mg of cordycepin, and about 4 to about 50 mg of adenosine for a person weighing 70 kg. The appropriate daily dose can be calculated per kg of body weight for any particular individual. For example, a 200 to 400 mg capsule may contain 1.2 to 15.0 mg of adenosine, 5.0 to 15.0 mg of cordycepin, and 50 to 200 mg of polysaccharides. As is known in the art, the daily dose may be taken as a single dose, or divided and taken two, three, or more times a day.

[0047] Suitable oral dosage forms are well known in the art and include capsules, tablets, solutions, suspensions, powders, and nanoencapsulated dosage forms.

[0048] In some preferred embodiments, the ratio of polysaccharide to cordycepin may be about 3:1 to about 40:1, and the ratio of cordycepin to adenosine may be about 1:5 to about 10:1.

[0049] Pharmaceutical compositions may include pharmaceutically acceptable carriers, as is known to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp. 1289-1329), including all kinds of solvents, dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and similar materials and combinations thereof. The use of conventional carriers is envisioned in the therapeutic or pharmaceutical compositions disclosed herein, unless incompatible with the active ingredient.

[0050] Exemplary compositions were developed using a systems biological approach developed by Tam, Tseng et al. (2022). The following examples describe the sequence of the formulation development process and human trials. In summary, the chemical profile of Cordyceps sinensis / Cordyceps militaris was compiled and potential active compounds were identified. [Examples]

[0051] We identified compound-target interactions in the COVID disease pathway described in the literature. The number of bioactive compounds with potential therapeutic value was narrowed down using criteria based on drug-like properties and the abundance of active compounds in fungi. As a result, three bioactive compounds were identified: adenosine, cordycepin, and a polysaccharide.

[0052] This specification describes the antiviral, anti-inflammatory, and immunomodulatory activities of cordycepin, as well as their mechanisms of action.

[0053] This specification describes the antiviral activity of adenosine.

[0054] This specification describes the antiviral effects of cordyceps polysaccharides through their immunomodulatory and anti-inflammatory activities.

[0055] In this specification, the drug-like properties of cordycepin, adenosine, and polysaccharides are described in humans using in silico estimation or human pharmacokinetic data.

[0056] In this specification, the effective oral dose of combination therapy is determined for the treatment of COVID and long-term COVID.

[0057] The anti-COVID and anti-long-COVID effects of SCI-2213 were tested in 40 human subjects. SCI-2213 showed significant effects in alleviating 11 common symptoms of COVID and long-COVID.

[0058] The following examples are for illustrative purposes only and do not limit the claimed inventions.

[0059] Example 1 The objective of this example is to first elucidate the disease network of COVID-19 using the methodology developed by Tam, Tseng et al. (2022), and then evaluate Cordyceps sinensis / Cordyceps militaris compounds that may influence the disease-carrying targets and pathways. Subsequently, the compound-target network is mapped, and thus the potential mechanisms of action of these compounds are derived.

[0060] While the symptoms of long-term COVID are thought to be similar to those of COVID, there are differences in the severity of the disease and symptoms. Long-term COVID patients experience more pronounced physical damage from the virus, but with a lower viral load.

[0061] COVID and the Long COVID Disease Network The COVID disease network is summarized in KEGG (KEGG ID, HSA:05171). Its pathways are described in Figures 1A and 1B. The network consists of 14 signaling pathways through which SARS-CoV-2 viral proteins trigger inflammatory cytokines, cytokine storms, inflammation, coagulation, evasion of the innate immune system, and inhibition of the establishment of the antiviral state. In addition to the primary pathways described in KEGG, targets directly or indirectly involved in modulating the primary pathways of the COVID network are also considered.

[0062] The targets involved in the disease process are outlined below. • Immunomodulation targets include interleukin-12 (IL-12) and interferon-gamma (IFNγ). Inflammation targets include IL-1α, IL-1β, IL-6, IL-10, tumor necrosis factor α (TNFα), transforming growth factor β (TGFβ1), and IFNγ. • Targets of hypoxia include nuclear factor erythroid 2-related factor 2 (Nrf2), hypoxia-inducible factor 1 (HIF1), nuclear factor kappa B subunit 1 (NFκB), and angiotensin-converting enzyme 2 (ACE2). • For fibrosis such as pulmonary fibrosis, targets include two families: matrix metalloproteinase (MMP) / metalloproteinase tissue inhibitor (TIMP), IFNγ, CSF2, ELANE, CCL2, CXCR4, and IL-1α.

[0063] The targets associated with the severity of COVID-19 infection are as follows (Karlowitz, Stanifer et al. 2022, Lu, Shirvani et al. 2022, Namkoong, Edahiro et al. 2022, Redin, Thorball et al. 2022). • A key target is cytokinesis regulator 2 (DOCK2) (Namkoong, Edahiro et al. 2022). ·IFNα, IFNl, IFNαR, IFNαR2, IFNlR • Non-receptor tyrosine protein kinase (TYK2) • Toll-like receptors TLR3 and TLR7 Neutrophils / lymphocytes, with folate receptor γ (FOLR3) and G protein signaling regulator 1 (RGS1) as targets, are among the most important.

[0064] Identification of active compounds in Cordyceps sinensis / Cordyceps militaris In this example, the inventors focus on Cordyceps sinensis / Cordyceps militaris, which have been reported to have multiple functions in immunomodulation, antiviral, anti-inflammatory, and antioxidant activity. Therefore, these two fungi may contain components active against COVID-19. Chemical profiles of Cordyceps were collected from TM-MC (https: / / informatics.kiom.re.kr / compound / search.do). A total of 91 compounds were identified in Cordyceps (Table 1). [Table 1]

[0065] Using PK / PD criteria for hit analysis, 17 chemicals from cordyceps that may interact with targets involved in the COVID-19 disease network were identified. These include cordycepin, adenosine, 2-chloroadenosine, 2'-O-methyladenosine, adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), campesterol, cholesterol, lovastatin, and eight fatty acids: pentadecanoic acid, docosanic acid, lauric acid, myristic acid, lignoceric acid, stearic acid, palmitic acid, and linoleic acid. Before constructing a compound-target interaction network, the results of biological assays of these 17 compounds were reviewed. Eight fatty acids reported in PubChem were excluded because their activity lies in the chemical derivatives of these compounds.

[0066] Yang et al. reported that Cordyceps militaris contains small amounts of AMP and GMP in amounts of approximately 100–700 μg / g (Yang, Li et al. 2010). While studies suggested that AMP and GMP have pharmacologically promising effects, they were not included in the network construction due to their negligible presence. These compounds are also not detected in the extracts disclosed in this invention.

[0067] Similarly, 2-chloroadenosine, 2'-O-methyladenosine, campesterol, cholesterol, and lovastatin are not detected in the cordyceps extract disclosed in this invention. Therefore, these compounds are also excluded from the compound target network construction.

[0068] Although our automated data mining did not include polysaccharides, many studies suggest that these compounds have potential activity against Covid-19 (Kuo, Chen et al. 2007, Ohta Y. 2007, Lee, Kwon et al. 2015, Liu, Feng et al. 2016, Mao, Song et al. 2022). These studies have used polysaccharides including extracellular polysaccharides, intracellular polysaccharides, and acidic polysaccharides. Therefore, we have included all of these polysaccharides in our investigation. However, in addition to the remaining two chemicals, we have simply labeled them as polysaccharides to construct an interaction network. The roles of these three chemicals are summarized in Table 2. [Table 2]

[0069] Cordyceps-COVID-19 Interaction Network. In addition to Table 2, Figures 1(A) and (B) show the interaction network of three compounds and their potential targets in the COVID-19 disease network. Figure 1(A) shows the pathways that modulate cytokine and hypoxia production. Figure 1(B) shows the pathways that modulate the innate immune system, coagulation, and platelet activity. In both figures, the three compounds are represented by gray ovals labeled with their generic names. Potential targets previously shown are highlighted in light gray. Larger font sizes are used for target genes associated with pathways that may be modulated by these compounds. Lines connecting two boxes with an arrow at the end indicate activation. Lines with a T at the end indicate inhibition. Solid lines without a trailing symbol indicate binding / association. Dashed lines with arrows indicate indirect interactions, and dotted lines indicate undetermined interactions. The width of the line between the compound (oval) and the target (rectangular box) represents the activity value in the range of 1 (thickest) μM to 30 (thinnest) μM. In other words, the thicker the line, the stronger the compound's activity. Finally, ten gray solid and dashed lines are used to highlight the downstream signaling pathways of targets that are likely regulated by the three compounds.

[0070] Potential mechanisms of action of cordyceps in the treatment of COVID-19. Based on this interaction network (Figure 1(A) and Figure 1(B)), a comprehensive overview of how these three chemicals may modulate cellular functions involved in the treatment of COVID-19 is presented. The following are the potential roles of these three chemicals in the major pathogenesis of COVID-19. • Regulation of inflammatory cytokines. The SARS-CoV-2 virus uses ACE2 to enter cells, directly affecting the renin-angiotensin system. One of its downstream targets, NFκB1, leads to the expression of several inflammatory cytokines (pathway 1 in Figure 1(A)), providing one pathway that triggers a cytokine storm. Two chemicals, cordycepin and adenosine, likely inhibit NFκB1 and can reduce the expression of inflammatory cytokines. • Regulation of cytokine storms. Several pathways exist that can regulate cytokine storms. These include the renin-angiotensin system, the TNF signaling pathway, FcγR-mediated phagocytosis, the Toll-like receptor signaling pathway, and the Nod-like receptor signaling pathway. The renin-angiotensin system has already been described, so the following disclosure will focus on the remaining pathways. It has also been found that polysaccharides can increase IL-1β expression. Regarding the TNF signaling pathway, it primarily mediates the innate immune system by promoting the expression of several inflammatory cytokine genes via NFκB1 (pathways 1, 2, and 3 in Figure 1(A)). Two chemicals, cordycepin and adenosine, are likely to inhibit NFκB1 and reduce the expression of inflammatory cytokines. However, polysaccharides have been shown to increase TNF and IFNγ gene expression and promote NFκB1 expression. Cordycepin, on the other hand, inhibits both. Regarding FcγR-mediated phagocytosis, it promotes the expression of inflammatory cytokine genes via MAPK (pathway 6 in Figure 1(A)). Adenosine can inhibit MAPK, which mediates cytokine storms. Regarding the Toll-like receptor signaling pathway (pathway 7 in Figure 1(A)), there is a combined effect of the three compounds. Cordycepin was found to inhibit JNK and JUN (AP-1 transcription factor), suppressing cytokine storms. On the other hand, polysaccharides were found to increase the expression of TLR-2, which can promote the expression of downstream cytokine genes. Regarding the Nod-like receptor signaling pathway, it promotes the innate immune system through indirect activation by NFκB1, which can be downregulated via the Toll-like receptor pathway (pathway 3 in Figure 1(A)). Here again, two chemicals that inhibit NFκB1, adenosine and cordycepin, can also mediate the innate immune system through this pathway. • Regulation of phagocytosis. FcγR-mediated phagocytosis is one of the immune responses in humans to eliminate viral infection. Our mining shows that adenosine can suppress FcγR-mediated phagocytosis by inhibiting PLCγ1 (pathway 5 in Figure 1(A)). • Regulation of fibrosis. As previously mentioned, several targets, including MMP, CSF2, and CCL2, are associated with fibrosis (Kaymakci and Guler 2020). Our analysis has shown that cordycepin and adenosine can directly or indirectly repress the expression of these genes by inhibiting several upstream targets in the renin-angiotensin system, FcγR-mediated phagocytosis, and Nod-like receptor signaling pathways (pathways 1, 2, 3, and 6 in Figure 1(A)). Therefore, they may be able to regulate fibrosis. • Regulation of hypoxia. Several targets, including Nrf2, HIF1, NFκB1, and ACE2, are associated with hypoxia tolerance, as shown in pathway 4 in Figure 1(A). As suggested by an in vitro study (Singh, Tulsawani et al. 2013), cells treated with Cordyceps sinensis resulted in increased Nrf2 and HIF1 levels and decreased NFκB1, which significantly improved hypoxia tolerance. Based on the data mining disclosed herein, NFκB1 is the only target found to be directly inhibited by cordycepin and adenosine. However, it has been suggested that polysaccharides may increase NFκB1 expression by activating IFNγ and IL6. However, inhibition of NFκB1 may decrease HIF1 expression. Furthermore, HIF1 may be regulated by adenosine via inhibition of MAPK1, as shown in pathway 4 in Figure 1(A). However, since MAPK1 also inhibits HIF1 translation through the inhibition of EIF4EBP1, the overall activity of these two chemicals in increasing HIF1 expression requires further investigation. The effects of the three active compounds on hypoxia are not clear, but the potential effect of polysaccharides on preventing hypoxia has been reported by Dong, Hu et al. (2015). • Regulation of the innate immune system. Several SARS-CoV-2 viral proteins, including nsp3, nsp6, and ORF6, have been shown to help the virus escape the innate immune system by suppressing several targets in the cytoplasmic DNA sensing pathway (pathway 8 in Figure 1(B)). Unfortunately, in this disclosure, we were unable to obtain any compounds that could potentially prevent the SARS-CoV-2 virus from escaping the immune system. • Regulation of antiviral state establishment. The JAK-STAT signaling pathway plays a crucial role in regulating inflammation in response to viral infection. In particular, interferon-stimulated genes (ISGs) contain the DNA-binding component IRF9 (Figure 1(B)). These ISGs act at various stages of the viral life cycle, resulting in an antiviral state that provides sufficient immunity against the virus. Lack of IRF9 has been shown to result in impaired antiviral state (Wang, Xu et al. 2017). As shown in Figure 1(B), the SARS-CoV-2 virus inhibits the establishment of antiviral state by inhibiting STAT and IRF9. Through our mining, we identified one chemical, adenosine, that may modulate the JAK-STAT signaling pathway (pathway 9 in Figure 1(B)). Unfortunately, adenosine has been found to inhibit IFNβ, which may suppress the establishment of antiviral state. • Regulation of thrombosis. One of the complications associated with COVID-19 is a tendency towards thrombosis (Badulescu, Sirbu et al. 2022). As shown in the complement cascade at the bottom of Figure 1(B), the SARS-CoV-2 virus may promote the overexpression of thrombin in the blood system, increasing the risk of thrombosis. Polysaccharides have been found to inhibit platelet aggregation by inhibiting CD62p and ITGA2B, thereby reducing the risk of thrombosis (pathway 10 in Figure 1(B)). The antiplatelet activity of polysaccharides in cordyceps has been demonstrated by Mao, Song et al. (2022).

[0071] Cordyceps has been shown to have beneficial effects in the treatment of COVID-19. The inventors identified three compounds that may be involved in these effects. The potential mechanisms of action of these compounds were investigated using a compound-target interaction network (Figure 1(A) and Figure 1(B)). The inventors' findings suggest that these compounds may modulate intracellular signaling in patients to activate the immune system against the SARS-CoV-2 virus and / or restore several functions affected by the virus. The potential mechanisms of action of these compounds against COVID-19 can be summarized by five key elements: 1. modulation of cytokine storm, 2. enhancement of the immune system, 3. increased hypoxia tolerance, 4. reduced risk of thrombosis, and 5. reduced risk of fibrosis. These five key elements are detailed below, as shown in Table 2. • Regulation of cytokine storms: Several pathways regulate cytokine storms. Cordycepin and adenosine can suppress cytokine storms by inhibiting two targets, NFκB1 and MAPK. These chemicals have shown potential to reduce inflammatory responses by downregulating various pathways and suppressing cytokine expression. • Enhancement of the immune system: Our findings suggest that polysaccharides are promising compounds that enhance the immune system by regulating the TNF and Toll-like receptor pathways through the activation of TNF, IL6, TLR2, and IFNγ. • Increased Hypoxia Tolerance: Our findings indicate that cordycepin and adenosine may have a dual effect in regulating the expression of HIF1, an important biomarker in the regulation of hypoxia. Depending on the specific pathway involved, these two chemicals can activate or inhibit hypoxia-related pathways. By better understanding these mechanisms, we believe it may be possible to potentially increase hypoxia tolerance. Furthermore, the contribution of polysaccharides to increasing hypoxia tolerance needs to be elucidated. • Reduced risk of thrombosis: Polysaccharides may reduce the risk of thrombosis by inhibiting platelet activation through the inhibition of integrin alpha-II-beta. Here again, a better understanding of these mechanisms may reduce the potential risk associated with thrombosis. • Reduced risk of fibrosis: Our findings indicate that the compound could not directly inhibit MMP3, one of the fibrosis-related genes. Other fibrosis-related genes, such as CSF2 and CCL2, can only be regulated indirectly by inhibiting NFκB1 and MAPK with cordycepin and adenosine.

[0072] In summary, our research suggests that cordycepin and adenosine play a dual role in cytokine production and immune system regulation, which are key components of COVID-19 treatment. On the one hand, cordycepin and adenosine can suppress cytokine storms, while on the other hand, they can also suppress the adaptive immune system. Meanwhile, polysaccharides can activate TNFα, IL-6, TLR2, and IFNγ to counteract the effects of cordycepin and adenosine and achieve immune system balance. Furthermore, polysaccharides may also play a role in regulating platelet activity and reducing the risk of thrombosis. It is believed that by combining cordycepin, adenosine, and cordyceps polysaccharides, preferably in effective proportions and effective doses, the immune system can be regulated to effectively cope with COVID-19 and long COVID, without being limited by theory.

[0073] Example 2 The purpose of this example is to estimate the minimum clinically effective dose of cordycepin in humans using the in silico method.

[0074] Recent meta-analyses have shown that remdesivir reduces mortality in hospitalized COVID-19 patients who do not require standard oxygen therapy (Amstutz, Speich et al. 2023). Both in vitro and in silico studies have shown that cordycepin exhibits greater inhibitory effects against key SARS-CoV-2 protein targets, including the spike (S) protein, the main protease (M(pro)) enzyme, and the RNA-dependent RNA polymerase (RdRp) enzyme, compared to remdesivir (Rabie 2022). However, the pharmacokinetics of cordycepin in humans are not well understood. The aim of this study is to computationally estimate the pharmacodynamic (PD) and pharmacokinetic (PK) parameters of cordycepin, with the aim of estimating, through PK / PD simulations, human doses that can be used to efficiently control COVID-19 and provide relief from long-term COVID-19.

[0075] Using Vero E6 cells containing the first variant of the SARS-CoV-2 strain from December 2020, Rabie (2022) found that cordycepin was administered at an IC50 level of 2 μM or 0.502 μg / ml. 50 This IC is reporting a value. 50 The value is adopted as the PD parameter for current dose estimation. The remaining goal is to estimate the minimum effective dose of cordycepin required to inhibit SARS-CoV-2.

[0076] PK Simulation. The inventors performed PBPK (physiology-based pharmacokinetic) simulations using the PK-Sim program from Open Systems Pharmacology(C) Suite. At the time of this simulation, the inventors identified only one clinical report using the herbal formulation HAD-B1 containing cordycepin. One objective in this study was to determine the pharmacokinetics of cordycepin in the HAD-B1 formulation. Changes in cordycepin concentration over 24 hours after a single oral administration of the formulation in the subjects were collected and analyzed. Unfortunately, data other than the time to peak concentration (1.5 hours) and half-life (8.62 hours) have not yet been published. In addition, the inventors obtained experimental solubility in water at 1 mg / ml and 1.1 × 10⁻⁶ from Sigma's product information sheet. -7 CaCO2 permeability data in cm / s (Lee, Radhi et al. 2019) were also obtained. Therefore, the inventors use these data to prepare the input parameters necessary to establish a PK model in humans. The tissue-to-plasma coefficient was estimated using the method of Poulin and Theil (2002). For the remaining physiological parameters for a 30-year-old male weighing 73 kg, default values ​​from PK-Sim were used. In the simulation, a single dose of 1 mg of cordycepin was administered orally. 24-hour concentration profiles of cordycepin in plasma and lung tissue were created and replotted using Originpro.

[0077] Method for estimating the minimum effective dose. Effective dose EC of the target compound. 50 (ng / ml) can be interpreted as the concentration required to have a 50% chance of activating or inhibiting a specific cellular function, so in order to exert an effect on target cells at the site of action, the concentration level of the compound in plasma over time T must be EC 50 *It is suggested that it must be greater than T. That is, given a target compound with an initial dose D0 (mg) that would have a 50% chance of activating or inhibiting the target protein in humans over a T time, in plasma

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[0078] Simulated concentration profiles of cordycepin in humans. PK-Sim was used to estimate the PK profile of cordycepin in human target organs. Figure 2 shows the 24-hour concentration profiles of cordycepin in plasma, lung, and muscle tissue (for illustrative purposes only). The complete set of organ-specific PK data, expressed in AUC, is for brain, fat, heart, kidney, liver, lung, muscle, as well as the small and large intestinal lumen, and is shown in Table 3. Note that the AUC units for the intestinal lumen are mg*h / ml, and the rest are ng*h / ml. By a brief comparison, our simulation (shown by PK-Sim) is similar to the clinical study reported by Kim et al. 2022. max = 1.85 hours and t 1 / 2 The study shows that a time of 7.93 hours is obtained. Furthermore, simulations using a rat model produce plasma concentration profiles similar to those in the in vivo study by Lee et al. (Lee, Radhi et al. 2019) (data not shown). Therefore, although the simulation results cannot be fully validated, these results are considered reasonable.

[0079] Cordycepin appears to be stored in the muscles. Figure 2 shows an interesting feature. Simulations suggest that cordycepin is stored in the muscles and released slowly. The half-life of cordycepin in muscles is 16.31 hours, while in plasma it is 7.93 hours.

[0080] Estimated minimum effective dose of purified cordycepin required to inhibit the SARS-CoV-2 virus. IC for cordycepin against SARS-CoV-2 virus (Rabie 2022). 50Based on the AUC of all organs in Equation (4) and Table 3, the inventors calculated the minimum effective oral dose of cordycepin that can inhibit 50% of the infectious virus in the corresponding organs. The results are shown in Figure 3. Figure 3 shows that 4905.66 mg of cordycepin is required to inhibit 50% of the virus in the brain tissue, about 1200.4 mg for fat and liver, 355 mg for plasma, 463 mg for the heart, 605 mg for the kidney, 414 mg for the lung, and 400 mg for muscle.

[0081] Therefore, we speculate that a formulation consisting of at least 4905.66 mg of cordycepin is required to achieve an effect that acts on the whole human body in order to address various complications caused by COVID-19. However, at a dose of 400 mg to 600 mg of cordycepin, effective concentrations are achieved in plasma, fat, heart, kidney, liver, lung, muscle, and the lumens of the small and large intestines, and a sufficient clinical response is obtained.

Table 3

[0082] In summary, the inventors established a PK model using in vitro and in silico data and studied the ADME of cordycepin in humans. Subsequently, the inventors estimated the AUC of cordycepin in each organ of humans using a pharmacokinetic (PK) model. Furthermore, the inventors predicted and calculated the minimum effective dose of cordycepin required to inhibit 50% of the SARS-CoV-2 virus using the in vitro IC 50 value (Equation (4)). The calculations of the inventors suggest that the minimum clinical effective dose of cordycepin is approximately 500 mg.

[0083] Example 3 The purpose of this example is to measure the chemical profile of an extract of Cordyceps militaris evaluated in a preliminary study in humans.

[0084] Analysis of nucleosides and other bases in the extracts: 100 mg of each sample was dissolved in 0.3% formic acid at a concentration of 10.0 mg / ml while sonicating at room temperature for 1 hour. The sample was then clarified by centrifugation, filtered, and directly injected into HPLC. Detailed procedure is described in SCI-TMS 001. Samples were run in double cycles. The amounts of uracil, hypoxanthine, uridine, thymine, adenine, adenosine, and cordycepin were determined by HPLC-DAD absorbance detection at 260 nm using external standards (obtained from Sigma), and identity was confirmed by the retention time of each standard (Figure 4 and Table 4). [Table 4]

[0085] Quantitative determination of polysaccharides Extraction: 100 mg of each sample was dissolved in hot water (LC-MS grade) at a concentration of 10.0 mg / ml and sonicated at 80°C for 1 hour. The samples were then clarified by centrifugation and filtered as necessary. Hydrolysis: The sample was hydrolyzed with concentrated HCl at 100-105°C for 4 hours to obtain the corresponding monosaccharide. Derivatization: All samples were treated with PMP and 1-phenyl-3-methyl-5-pyrazolone as derivatizing agents at 68-70°C for 4 hours. HPLC-DAD: All samples were injected after filtration and run in two cycles. The amounts of mannose, glucuronic acid, galacturonic acid, rhamnose, glucose, galactose, and arabinose were determined by HPLC-DAD absorbance detection at 250 nm using an external standard (obtained from Sigma), compared to a control sample (a normal sample but not hydrolyzed). Identity was confirmed by the retention time (SCI-TMS-009-v2) of each standard (after the same treatment as the sample). Total polysaccharides were calculated as the sum of all monosaccharides, and the value was corrected for polysaccharide hydrolysis (Figure 5 and Table 5). [Table 5]

[0086] The results from these studies clearly showed that adenosine, cordycepin, and polysaccharides are the major components in the extract. Based on computer analysis, it is reasonable to assume that these three compounds are responsible for the anti-COVID and anti-long-COVID activity of Cordyceps militaris.

[0087] Separation of polysaccharides

[0088] Polysaccharides from Cordyceps militaris were isolated using polyacrylamide gel electrophoresis (PS-PAGE). Briefly, a crude Cordyceps militaris extract (CME) was prepared from raw, dried mushroom powder using a slightly modified procedure described by Dong, Hu et al. (2015).

[0089] A polysaccharide extract was prepared using the crude extract. This was achieved by separating the protein using the SEVAG reagent and then precipitating the polysaccharides with ethanol (Dong, Hu et al. 2015). The polysaccharide residue was freeze-dried and stored in a freezer before analysis.

[0090] The polysaccharides were derivatized using the fluorescent agent 2-aminoacridone (AMAC). A slightly modified version of the procedure described by Calabro, Benavides et al. (2000) was used.

[0091] PS-PAGE was performed following the procedure described by ALCunha et al. (2015), with slight modifications.

[0092] Polysaccharides from six Cordyceps militaris samples collected from various sources were separated using this method (Figure 6). This method is used as a quality control measure to ensure uniformity of polysaccharide distribution.

[0093] Example 4 The purpose of this study is to evaluate the potential increase in the oral bioavailability of cordycepin due to the components of the extract.

[0094] The oral bioavailability of cordycepin, when given as a pure compound, is extremely low (Lee, Radhi et al. 2019). This is at least in part due to rapid removal by deaminases in intestinal cells.

[0095] Method: Pore size 1 μm and 0.3 cm 2 Transepithelial wells with a surface area were pre-incubated for 1 hour at 37°C in 5% CO2 with DMEM-F12 medium containing 10% FBS and 10 μg / ml penicillin / streptomycin. CaCO2 cells were seeded in the same medium at a density of 50,000 cells. The cells were maintained under these conditions throughout the experiment. The medium was changed every 2-3 days, and the TEER value was monitored from day 10 onwards. On day 18, the TEER value was 200 ohms / cm². 2 Cells with TEER values ​​exceeding a certain threshold were incubated in PAB buffer for 20 minutes: 1 ml in the basolateral and 0.5 ml in the apical. Samples of 200 μg / ml cordycepin and 200 μg / ml cordycepin in standardized cordyceps extract were prepared in PAB buffer, and the PAB buffer was replaced with apical buffer. 50 μl samples were taken from each of the apical and basolateral compartments, initially and at 1-hour intervals. The samples were immediately added to 450 μl of methanol and quantified using an Agilent 4910 LC-mass spectrometer. Cordycepin was measured by direct injection in SMS mode, and the integrated values ​​were compared to a standard curve prepared in the same manner as the test samples.

[0096] Results: As shown in Figure 7, no detectable cordycepin was measured at the basal level, suggesting that the permeability of cordycepin is negligible when evaluating a pure cordycepin solution. This observation is consistent with that reported by Lee, Radhi et al. (2019). Rapid deamination during the permeation process may be one of the main reasons. This observation indicates that cordycepin does not have significant bioavailability when administered orally to humans, suggesting that cordycepin is not very effective in systemically eradicating viruses that come into contact with it.

[0097] On the other hand, the extract showed significant permeability (3.76 × 10⁻⁶). -6 P in cm / s app The values ​​indicated that the oral bioavailability of cordycepin was enhanced by the components of the extract.

[0098] These results suggest that, in addition to the potential pharmacodynamic interaction between cordycepin and adenosine, other components may exist that could reduce the elimination of cordycepin and increase its bioavailability in humans. These may include adenosine and other adenosine analogs.

[0099] Since cordycepin needs access to the site of action to induce its pharmacological activity, including antiviral activity in the lungs, systemic availability of cordycepin is important.

[0100] Example 5 The purpose of this study is to evaluate the efficacy of cordyceps militaris extract in human subjects tested in either COVID-positive (within 30 days) or with prolonged COVID symptoms three months after a positive COVID diagnosis (WHO definition of long COVID).

[0101] Specifically, the amounts of the three main components—adenosine, cordycepin, and polysaccharides—are 1.3%, 3.0%, and 38% (by weight), respectively.

[0102] This extract (SCI-2213) was formulated into 300 mg capsules. Each capsule contains 4.0 mg of adenosine, 9.0 mg of cordycepin, and 114 mg of polysaccharides.

[0103] The preliminary study enrolled 40 participants, 18 men and 22 women, ranging in age from 20 to over 80 years old (Figure 8).

[0104] Figure 9 shows the distribution of subjects regarding the time elapsed since a positive COVID test. These participants consist of individuals with acute COVID-19 and those with long-term COVID-19 illness.

[0105] Participants were instructed to take two capsules twice daily on an empty stomach for at least seven days. Each participant was required to complete a questionnaire before and daily during the study. The questionnaire included descriptions of 11 COVID symptoms and long-term COVID symptoms. These symptoms included fever, cough, sputum, headache, body aches, fatigue, brain fog, sleep quality, hearing, taste, and smell. On a scale of 1 to 10, 10 indicated no symptoms, and 1 indicated the most severe discomfort experienced by the participant.

[0106] Data Analysis The data was preprocessed before the final analysis. For each symptom, if the score was 5 or higher, the data was excluded from the analysis because it was determined that the potential for improvement was not large enough to warrant statistical analysis.

[0107] The mean scores for each symptom before subjects began taking the extract ranged from 3 to 5. The mean response to all symptoms after treatment was above 8 (Figure 10). The data demonstrated that SCI-2213 has a significant effect in alleviating symptoms associated with COVID and long-term COVID. The time elapsed since a positive COVID diagnosis appears to have little impact on the effectiveness of SCI-2213.

[0108] The average dose required to achieve this significant response was 5.29, suggesting that participants experienced significant symptom relief on average in less than two days (Figure 11).

[0109] A male subject began taking SCI-2213 one day after a positive COVID-19 diagnosis. For the first two days, this subject showed no response to treatment; in fact, his condition worsened. Specifically, he developed a higher fever, limb pain, and a worsening cough. After increasing the dose to four capsules twice daily, the subject's condition significantly improved within 24 hours. This observation suggests a dose-related response to SCI-2213.

[0110] Given the current lack of a diagnosis or even a treatment for long-term COVID-19, SCI-2213 could be a solution to provide relief to people suffering from mild to moderate COVID-19 symptoms and long-term COVID-19 symptoms. SCI-2213 will fill a long-awaited and unmet need worldwide.

[0111] conclusion This disclosure describes the development of a combination therapy for treating COVID and long COVID using a process developed by Tam, Tseng et al. (2022). The response rate for major symptoms is high at 90%, making initial observations in patients promising. In one embodiment, the combination comprises SCI-2213 having a synergistic combination, with each component formulated in a desired dosage. References The following references indicate the level of skill of those skilled in the art, and where permitted, they are incorporated herein by reference in their entirety. JPEG2026514740000015.jpg191168 JPEG2026514740000016.jpg198168

Claims

1. A composition for the treatment of COVID and / or long COVID, comprising cordycepin, adenosine, and a cordyceps fungal extract containing polysaccharides.

2. The composition according to claim 1, wherein the ratio of the polysaccharide component to cordycepin is about 3:1 to about 40:

1.

3. The composition according to claim 1 or 2, wherein the ratio of cordycepin to adenosine is about 1:5 to about 10:

1.

4. The composition according to any one of claims 1 to 3, wherein either or both of cordycepin and adenosine are extracted from cordyceps.

5. The composition according to any one of claims 1 to 3, wherein either or both of cordycepin and adenosine are produced synthetically.

6. The composition according to any one of claims 1 to 5, in the form of a pure form or a standardized extract.

7. An oral formulation comprising the composition according to any one of claims 1 to 6, and optionally comprising a pharmaceutically acceptable carrier.

8. The oral formulation according to claim 5, which is a capsule, tablet, solution, suspension, powder, or nanoencapsulated formulation.

9. A method for treating COVID-19 or long-term COVID-19 by administering a therapeutically effective amount of the composition according to any one of claims 1 to 6 or the oral formulation according to claim 7 or 8.

10. The method according to claim 9, wherein the polysaccharide and cordycepin are administered in a ratio of about 3:1 to about 40:1, for example, about 20:1 to about 35:

1.

11. The method according to claim 10, wherein the ratio is approximately 28.5:

1.

12. The method according to any one of claims 9 to 11, wherein cordycepin and adenosine are administered in a ratio of about 1:5 to about 10:1, for example, about 2:1 to about 5:

1.

13. The method according to claim 12, wherein the ratio is approximately 2.25:

1.

14. The method according to any one of claims 9 to 13, wherein the daily dose comprises, for a person weighing 70 kg, a daily dose of approximately 200 to approximately 800 mg of polysaccharides, a daily dose of approximately 15 to approximately 50 mg of cordycepin, and / or a daily dose of approximately 4 to approximately 50 mg of adenosine, or an equivalent daily dose calculated per kg of human body weight.

15. The method according to claim 12, wherein the daily dose comprises one or more administrations of an oral formulation containing approximately 1.0 to approximately 15.0 mg of adenosine, approximately 5.0 mg to approximately 15.0 mg of cordycepin, and / or approximately 50 mg to approximately 200 mg of polysaccharides in an oral formulation of 200 to 400 mg.

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