Use of cyanobacterial biomass in the treatment of hepatitis B virus infection

Abstract

Disclosed herein is the use of cyanobacterial biomass for treating hepatitis B virus (HBV) infection, particularly chronic HBV infection. According to various embodiments of the present disclosure, the cyanobacterial biomass, when administered for at least one month, significantly reduces detectable levels of hepatitis B virus surface antigen (HBsAg) and / or alleviates insomnia associated with chronic HBV infection in a subject receiving treatment.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims the benefit of U.S. Provisional Application No. 62 / 587,488, filed November 17, 2017, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] FIELD OF THEINVENTION The present disclosure relates generally to the field of treatment of Hepatitis B virus (HBV) infection. More specifically, the present disclosure relates to the use of cyanobacterial biomass in the treatment of HBV infection.

[0003] Description of Related Art

[0004] HBV is a small, double-shelled virus in the Hepadnaviridae family. HBV can cause both acute and chronic infections. Acute infection with HBV is associated with acute viral hepatitis, a disease characterized by fatigue, nausea, vomiting, headache, fever, skin rash, joint pain, arthritis, myalgia, and / or dark urine before the appearance of jaundice. The disease can last for several weeks and progresses to fulminant hepatic failure (ALF) in most affected individuals, but only about 1%-2% of acutely infected people develop fulminant hepatic failure (ALF).

[0005] The proportion of patients with acute HBV infection who progress to chronic infection usually varies with age and immune status. It has been reported that approximately 90% of infants who acquire HBV infection from their mothers at birth or during infancy become chronically infected. Among children infected with HBV between 1 and 5 years of age, 30% to 50% become chronically infected. By adulthood, the risk of acquiring chronic HBV infection is approximately 5%, and people with weakened or deficient immune systems are more likely to develop chronic HBV infection than adults with normal immune systems. Chronic infection is responsible for most HBV-related morbidity and mortality, including chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma.

[0006] The diagnosis of HBV infection is usually based on serological tests for HBV antigens and antibodies. HBV contains multiple antigenic components, including hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and hepatitis B e antigen (HBeAg), among which HBsAg, a protein involved in HBV replication and infectivity, is the most commonly used marker for the diagnosis of HBV infection. Generally, HBsAg can be identified in serum 30 to 60 days after infection and may persist for an indefinite period. Antibodies against HBsAg (anti-HBs or HBsAb) usually develop during the convalescent phase after HBV infection that neutralize HBV infectivity, clearing circulating HBsAg and infectious HBV particles from the peripheral blood. Persistence of HBsAg for more than a 6-month period indicates a chronic carrier state or the development of chronic HBV infection.

[0007] Currently, there are two main categories of antiviral therapy approved for the treatment of chronic HBV infection in many countries. The first is interferon alpha (IFN-α) or pegylated IFN-α (PEG-IFNα), and the second includes various nucleoside or nucleotide analogs (e.g., lamivudine, adefovir, entecavir, telbivudine, and tenofovir). However, these drugs simply stop or reduce viral replication, but none of them can eliminate the infection. Therefore, the majority of patients with chronic HBV infection require long-term treatment to continue to suppress viral replication. Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the foregoing, there exists a need in the art for new drugs useful in the treatment of chronic HBV infection. [Means for solving the problem]

[0009] (overview) The following presents a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary is not an extensive overview of the disclosure and does not identify key / critical elements of the invention or delineate the scope of the invention. The sole purpose of this summary is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description presented later.

[0010] In one aspect, the disclosure is directed to a method for treating an infection caused by Hepatitis B Virus (HBV) in a subject in need of treatment. As embodied and broadly described herein, the method of treatment is effective in treating HBV infection, including chronic HBV infection. The method is also effective in reducing detectable Hepatitis B Virus Surface Antigen (HBsAg) in the subject, and in some cases, HBsAg seroclearance. The present invention is also effective in preventing or delaying the onset of liver disease (e.g., liver fibrosis, cirrhosis, or liver decompensation) associated with chronic HBV infection. Additionally, the method can effectively alleviate insomnia associated with chronic HBV infection.

[0011] According to some embodiments of the present disclosure, the method includes administering an effective amount of a cyanobacterial biomass to a subject.

[0012] According to certain embodiments of the present disclosure, the cyanobacterial biomass is derived from Arthrospira maxima.

[0013] In various embodiments, the subject is a mammal, including a human.

[0014] In some embodiments, the subject suffers from chronic HBV infection. In some other embodiments, the subject suffers from cirrhosis or hepatic decompensation associated with chronic HBV infection. In yet some other embodiments, the subject suffers from insomnia associated with chronic HBV infection. In some other embodiments, the subject suffers from liver fibrosis associated with chronic HBV infection.

[0015] In any embodiment, the effective amount is 1-1,000 mg / Kg body weight / day, preferably 20-200 mg / Kg body weight / day. Still optionally, the effective amount is 1-20 grams / day. According to yet another embodiment, the cyanobacterial biomass is administered orally.

[0016] According to some embodiments of the present disclosure, the cyanobacterial biomass is administered daily for at least one month. Optionally, the cyanobacterial biomass is administered daily for at least six months.

[0017] In some optional embodiments, the method for treating HBV infection further comprises administering an effective amount of an antiviral agent, such as interferon alpha (IFN-α), pegylated IFN-α, a nucleoside analog, or a nucleotide analog. Examples of nucleoside or nucleotide analogs include, but are not limited to, acyclovir, famciclovir, ganciclovir, valacyclovir, vidarabine, ribavirin, zoster immune globulin (ZIG), lamivudine, adefovir, dipivoxil, entecavir, telbivudine, clevudine, and tenofovir.

[0018] According to certain embodiments of the present disclosure, the subject has a chronic HBV infection and the cyanobacterial biomass is administered for at least one month to reduce detectable levels of Hepatitis B virus surface antigen (HBsAg) in the subject.

[0019] According to some other embodiments of the present disclosure, the subject suffers from insomnia associated with chronic HBV infection, and the cyanobacterial biomass is administered for at least three months to alleviate the insomnia.

[0020] According to some other embodiments of the present disclosure, the subject suffers from liver fibrosis associated with chronic HBV infection and the cyanobacterial biomass is administered for at least three months to reduce liver fibrosis. According to some other embodiments of the present disclosure, the subject suffers from or is susceptible to liver disease associated with chronic HBV infection (e.g., cirrhosis or hepatic decompensation) and the cyanobacterial biomass is administered for at least one month to prevent or delay the onset of liver disease.

[0021] In yet another aspect, the present disclosure is directed to a pharmaceutical or nutraceutical composition for treating HBV infection, including chronic HBV infection. Subject matter included in other aspects of the present disclosure includes the use of cyanobacterial biomass in the manufacture of a pharmaceutical or nutraceutical composition for use in treating HBV infection, including chronic HBV infection.

[0022] The pharmaceutical or nutraceutical compositions are also useful in treating insomnia or liver disease associated with chronic HBC infection. In some embodiments, administration of the pharmaceutical or nutraceutical compositions effectively reduces HBsAg levels in a subject and may even eliminate HBsAg in some subjects.

[0023] According to some embodiments, the pharmaceutical or nutraceutical composition comprises an effective amount of a cyanobacterial biomass and a pharma- ceutically or nutraceutical acceptable excipient.

[0024] According to certain embodiments of the present disclosure, the cyanobacterial biomass is derived from Arthrospira maxima.

[0025] According to some embodiments, the pharmaceutical or nutraceutical composition is formulated into a preparation suitable for oral administration, such as a tablet, caplet, capsule, or liquid dosage form (e.g., dispersion or suspension). According to some embodiments, the pharmaceutical or nutraceutical composition is formulated as a tablet, and the pharma- ceutical or nutraceutical acceptable excipient is silicon dioxide or a sucrose ester of a fatty acid.

[0026] Many of the attendant features and advantages of the present disclosure will be better understood by reference to the following detailed description taken in conjunction with the accompanying drawings.

[0027] The specification will be better understood from the following detailed description read in light of the accompanying drawings. [Brief description of the drawings]

[0028] [Figure 1] 1 is a line graph depicting the change in qHBsAg in subjects treated with specific treatments. [Diagram 2] 13 is data depicting changes in liver stiffness in subjects treated with specific treatments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description of the Invention The detailed description set forth below in conjunction with the accompanying drawings is intended to illustrate the present embodiment and is not intended to represent the only manner in which the present embodiment may be constructed or utilized. The description sets forth the functions of the embodiment and the sequence of steps for constructing and executing the embodiment. However, the same or equivalent functions and sequences may be accomplished by different embodiments.

[0030] For convenience, certain terms used herein, examples, and the appended claims are summarized here. Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood and used by those of ordinary skill in the art. It is also understood that singular terms include the plural of the same term, and plural terms include the singular, unless otherwise required by context. Specifically, as used in this specification and claims, the singular forms "a" and "an" include plural references unless the context clearly indicates otherwise. Also, as used in this specification and claims, the terms "at least one" and "one or more" have the same meaning, including one, two, three, or more.

[0031] Notwithstanding that the numerical ranges and parameter settings setting forth the broad scope of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of what one of ordinary skill in the art would consider. Unless otherwise indicated in the working / performing examples or otherwise expressly indicated, all numerical ranges, amounts, values, and percentages for amounts of materials, time periods, temperatures, operating environments, proportions of amounts, and the like disclosed herein are to be understood as modified in all instances by the term "about." Thus, unless specifically indicated to the contrary, the numerical parameters set forth in the present disclosure and the appended claims are approximations that may be varied as desired. At the very least, each numerical parameter should be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques. Ranges may be expressed herein as from one endpoint to another endpoint or between two endpoints. All ranges disclosed herein include the endpoints unless otherwise specified.

[0032] The term "nucleoside analog" or "nucleotide analog" as used herein refers to a molecule that is similar to a nucleoside or nucleotide and can perform at least some of the biochemical functions of a nucleoside or nucleotide. Specifically, the term "nucleotide analog" refers to a molecule that is structurally similar to a natural nucleotide and can function in a manner similar to a naturally occurring nucleotide (e.g., exhibits a similar ability to base pair with one of the naturally occurring bases). The term "nucleoside analog" as used herein refers to a molecule that is structurally similar to a natural nucleoside and can function in a manner similar to a naturally occurring nucleoside (e.g., exhibits a similar ability to be incorporated into DNA by DNA replication).

[0033] The terms "treatment" and "treating" as used herein may refer to prophylactic (e.g., preventative), curative or palliative measures. In particular, the term "treating" as used herein refers to the application or administration of the cyanobacterial biomass or a pharmaceutical or nutraceutical composition comprising the same to a subject with a medical condition (i.e., HBV infection or chronic HBV infection), a symptom associated with a medical condition (insomnia, fatigue, malaise, muscle pain, joint pain), a disease or disorder secondary to a medical condition (e.g., cirrhosis or hepatic decompensation), or a predisposition to a medical condition, with the purpose of partially or completely alleviating, ameliorating, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of the particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of a disease, disorder, and / or condition, and / or who show only early signs of a disease, disorder, and / or condition, with the objective of reducing the risk of developing pathologies associated with the disease, disorder, and / or condition. In a preferred embodiment, the cyanobacterial biomass may be used to ameliorate or prevent insomnia or liver cirrhosis in vivo. Administration of the cyanobacterial biomass also results in seroclearance of HBsAg in the subject. Thus, the method of treatment provides a means for substantially eradicating infectious pathogens in a host organism, such that the pathogen is not detectable in the host organism.

[0034] The terms "subject" and "patient" are used interchangeably herein and are intended to refer to animals, including the human species, treatable by the cyanobacterial biomass, pharmaceutical or nutraceutical compositions and / or methods of the present invention. The term "subject" or "patient" is intended to refer to both males and females, unless one gender is specifically indicated.

[0035] The terms "application" and "administration" are used interchangeably herein and refer to the application of the cyanobacterial biomass or the pharmaceutical or nutraceutical composition of the invention to a subject in need of treatment. For example, the subject may be one having or suspected of having a chronic HBV infection.

[0036] The term "effective amount" as used herein refers to an amount of the cyanobacterial biomass sufficient to produce a desired therapeutic effect. For therapeutic purposes, an effective amount is also an amount in which any toxic or deleterious effects of the component are outweighed by the therapeutically beneficial effects. An effective amount of a drug is not necessary to cure a disease or condition, but provides treatment for the disease or condition, such as delaying, interfering with, or preventing the onset of the disease or condition, or ameliorating the symptoms of the disease or condition. An effective amount may be divided into one, two or more doses in a form suitable for administration once, twice or more times over a specified period of time. The specific effective or sufficient amount will vary depending on factors such as the particular condition being treated, the physical condition of the patient (e.g., the patient's weight, age or sex), the type of mammal or animal being treated, the duration of the treatment, the characteristics of the concomitant therapy (if any), and the particular dosage form and structure of the active ingredient or its derivatives used. Effective amounts may be expressed, for example, as the total mass of cyanobacterial biomass (e.g., in grams, milligrams, micrograms) or as a ratio of mass of cyanobacterial biomass to body weight (e.g., milligrams per kilogram (mg / kg)). Alternatively, effective amounts can be expressed as a concentration of the active ingredient (e.g., the cyanobacterial biomass of the present disclosure), such as molar concentration, mass concentration, volume concentration, molar concentration, mole fraction, mass fraction, and mixture ratios.

[0037] The term "nutraceutically or pharma- ceutical acceptable excipient" as used herein means a substance, composition or vehicle, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material, involved in carrying or transporting a subject drug from one organ or part of the body to another organ or part of the body. Each excipient is "acceptable" in the sense that it is compatible with the other ingredients of the formulation. The nutraceutical or pharmaceutical formulation contains the cyanobacterial biomass of the invention in combination with one or more nutraceutically or pharma- ceutical acceptable ingredients. The excipient may be in the form of a solid, semi-solid or liquid diluent, cream or capsule. These nutraceutical or pharmaceutical preparations are further objects of the present invention. Usually, the amount of cyanobacterial biomass is 0.1-95% by weight of the preparation, preferably 0.2-20% by weight of the preparation for parenteral use and 1-50% by weight of the preparation for oral administration. For clinical use of the method of the present invention, the nutraceutical or pharmaceutical composition of the present invention is formulated into a dosage form suitable for the intended route of administration, such as oral administration.

[0038] The term "biomass" as used herein means biomass derived from a culture containing A. maxima. This term includes live and dead organisms, as well as preformed, dried, frozen, or previously functioning biomass.

[0039] Spontaneous HBsAg seroclearance is a rare event in chronic HBV infection. Previous studies have shown that the annual incidence of spontaneous HBsAg seroclearance ranges from approximately 0.1 to 2.5% in various countries. Patients with chronic HBV infection need to take antiviral agents continuously to inhibit the replication of HBV virus in the patient's body. In the early stages of antiviral treatment, antiviral agents can effectively reduce the detectable HBsAg levels in patients, which vary from patient to patient due to a wide variety of factors. However, HBsAg levels remain constant during subsequent treatments, and it is often very difficult to further reduce HBsAg levels in patients receiving currently available antiviral treatments after HBsAg levels reach an individual constant level.

[0040] The present disclosure is based, at least in part, on the discovery that the cyanobacterial biomass, when administered orally with an antiviral agent for an extended period of time (e.g., at least one month), can further reduce HBsAg levels in a subject and potentially achieve seroclearance of HBsAg in patients suffering from chronic HBV infection. By reducing HBsAg levels and clearing HBsAg in the serum of a patient, the risk of the patient developing liver cirrhosis, liver decompensation, and / or hepatocellular carcinoma can be reduced. In view of the above, the present disclosure proposes a method for treating HBV infection, including chronic HBV infection. Some embodiments of the present disclosure are directed to methods for treating symptoms or disorders associated with, secondary to, or caused by such HBV infection. Also provided herein is the use of the cyanobacterial biomass for use in the treatment of said HBV infection and for use in the manufacture of a pharmaceutical or nutraceutical preparation for said therapeutic purpose. Pharmaceuticals (i.e., pharmaceutical compositions) or nutraceutical preparations are of course subject matter covered by the scope of the present application.

[0041] In one aspect, the present disclosure is directed to a method for treating an infection caused by HBV, or a symptom or disorder associated with, secondary to, or caused by said HBV infection, in a subject in need of treatment.

[0042] Cyanobacteria are microscopic bacteria found on land and in fresh, brackish or marine waters. Cyanobacteria perform oxygenic photosynthesis. Because they utilize photosynthesis, aquatic cyanobacteria are commonly referred to as blue-green algae. Currently, over 2,000 species have been described in the phylum Cyanobacteria. Cyanobacteria have been identified as a rich source of biologically active compounds with antiviral, antibacterial, antimicrobial and anticancer activity. Compounds isolated from cyanobacteria belong to the groups of polyketides, amides, alkaloids, fatty acids, indoles and lipopeptides. Efforts are being made to identify active extract fractions or compounds with the desired therapeutic effect.

[0043] Spirulina refers to a dietary supplement made from the dried biomass of Arthrospira platensis and A. maxima. A. maxima (or Spirulina maxima) is found in tropical or subtropical regions with saline and alkaline waters. For example, it is common in Lake Chad in Africa and Lake Texcoco in Mexico. These two species were once classified in the genus Spirulina. In current taxonomy, there is agreement that the name Spirulina for these two strains is inappropriate and that the genus Arthrospira includes A. platensis and A. maxima, but the outdated taxonomy is still used today and dietary supplements made from them are most often referred to by the common name Spirulina. Spirulina has been used as a food source since the ancient Aztecs. Nonetheless, the present invention is the first to discover the ability to further reduce HBsAg levels in subjects with chronic HBV infection, and to apply such findings to practical use.

[0044] According to certain embodiments of the present disclosure, the cyanobacterial biomass is from Arthrospira maxima. According to yet other embodiments, the cyanobacterial biomass is administered orally. According to some embodiments of the present disclosure, the cyanobacterial biomass is provided as a dried powder, although the present disclosure is not limited thereto. Alternatively, the dried powder may be further processed into tablets, caplets, or capsules. Additionally or alternatively, the cyanobacterial biomass may be formulated into a liquid preparation, such as a dispersion or suspension having an effective amount of the cyanobacterial biomass.

[0045] In various embodiments, the subject is a mammal that can benefit from the therapeutic methods of the present disclosure. As used herein, "mammal" refers to all members of the class Mammalia, including humans, primates (e.g., monkeys and chimpanzees), domestic and farm animals such as dogs, cats, rabbits, pigs, sheep, goats, cows, horses, and cattle, zoo, sports, and pet animals, and rodents such as mice, rats, and guinea pigs. In one exemplary embodiment, the patient is a human.

[0046] In some embodiments, the subject suffers from chronic HBV infection. Patients with chronic HBV infection are at high risk of developing liver cirrhosis, and administering a cyanobacterial biomass to such patients can reduce the risk of the patient developing liver disease (e.g., liver cirrhosis or liver decompensation). In some other embodiments, the subject suffers from liver cirrhosis associated with chronic HBV infection, and the cyanobacterial biomass is administered for a period of time sufficient to inhibit or slow the progression of liver cirrhosis (e.g., at least one month).

[0047] It has been reported that patients with progressive liver disease (e.g., cirrhosis or hepatic decompensation) usually exhibit disturbed sleep patterns (e.g., insomnia). In yet some other embodiments, a subject suffers from insomnia or disturbed sleep patterns associated with chronic HBV infection, and a cyanobacterial biomass is administered to reduce the insomnia or improve the quality of the subject's sleep. In some embodiments, a cyanobacterial biomass is administered to a patient with chronic HBV infection to reduce the patient's risk of developing hepatocellular carcinoma. In view of the above, the present methods relating to administering a cyanobacterial biomass to a patient with HBV infection or chronic HBV infection may be used as a therapeutic measure, a preventative measure, or for nutritional support.

[0048] In any embodiment, the effective amount of cyanobacterial biomass is about 1-1,000 mg / Kg body weight / day, preferably about 20-200 mg / Kg body weight / day, more preferably about 40-120 mg / Kg body weight / day. For example, the effective amount may be 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 2 00, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, It may be 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 or 1,000 mg / Kg body weight / day. More optionally, the effective amount is between 1 and 20 grams per day, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 g / day. In some other embodiments, the cyanobacterial biomass is administered once, twice or three times per day.

[0049] According to some embodiments of the present disclosure, the cyanobacterial biomass is administered daily for at least one month. Optionally, the cyanobacterial biomass is administered daily for at least six months. Further optionally, the cyanobacterial biomass is administered daily for at least nine months or one year. According to certain embodiments of the present disclosure, the cyanobacterial biomass is administered daily to a subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more. As will be appreciated, the safety of long-term administration of cyanobacterial biomass has been recognized, since cyanobacterial biomass has been used as a dietary supplement for a long time. Thus, the cyanobacterial biomass may be used for long-term therapeutic, preventative or dietary supplemental purposes without posing substantial safety concerns.

[0050] In some optional embodiments, the method of treating HBV infection further comprises administering an effective amount of an antiviral agent, such as interferon alpha (IFN-α), pegylated IFN-α, a nucleoside analog, or a nucleotide analog. Examples of nucleoside or nucleotide analogs include, but are not limited to, acyclovir, famciclovir, ganciclovir, valacyclovir, vidarabine, ribavirin, zoster immune globulin (ZIG), lamivudine, adefovir, dipivoxil, entecavir, telbivudine, clevudine, and tenofovir. Alternatively, the antiviral agent may be (1) a small interfering RNA (siRNA) that interferes with and / or inhibits the replication of HBV, (2) a molecule that interferes with and / or inhibits the entry of HBV into hepatocytes (e.g., myrcludex B), (3) a molecule that interferes with and / or inhibits the formation of viral capsids (e.g., morphothiadin (GLS4), NVR 3-778, JNJ56136379, and AB-423, etc.), or (4) a molecule that interferes with and / or inhibits the production or expression of viral antigens (e.g., Rep 2139, Rep 2165, and RO7020322 (RG7834), etc.). The cyanobacterial biomass may be administered before, with, or after administration of the antiviral agent. Alternatively, the cyanobacterial biomass and the antiviral agent may have different administration regimens such that the two are taken at different time intervals during the course of treatment.

[0051] In yet another aspect, the present disclosure is directed to a pharmaceutical or nutraceutical composition for treating HBV infection or chronic HBV infection. According to various embodiments, the pharmaceutical or nutraceutical composition is also useful for treating or preventing insomnia or cirrhosis associated with chronic HBV infection. According to any embodiment, the pharmaceutical or nutraceutical composition may also be used to reduce a patient's risk of developing hepatocellular carcinoma. In some embodiments, administering the pharmaceutical or nutraceutical composition in combination with an antiviral agent effectively reduces the subject's HBsAg level compared to a patient receiving only an antiviral agent. Long-term administration of the pharmaceutical or nutraceutical composition is expected to result in seroclearance of HBsAg in some subjects.

[0052] According to some embodiments, the pharmaceutical or nutraceutical composition comprises an effective amount of cyanobacterial biomass (eg, died biomass from A. maxima) and a pharma- ceutically or nutraceutical acceptable excipient.

[0053] In some embodiments, the pharmaceutical or nutraceutical compositions are formulated into preparations suitable for oral administration, such as solid dosage forms (e.g., capsules, sachets, tablets, pills, lozenges, powders or granules) and liquid dosage forms (e.g., solutions, suspensions, emulsions, microemulsions or dispersions).

[0054] Some of the described solid dosage forms may optionally include coatings or shells, such as enteric coatings, as well as coatings to modify the release rate of any component. Examples of such coatings are well known in the art. In one example, the pharmaceutical composition of the present disclosure is a tablet, such as a fast-release tablet. In yet another example, the pharmaceutical composition of the present disclosure is formulated into a sustained-release formulation. In another example, the pharmaceutical composition of the present disclosure is a powder that is encapsulated in soft and hard gelatin capsules.

[0055] According to the desired purpose, the pharma- ceutically or nutraceutical acceptable excipient of the pharmaceutical composition may be water-soluble or water-insoluble excipient.Exemplary water-soluble excipients include, but are not limited to, mannitol, lactose, sucrose, glucose, maltose, hydrogenated maltose, maltotetraose, fructose, lactulose, lactitol, sorbitol, maltitol, erythritol, xylitol, and combinations thereof.Exemplary water-insoluble excipients include, but are not limited to, cellulose derivatives, microcrystalline cellulose, cellulose, ethyl cellulose, silicon dioxide, crospovidone, sodium starch glycolate, croscarmellose sodium, methacrylic acid polymers, corn starch, dibutyl phthalate, diethyl phthalate, dibutyl sebacate, triacetin, and combinations thereof.

[0056] Liquid formulations may further include a buffer to maintain a desired pH.Liquid dosage formulations may also be encapsulated in a soft gelatin capsule.

[0057] Preferably, the pharma- ceutically or nutraceutical acceptable excipient comprises from about 0.01% to about 85% by weight (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 9 , 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85% by weight), preferably about 0.01% to about 50% by weight, and more preferably about 0.01% to about 10% by weight.

[0058] According to one particular example of the present disclosure, the pharma- ceutically or nutraceutical acceptable excipient is silicon dioxide or sucrose esters of fatty acids, which are included in the pharmaceutical or nutraceutical composition in an amount of about 0.01% to about 10% by weight based on the total weight of the composition.

[0059] The compositions of the present invention may also include various additives known to those skilled in the art. For example, solvents, including relatively small amounts of alcohol, may be used to solubilize certain active ingredients. Any other pharma- ceutically or nutraceutical acceptable additives may include opacifiers, antioxidants, flavors, colorants, gelling agents, thickeners, stabilizers, surfactants, etc. Other agents, such as antibacterial agents, may be added to prevent spoilage during storage (e.g., to inhibit the growth of other microorganisms, such as yeasts and molds). Permeation enhancers and / or irritation reducing additives may be included in the compositions of the present invention. Other examples include solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and combinations thereof, all of which are known to those skilled in the art.

[0060] The composition comprising the cyanobacterial biomass may be formulated into a dietary supplement, which may further comprise one or more additional nutritional components, such as vitamins, minerals, dietary fiber, fatty acids, or amino acids. Alternatively, the dietary supplement may comprise one or more edible carriers that may provide one or more benefits to the cyanobacterial biomass. Examples of such edible carriers include starch, cyclodextrin, maltodextrin, methylcellulose, carbomethoxycellulose, xanthan gum, and aqueous solutions thereof.

[0061] Yet another aspect of the present disclosure is directed to the use of a cyanobacterial biomass in the manufacture of a pharmaceutical or nutraceutical composition for use in the treatment of HBV infection or symptoms or disorders associated with, secondary to, or caused by HBV infection. Yet another aspect of the present disclosure is directed to the use of a cyanobacterial biomass in the treatment of HBV infection or symptoms or disorders associated with, secondary to, or caused by HBV infection. Similarly, the various cyanobacterial biomasses described above are also applicable to these aspects.

[0062] The following examples are provided to illustrate certain aspects of the present invention and to aid those skilled in the art in practicing the present invention. These examples are not to be considered in any way as limiting the scope of the present invention. Without further elaboration, it is believed that those skilled in the art can utilize the present invention to its fullest extent based on the description herein. All publications cited herein are incorporated herein by reference in their entirety. EXAMPLES

[0063] Materials and Methods

[0064] Characterization of strain FEM102

[0065] Chromosomal DNA was extracted from strain FEM102. After amplification by polymerase chain reaction (PCR), the sequence of 16S-23S rDNA was analyzed on an ABI 3730 DNA Analyzer using ABI BigDye V3.1. The analysis results showed that the 16S-23S ribosomal RNA (rRNA) gene of strain FEM102 has the nucleotide sequence set forth in SEQ ID NO: 1. A sequence query using the nucleotide BLAST database provided online by the National Center for Biotechnology Information (NCBI) shows that this sequence is highly conserved among various Arthrospira spp., including A. maxima (also known as Spirulina maxima), A. platensis (also known as S. platensis), A. erdosensis, A. fusiformis, A. indica, and A. terrebriformis.

[0066] Preparation of FEM102 tablets

[0067] FEM102 dry powder was prepared as follows. First, FEM-102 strain (stock culture: 350L; OD0.2) was cultured in the medium as shown in Tables 1-3 (350,000L) at 25-32°C for 20-28 days. The biomass was collected using a 50-μm filter and washed five times with clean water to reduce the salt content and other contaminants of the biomass. After filtration using a 50-μm filter, the biomass was centrifuged to remove water (<95% by weight), and the precipitate was spray-dried. Approximately 350,000 liters of culture yielded approximately 120-180 kg of FEM102 powder. As can be understood by those skilled in the art, the contents of the culture medium described herein are provided merely for illustrative purposes and should not be construed as limiting the scope of the present invention. Rather, those skilled in the art may adjust or modify the culture medium as they deem appropriate.

[0068] The FEM102 powder was then mixed with silicon dioxide or sucrose esters of fatty acids and compressed to form tablets (hereinafter "FEM102 tablets"), where each FEM102 tablet (500 mg) contained 495 mg of the FEM102 strain and 5 mg of silicon dioxide / sucrose esters of fatty acids.

[0069] [Table 1] *The components of A5 metal stock are summarized in Table 2. **The components of the micronutrient solution are summarized in Table 3.

[0070] [Table 2]

[0071] [Table 3]

[0072] Clinical Trials

[0073] A single-center, randomized, open-label clinical trial was designed to determine whether the combined administration of cyanobacterial biomass and antiviral drugs could reduce detectable serum levels of HBsAg in patients with chronic HBV infection.The clinical trial protocol and consent form were approved by the Ethics Committee of Taipei Municipal Wanfang Hospital.

[0074] Inclusion criteria for patients included men and women aged 20-75 years, documented chronic hepatitis B, receiving oral antiviral treatment with either entecavir, telbivudine or tenofovir for at least 1 year, and HBV DNA levels less than 20 IU / mL. Patients with seafood allergies were excluded from the study.

[0075] A total of 350 patients were assessed for eligibility. 247 were excluded and 43 refused to participate. Sixty patients who met the inclusion and exclusion criteria and signed the consent form were randomized to receive antiviral (20 patients, control group), antiviral plus 3 grams of FEM102 tablets (20 patients, low dose group), or antiviral plus 6 grams of FEM102 tablets (20 patients, high dose group) daily for at least 6 months. Liver and renal function, qHBsAg, HBeAg, B12 levels, and stenosis / fibrosis were investigated at baseline and at 1, 3, and 6 months. Rank sum tests were used for statistical analysis. Patient demographics and baseline characteristics are shown in Table 4.

[0076] [Table 4]

[0077] As can be seen in Table 4, there were no significant differences between the control, low-dose and high-dose groups with respect to age, sex, ALT (alanine aminotransferase), quantitative HBsAg levels, HBeAg status, and severity of fibrosis and stenosis at the start of the study.

[0078] Specifically, patients assigned to each group were treated with the respective initial antiviral agent at the recommended dose: entecavir (500 mg / tablet; 1 tablet per day), telbivudine (600 mg / tablet; 1 tablet per day) or tenofovir (300 mg / tablet; 1 tablet per day). For patients assigned to the low-dose group and the high-dose group, 6 tablets of FEM102 500 mg and 12 tablets of FEM102 500 mg were administered three times per day. Specifically, patients in the low-dose group took 2 tablets before each meal, and patients in the high-dose group took 4 tablets before each meal.

[0079] During the study, 12 patients in the control group discontinued the intervention, and 4 and 8 patients in the low-dose and high-dose groups, respectively, discontinued the intervention.

[0080] Results: Biological effects of FEM102 tablets

[0081] At 1, 3, and 6 months after the first dose, blood samples from patients in each group were analyzed by enzyme-linked immunosorbent assay (ELISA) to determine HBsAg levels. The change in qHBsAg levels in each group (each patient's qHBsAg level measured at a given time point minus the patient's qHBsAg level measured at the start of the study) is summarized in Figure 1 and Table 5.

[0082] The data presented in Figure 1 and Table 5 showed that the levels of qHBsAg in patients treated with FEM102 tablets changed in a dose-dependent manner compared to those in the control group. Significant differences were observed between the high-dose group and the control group at 1, 3, and 6 months.

[0083] [Table 5]

[0084] The liver stiffness in patients was evaluated by Fibroscan®, a non-invasive device for evaluating fibrosis and stenosis, and the results are summarized in Figure 2 and Table 6. Fibroscan value (FS) can serve as an index of the severity of liver fibrosis. In general, FS value higher than 7.0 kPa indicates that the patient has significant fibrosis, FS value higher than 9.1 kPa indicates that the patient has severe fibrosis, and FS value higher than 12.5 kPa indicates that the patient has cirrhosis. The data summarized in Table 6 showed that the combined administration of antiviral drugs (e.g., entecavir, telbivudine or tenofovir) and FEM102 tablets (6 grams) significantly reduced the patients' FS values, suggesting that the patients' liver condition was improving, and liver fibrosis seemed less severe (7.521 KPa (6 months) vs. 9.242 KPa (baseline), p = 0.005).

[0085] [Table 6]

[0086] Taken together, the above data confirmed that administration of cyanobacterial biomass (e.g., FEM102 tablets derived from dried A. maxima) may help to clear HBV particles and improve liver fibrosis in patients with chronic HBV infection. Since HBV particles and liver fibrosis have been implicated as contributing factors to disturbed sleep patterns and insomnia in these patients, the inventors further evaluated whether FEM102 tablets could improve the quality of sleep in such patients.

[0087] Sleep quality was assessed and scored according to several sleep-related parameters, such as time taken to fall asleep, duration of wakefulness during sleep, total sleep time, and daytime mental performance (such as memory, cognition, and attention). The score was inversely proportional to sleep quality, in other words, the worse the sleep quality, the higher the calculated score. The results are summarized in Table 7.

[0088] The data in Table 7 showed that the sleep quality of patients treated with a combination of antiviral agents and FEM102 tablets was obviously better than that of patients treated with antiviral agents alone.

[0089] [Table 7]

[0090] In conclusion, the present disclosure provides a cyanobacterial biomass (e.g., a biomass from A. maxima), a pharmaceutical composition comprising the same (e.g., FEM102 tablets, etc.), and its use in treating chronic HBV infection. Specifically, the cyanobacterial biomass is useful for reducing HBV particles (as indicated by detectable HBsAg levels in the serum of patients) and improving the quality of sleep in patients with liver fibrosis conditions and chronic HBV infection.

[0091] The above description of the embodiments is provided by way of example only, and it will be understood that various modifications may be made by those skilled in the art. The above specification, examples and data provide a complete description of the structure and use of the exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a degree of particularity or with reference to one or more individual embodiments, those skilled in the art may make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. A pharmaceutical composition for reducing detectable levels of Hepatitis B surface antigen (HBsAg) in a subject, wherein the subject is suffering from a chronic HBV infection, the pharmaceutical composition comprising an effective amount of a cyanobacterial biomass and a pharma- ceutical acceptable excipient, wherein the cyanobacterial biomass is derived from the Arthrospira FEM102 strain, and the pharmaceutical composition is co-administered with an antiviral agent.

2. The method of claim 1 , wherein the subject suffers from insomnia associated with chronic HBV infection.

3. The pharmaceutical composition of claim 1 , wherein the subject suffers from liver fibrosis associated with chronic HBV infection.

4. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is formulated into a solid dosage form.

5. 5. The pharmaceutical composition of claim 4, wherein the pharma- ceutically acceptable excipient is silicon dioxide or a sucrose ester of a fatty acid.

6. The pharmaceutical composition according to claim 1, wherein the effective amount is 1 to 1,000 mg / Kg body weight / day.

7. 10. The method of claim 1, wherein the pharmaceutical composition is administered daily for at least one month.

8. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is administered orally.

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