WO2019204471A1

WO2019204471A1 - Treatment of hepatitis c

Info

Publication number: WO2019204471A1
Application number: PCT/US2019/027908
Authority: WO
Inventors: YungChi CHENG
Original assignee: Yale University
Priority date: 2018-04-20
Filing date: 2019-04-17
Publication date: 2019-10-24

Abstract

The present invention provides a method of treating or preventing hepatitis C virus (HCV) infection in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising one or more antiviral active herbal extracts derived from herbs selected from the group consisting of Vaccaria segetalis, Albizia julibrissin, Areca catechu and Ficus pumila Linn. The present invention further includes methods of treating or preventing HCV infection in a subject, wherein the aforementioned herbs are administered in combination with herbal extracts of Rheum palmatum L. and/or Polygala tenuifolia or a direct acting antiviral agent. The present invention also includes methods for preparing these extracts and methods for evaluating their efficacy in treating HCV.

Description

TITLE OF THE INVENTION

Treatment of Hepatitis C

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/660,486, filed April 20, 2018, which is hereby incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under grant No. AI038204 awarded by National Institutes of Health (NIH). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is a positive single-stranded RNA virus of Hepacivirus genus in the Flaviviridae family. The HCV genome encodes two glycoproteins and two core proteins as structural proteins, and six non- structural (NS) proteins that are essential for HCV replication. Hepatitis C virus has spread to about 200 million people worldwide; 25% of HCV carriers develop end-stage liver diseases such as liver fibrosis, cirrhosis, and

hepatocellular carcinoma (HCC) leading to about 500,000 deaths each year. HCV genotype 1 (subtypes la or lb) accounts for 46% of all HCV infections and 70-75% of HCV patients in America, China and Japan. HCV genotypes 2 and 3 account for 20-25% of all HCV infections and infections by HCV genotypes 4, 5, or 6 make up the remainder.

There is currently no effective vaccine for HCV. The present standard-of-care for HCV treatment involves administering peg-interferon a and ribavirin to a patient, often supplemented with administration of other direct-acting antiviral agents (DAAs). The combined treatment with peg-interferon a and ribavirin exhibits nearly an 80% eradication rate for patients with HCV genotype 2 and 3 infection, and 40% to 70% for infection with the other HCV genotypes. The standard treatment period with peg-interferon a and ribavirin may range from 24 to 48 weeks depending on the HCV genotype.

The FDA has approved several DAAs for the treatment of HCV which mainly target non- structural proteins. Currently marketed DAAs can shorten the standard peg-interferon a and ribavirin treatment duration to 8-12 weeks with a relative high success rate. However, the cost of such a treatment cycle can be as high as $63,000-$300,000 (USD).

There remains an unmet need in the art for novel compositions and methods for the treatment of HCV, especially compositions and methods that may shorten a standard treatment cycle. The present invention satisfies this unmet need.

BRIEF SUMMARY OF THE INVENTON

In one aspect, the invention provides a method of treating or preventing hepatitis C virus (HCV) infection in a subject by administering to the subject in need thereof a therapeutically effective amount of a composition comprising one or more antiviral active herbal extracts derived from the herbs selected from the group consisting of Vaccaria segetalis , Albizia julibrissin, Areca catechu and Ficus pumila Linn.

In another aspect, the invention provides a method of determining the anti-HCV activity of a specific batch of an herbal extract.

In certain embodiments, the composition further comprises a therapeutically effective amount of one or more antiviral active herbal extracts derived from herbs selected from the group consisting of Rheum palmatum L. and Poly gala tenuifolia.

In certain embodiments, a therapeutically effective amount of at least one additional agent useful for treating HCV infection is administered to the subject. In certain

embodiments, the additional agent is one selected from the group consisting of peg-interferon alfa-2a, ribavirin, elbasvir, grazoprevir, ledipasvir, sofosbuvir, paritaprevir, ritonavir, ombitasvir, dasabuvir, simeprevir, daclatasvir, dasabuvir and ledipasvir.

In certain embodiments, the composition is administered orally to the subject. In certain embodiments, the orally administered composition is in one or more forms selected from the group consisting of a pill, tablet, capsule, soup, tea, concentrate, dragees, liquids, drops, and gelcaps.

In certain embodiments, the therapeutically effective amount of the composition is about 0.001 mg/day to about 1,000 mg/day.

In certain embodiments, the herbal extracts are water herbal extracts prepared in a water extract preparation method that includes drying an amount of the herb or herbs, grinding the dried amount of the herb or herbs into an herb powder, adding the ground, dried amount of herb powder to an amount of water to form a mixture, heating the mixture at a first elevated temperature for a first amount of time to form a heated mixture, allowing the heated mixture to cool to room temperature to form a cooled mixture, and removing any undissolved solids in the cooled mixture from the cooled mixture to yield the water herbal extract.

In certain embodiments, the amount of dried herbal powder is 100 mg and the amount of water is 1 mL.

In certain embodiments, the first elevated temperature is about 80°C.

In certain embodiments, the mixture is heated for about 1 hr.

In certain embodiments, any undissolved solids are removed from the cooled mixture by centrifuging the cooled mixture and decanting the resulting solution.

In certain embodiments, the method of preparing water herbal extract further comprises adding an amount of the water herbal extract to an amount of a b-glucuronidase solution to form a b -glucuronidase treated solution, incubating the b-glucuronidase treated solution to a second elevated temperature for a second amount of time to form an incubated b-glucuronidase treated solution, heating the incubated b-glucuronidase treated solution to a third elevated temperature for a third amount of time to form a heated b-glucuronidase treated solution, and allowing the heated b-glucuronidase treated solution to cool to room

temperature to form a b-glucuronidase treated extract.

In certain embodiments, the amount of the water herbal extract is about 500 pL at a concentration of about 100 mg/mL (herb/water).

In certain embodiments, the amount of b -glucuronidase solution comprises about 475 pL of TrisHCl (lOOmM, pH 6.8) and about 25 pL of E.coli b-glucuronidase protein (2.5 mg/mL).

In certain embodiments, the second elevated temperature is about 37°C. In certain embodiments, the second amount of time is about 1 hour.

In certain embodiments, the third elevated temperature is about l00°C. In certain embodiments, the third amount of time is about 5 minutes.

In certain embodiments, the herbal extracts are honey treated herbal extracts prepared in a honey extract preparation method that includes the steps of adding an amount of a honey solution to an amount of the herb or herbs to form a honey mixture, stirring and heating the honey mixture until the honey mixture is dry to form a dry honey mixture, and allowing the dry honey mixture to cool to room temperature to form the honey treated herbal extract.

In certain embodiments, the amount of honey solution is about 30 mL of about 15% (w/v) honey in water and the amount of herb or herbs is about 20 g.

In certain embodiments, the herbal extracts are licorice treated herbal extracts prepared in a licorice extract preparation method that includes the steps of adding an amount of a licorice solution to an amount of the herb or herbs to form a licorice mixture, stirring and heating the licorice mixture until the licorice mixture is dry to form a dry licorice mixture, and allowing the dry licorice mixture to cool to room temperature to form the licorice treated herbal extract.

In certain embodiments, the amount of licorice solution is about 12 mL of about 10% (w/v) licorice in water and the amount of herb or herbs is about 20 g.

In certain embodiment, the subject is a mammal. In certain embodiments, the mammal is a human.

In certain embodiments, the anti-HCV activity of a specific batch of an herbal extract is determined by a process comprising drying an amount of the herb from a specific batch of herb, grinding the dried amount of the herb into an herb powder, adding the ground, dried amount of herb powder to an amount of water to form a mixture, heating the mixture at a first elevated temperature for a first amount of time to form a heated mixture, allowing the heated mixture to cool to room temperature to form a cooled mixture, removing any undissolved solids in the cooled mixture from the cooled mixture to yield the water herbal extract, diluting the water herbal extract to a range of concentrations, testing the water herbal extracts for inhibition of HCV in an HCV luciferase replicon cell assay for 72 h, determining the IC50 value of the water herbal extract, comparing the determined IC50 value to a standard IC50 value, wherein if the determined IC50 value is lower than the standard IC50 value then the specific batch of herb is considered to be active for the inhibition of HCV.

In certain embodiments, the herb is Ficus pumilaLinn. and the standard IC50 value is 10 pg/mL.

In certain embodiments, the herb is Areca catechu and the standard IC50 value is 10 pg/mL.

In certain embodiments, the herb is Albizia julibrissin and the standard IC50 value is 5 pg/mL.

In certain embodiments, the herb is Rheum palmatum L. and the standard IC50 value is 50 pg/mL.

In certain embodiments, the herb is Polygala tenuifolia and the standard IC50 value is 1 pg/mL.

In certain embodiments, the herb is Vaccaria segetalis and the standard IC50 value is 15 pg/mL.

BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings specific embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1 depicts a table reporting IC50 values of the herbal extracts of the invention against HCV at 72 hours using an HCV luciferase replicon assay and cell toxicity of the herbal extracts of the invention using methylene blue staining method. These values are reported for the water extract preparation method as well as the b -glucuronidase treated method.

FIGs. 2A-2B illustrate graphs reporting HCV RNA expression after treatment with the herbal water extracts and the herbal b -glucuronidase treated extracts of the invention at the determined IC50 concentration for 24h, 48h, 72h. The relative HCV RNA was quantified using qPCR assays.

FIGs. 3A-3D illustrate the anti -HCV activity of herbal extracts of MYZ (honey processed) and HHP against HCV genotype lb using an HCV luciferase replicon assay and against HCV genotype 2a using qPCR assays. FIG. 3 A depicts the MYZ (honey processed) extract against HCVlb using an HCV luciferase replicon assay; FIG. 3B depicts the MYZ extract against HCV2a using qPCR assays; FIG. 3C depicts the HHP extract against HCVlb using an HCV luciferase replicon assay; FIG. 3D depicts the HHP extract against HCV2a using qPCR assays.

FIGs. 4A-4D illustrate the anti -HCV activity of extracts of MYZ (honey processed). FIG. 4A depicts a graph reporting the anti -HCV activity of MYZ over a range of

concentrations as determined using an HCV luciferase replicon assay. FIG. 4B depicts a graph reporting the anti -HCV activity of MYZ over a range of concentrations as determined by HCV RNA expression levels using qPCR assays. FIG. 4C depicts Western blot analyses of the effect of MYZ extracts on the expression of HCV protein, NS5B, NS3 and NS5A at 72h after treatment. FIG. 4D is a graph reporting the quantified results depicted in FIG. 4C.

FIGs. 5A-5D illustrate the anti -HCV activity of extracts of HHP. FIG. 5 A depicts a graph reporting the anti -HCV activity of HHP over a range of concentrations as determined using an HCV luciferase replicon assay. FIG. 5B depicts a graph reporting the anti-HCV activity of HHP over a range of concentrations as determined by HCV RNA expression levels using qPCR assays. FIG. 5C depicts Western blot analyses of the effect of HHP extracts on the expression of HCV protein, NS5B, NS3 and NS5A at 72h after treatment. FIG. 5D is a graph reporting the quantified results depicted in FIG. 5C.

FIGs. 6A-6C illustrate the anti -HCV activity of extracts of MYZ (honey processed). FIG. 6A is a graph demonstrating the anti -HCV activity of different fractions of MYZ extract as determined using an HCV luciferase replicon assay. FIG. 6B is a graph demonstrating the anti -HCV activity of different fractions of MYZ extract at different concentrations using an HCV luciferase replicon assay. FIG. 6C depicts a chromatogram of the various fractions of an MYZ extract, reporting the molecular weights of significant chemical components of those fraction.

FIGs. 7A-7C illustrate the anti -HCV activity of extracts of HHP. FIG. 7A is a graph demonstrating the anti -HCV activity of different fractions of HHP extract as determined using an HCV luciferase replicon assay. FIG. 7B is a graph demonstrating the anti-HCV activity of different fractions of HHP extract at different concentrations. FIG. 7C depicts a chromatogram of the various fractions of an HHP extract, reporting the molecular weights of significant chemical components of those fraction.

FIG. 8 is a graph depicting the anti-HCV activity of extracts of a variety of batches of DH using an HCV luciferase replicon assay. As illustrated by this graph, not all batches of the herbal extract are equally effective for the treatment of HCV. The efficacy of a batch can be determined prior to administration by using an in vitro HCV luciferase replicon assay.

FIG. 9 is a table depicting the results of HCV luciferase replicon assays testing the efficacy of different batches of the herbs of the invention. Batches of herbs from different sources were prepared by the water extract, honey extract and licorice extract methods of the invention and tested for activity. Maximum IC50 values, which separated“active” batches from less active or inactive batches, were determined experimentally and reported in the bottom row of the table.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates in certain aspects to the unexpected discovery that extracts of the herbs Wang Bu Liu Xing (also known as Vaccaria segetalis or Cowherb seed),

Guangdong Wang Bu Liu Xing (also known as Ficus pumila Linn or creeping fig or climbing fig), Bing Lang (also known as Areca catechu or Areca seed) and He Huan Pi (also known as Albizia julibrissin or Silktree albizia bark) can be used to treat HCV infection. In certain embodiments, these herbal extracts can be administered to a subject suffering from HCV infection concurrently or in combination with other herbal extracts known to treat HCV.

In certain embodiments, these herbal extracts are administered to a subject suffering from HCV infection concurrently or in combination with additional extracts from the herbs Da Huang (also known as Rheum palmatum L. or Medicinal Rhubarb root and rhizome) and Mi Yuan Zhi (also known as Polygala tenuifolia or Prepared Thinleaf Milkwort root).

In certain embodiments, the herbal extracts are administered to the subject concurrently or in combination with an extract from the herb Tian Ji Huang (also known as Hypericum japonicum Thunb. exMurray or Japanese St. John's wort or all-grass of Madras Grangea).

In certain embodiments, these herbal extracts are administered to a subject suffering from HCV infection concurrently or in combination with direct acting antiviral agents commonly prescribed for the treatment of HCV. In certain embodiments, administration of the herbal extracts increases the efficacy of the direct acting antiviral agents. In certain embodiments, the administration of the herbal extracts decreases the required effective dose or shortens the required dosage period of the direct acting antiviral agent.

In certain embodiments, the antiviral efficacy of a specific batch of an herbal extract of the invention can be determined by testing the extract using an HCV luciferase replicon assay.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, pharmacology and organic chemistry are those well-known and commonly employed in the art.

Standard techniques are used for biochemical and/or biological manipulations. The techniques and procedures are generally performed according to conventional methods in the art and various general references ( e.g. , Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al ., 2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this document.

As used herein, the articles“a” and“an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

As used herein, the terms“analog,”“analogue,” or“derivative” are meant to refer to a chemical compound or molecule prepared from another compound or molecule by one or more chemical reactions. As such, an analog can be a structure similar to, or based on, the structure of any small molecule inhibitor described herein, and/or may have a similar or dissimilar metabolic behavior.

In one aspect, the terms“co-administered” and“co-administration” as relating to a subject refer to administering to the subject a compound and/or composition of the invention along with a compound and/or composition that may also treat or prevent a disease or disorder contemplated herein. In certain embodiments, the co-administered compounds and/or compositions are administered separately, or in any kind of combination as part of a single therapeutic approach. The co-administered compound and/or composition can be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.

A“disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a“disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

As used herein, the term“extract” refers to a concentrated preparation or solution of a compound or drug derived from a naturally occurring source, such as an herb or other plant material. Extracts can be prepared by a number of processes including steeping an herb in solution or drying and grinding an herb into a powder and dissolving the powder in a solution. An extract can be further concentrated by removing a portion of the solvent after dissolving an amount of the desired compound in the solution. An extract may also be strained or centrifuged to remove any solid material from the solution.

The phrase“inhibit,” as used herein, means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g, bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g. , antagonists.

As used herein, the term“pharmaceutical composition” or“composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject.

As used herein, the term“pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the invention, and is relatively non-toxic, i.e., the material can be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The terms“pharmaceutically effective amount” and“effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. By "pharmaceutical formulation" it is further meant that the carrier, solvent, excipient(s) and/or salt must be compatible with the active ingredient of the formulation (e.g. a compound of the invention). It is understood by those of ordinary skill in this art that the terms“pharmaceutical formulation” and“pharmaceutical composition” are generally interchangeable, and they are so used for the purposes of this application.

As used herein, the term“pharmaceutically acceptable carrier” means a

pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil;

glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid;

pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein,“pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The“pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention.

Other additional ingredients that can be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

As used herein, the language“pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof. Suitable pharmaceutically acceptable acid addition salts can be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4- hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic,

ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, b-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable

pharmaceutically acceptable base addition salts of compounds of the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.

Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N’ -dibenzyl ethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N methylglucamine) and procaine. All of these salts can be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

As used herein, the term“prevent,”“prevention,” or“preventing” refers to any method to partially or completely prevent or delay the onset of one or more symptoms or features of a disease, disorder, and/or condition. Prevention is causing the clinical symptoms of the disease state not to develop, i.e., inhibiting the onset of disease, in a subject that can be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state. Prevention can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition.

By the term“specifically bind” or“specifically binds” as used herein is meant that a first molecule preferentially binds to a second molecule ( e.g ., a particular receptor or enzyme), but does not necessarily bind only to that second molecule.

As used herein, the term“subject,”“patient” or“individual” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g, young adult, middle-aged adult or senior adult)) and/or other primates (e.g, cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and/or turkeys.

As used herein, the term“therapeutically effective amount” is an amount of a compound of the invention, that when administered to a patient, treats, minimizes and/or ameliorates a symptom of the disease or disorder. The amount of a compound of the invention that constitutes a“therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the age of the patient to be treated, and the like. The therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

The terms“treat,”“treating,” and“treatment,” refer to therapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, in need of such treatment, a composition of the present invention, for example, a subject afflicted a disease or disorder, or a subject who ultimately may acquire such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual and partial numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

The following abbreviations are used herein:

BL Bing Lang, also known as Areca catechu or Areca seed

DAA direct acting antiviral

DH Da Huang, also known as Rheum palmatum L. or Medicinal Rhubarb root and rhizome

DTT dithiothreitol

EC50 half maximal effective concentration

EtOH ethanol

IC50 half maximal inhibitory concentration

IgG Immunoglobin G

GWBL Guangdong Wang Bu Liu Xing also known as Ficus pumila Linn or creeping fig or climbing fig

HCV Hepatitis C virus

HCVlb Hepatitis C virus, genotype lb

HCV2a Hepatitis C virus, genotype 2a

HHP He Huan Pi also known as Albizia julibrissin or Silktree albizia bark

LC-MS liquid chromatography - mass spectrometry

MYZ Mi Yuan Zhi also known as Polygala tenuifolia or Prepared Thinleaf

Milkwort root

NS non- structural PBS phosphate-buffered saline

qPCR real-time PCR

RNA ribonucleic acid

SDS sodium dodecyl sulfate

TBS-T tris-buffered saline + tween

TJH Tian Ji Huang, also known as Hypericum japonicum Thunb. exMurray, or Japanese St. John's wort

WBL Wang Bu Liu Xing also known as Vaccaria segetalis or Cowherb seed

Compositions

The present invention relates to compositions comprising an extract of an herbal species selected from the group consisting of Wang Bu Liu Xing (also known as Vaccaria segetalis or Cowherb seed), Guangdong Wang Bu Liu Xing (also known as Ficus pumila Linn or creeping fig or climbing fig), Bing Lang (also known as Areca catechu or Areca seed) and He Huan Pi (also known as Albizia julibrissin or Silktree albizia bark).

The herbal extracts of the invention can be useful within the methods of the invention, namely for the treatment of HCV and HCV related diseases and disorders.

In certain embodiments, the herbal extracts of the invention can be water extracts.

The water extracts can be prepared through a method comprising drying the herbs of the invention, grinding the dried herbs into an herb powder, adding the herb powder to an amount of water to form a mixture, heating the mixture to an elevated temperature for a period of time, allowing the mixture to cool down to room temperature, removing and removing any undissolved solids.

In certain embodiments, the herbal powder is added to the water in a ratio of about 100 mg of herbs : 1 mL of water. In certain embodiments, the mixture is heated to a temperature of about 80°C for about 1 h. In certain embodiments, the undissolved solids are removed by centrifuging the mixture to form a pellet and then decanting and collecting the water extract, leaving behind the solid pellet.

In certain embodiments, the herbal extracts of the invention can be b -glucuronidase treated extracts wherein a water extract is further treated with b-glucuronidase. The b- glucuronidase treated extract can be prepared through a method comprising adding an amount of the water extract to an amount of a b-glucuronidase solution, incubating the combined solution at an incubating temperature for a period of time, heating the combined solution at a heating temperature for a period of time and then allowing the solution to cool. In certain embodiments, the amount of water extract added to the b -glucuronidase solution is about 500 pL. In certain embodiments, the amount of b-glucuronidase solution comprises about 475 pL of TrisHCl (lOOmM, pH 6.8) and about 25 pL of E.coli b- glucuronidase protein (2.5 mg/mL). In certain embodiments, the combined solution is incubated at about 37°C for about 1 h. In certain embodiments, the combined solution is then heated at about l00°C for about 5 min.

In certain embodiments, the herbal extracts of the invention can be honey extracts.

The honey extracts can be prepared through a method comprising adding an amount of a honey solution to an amount of the herbs of the invention, stirring and heating the honey-herb mixture over low heat in a pan until the mixture is dry and allowing the mixture to cool to room temperature. In certain embodiments, the amount of honey solution is about 30 mL of about 15% (w/v) honey in water and the amount of herbs is about 20 g.

In certain embodiments, the herbal extracts of the invention can be licorice extracts. The licorice extracts can be prepared through a method comprising adding an amount of a licorice solution to an amount of the herbs of the invention, stirring and heating the licorice- herb mixture over low heat in a pan until the mixture is dry and allowing the mixture to cool to room temperature. In certain embodiments, the amount of licorice solution is about 12 mL of about 10% (w/v) licorice in water and the amount of herbs is about 20 g.

In certain embodiments, the extracts of the invention are tested for antiviral activity in in vitro luciferase replicon assays before administering them to a patient. In further embodiments, batches of herbal extract that display limited or no antiviral activity are discarded and are not administered to the patient. In certain embodiments, batches of GWBL extract with an IC50 value less than 10 pg/mL are considered active. In certain

embodiments, batches of HHP extract with an IC50 value less than 5 pg/mL are considered active. In certain embodiments, batches of BL extract with an IC50 value less than 10 pg/mL are considered active. In certain embodiments, batches of WBL extract with an IC50 value less than 15 pg/mL are considered active.

Combination Therapies

In certain embodiments, the extracts of the invention are useful in the methods of present invention when used concurrently with at least one additional compound or composition useful for treating HCV.

In a non-limiting example, the compounds of the invention can be used concurrently or in combination with a therapeutically effective amount of at least one antiviral herbal extract. In a non-limiting example, the compounds of the invention can be used concurrently or in combination with a therapeutically effective amount of at least one direct acting antiviral agents, or salts, solvates, enantiomers, diastereoisomers, or tautomers thereof.

In certain embodiments, the one or more antiviral active herbal extracts is selected from the group consisting of Rheum palmatum L. and Polygala tenuifolia.

In certain embodiments, the direct acting antiviral agents is one selected from the group consisting of peg-interferon alfa-2a, ribavirin, elbasvir, grazoprevir, ledipasvir, sofosbuvir, paritaprevir, ritonavir, ombitasvir, dasabuvir, simeprevir, daclatasvir, dasabuvir and ledipasvir. In other embodiments, the direct acting antiviral agent can be any antiviral agent currently known or unknown in the art.

In certain embodiments, the amount of the herbal extract composition and the amount of the at least one antiviral drug administered to the subject are such that : (a) administration of the amount of the herbal extract composition is not therapeutically effective in treating the HCV infection in the absence of the co-administration of the at least one antiviral drug; and/or (b) administration of the amount of the at least one antiviral drug is not therapeutically effective in treating the HCV infection in the absence of the co-administration of the herbal extract composition.

In certain embodiments, administration of the herbal extracts increases the efficacy of the direct acting antiviral agents. In certain embodiments, the administration of the herbal extracts decreases the required effective dose or shortens the required dosage period of the direct acting antiviral agent.

In certain embodiments, the extracts of the invention are useful in the methods of present invention when used concurrently with at least one additional compound or composition useful for treating depression.

In a non-limiting example, the compounds of the invention are used concurrently or in combination with a therapeutically effective amount of at least one anti-depressant.

In certain embodiments, the at least one anti-depressant is at least one selected from the group consisting of selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), tricyclic antidepressants (TCAs), tetracyclic antidepressants, dopamine reuptake blockers, 5-HT1 A receptor antagonists, 5-HT2 receptor antagonists, 5-HT3 receptor antagonists, monoamine oxidase inhibitors (MAOIs), noradrenergic antagonist and anti -depressive herbal extracts.

In certain embodiments, the at least one anti-depressant is at least one selected from the group consisting of sertraline (Zoloft), fluoxetine (Prozac, Sarafem), citalopram (Celexa), escitalopram (Lexapro), paroxetine (Paxil, Pexeva, Brisdelle), fluvoxamine (Luvox), desvenlafaxine (Pristiq, Khedezla), duloxetine (Cymbalta), levomilnacipran (Fetzima), venlafaxine (Effexor XR), amitriptyline, amoxapine, clomipramine (Anafranil), desipramine (Norpramin), doxepin, imipramine (Tofranil), nortriptyline (Pamelor), protriptyline, trimipramine (Surmontil), maprotiline, bupropion, vilazodone, nefazodone, trazodone, vortioxetine, isocarboxazid (Marplan), phenelzine (Nardil), selegiline (Emsam),

tranylcypromine (Parnate), mirtazapine (Remeron), olanzapine/fluoxetine and St. John’s wort.

A synergistic effect can be calculated, for example, using suitable methods such as, for example, the Sigmoid-E_max equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to elsewhere herein can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to elsewhere herein are the concentration-effect curve, isobologram curve and combination index curve, respectively.

Methods of treatment

The present invention includes methods of treating or preventing HCV infection in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a composition comprising one or more antiviral active herbal extracts selected from the group consisting of Vaccaria segetalis , Albizia julibrissin,

Ficus pumila Linn and Areca catechu.

In certain embodiments, the administration of the compositions of the invention eliminates the HCV infection in the subject. In certain embodiments, the compositions of the invention ameliorate the symptoms of HCV infection. In certain embodiments, the compositions of the invention can be administered concurrently with at least one additional compound or composition useful for treating HCV. In certain embodiments, the methods of the present invention comprise using concurrently at least one additional compound or composition useful for treating HCV.

In a non-limiting example, the compounds of the invention are used concurrently or in combination with at least one antiviral herbal extract. In a non-limiting example, the compounds of the invention can be used concurrently or in combination with a

therapeutically effective amount of at least one direct acting antiviral agents, or salts, solvates, enantiomers, diastereoisomers, or tautomers thereof.

In certain embodiments, the composition is administered to the subject by at least one route selected from the group consisting of oral, nasal, inhalational, topical, buccal, rectal, pleural, peritoneal, intra-peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes

In certain embodiments, the one or more herbal extracts are administered to the subject at the same time as the antiviral agent. In other embodiments, the one or more herbal extracts are administered to the subject before the antiviral agent. In yet embodiments, the one or more herbal extracts are administered to the subject after the antiviral agent.

In certain embodiments, the therapeutically effective amount of the additional direct acting antiviral agent is lower when administered as part of the methods of the invention than if it was administered alone. In other embodiments, the therapeutically effective course of treatment with the direct acting antiviral agent is shorter when administered as part of the methods of the invention than if it was administered alone.

In certain embodiments, the therapeutically effective amount of the composition ranges from about 0.001 mg/day to about 1,000 mg/day.

In certain embodiments, the methods of the invention allow for treating drug-resistant HCV. In certain embodiments, the herbal extracts of the invention are effective in treating HCV clones resistant to treatment with one or more drugs selected from the group consisting of Daclatasvir and Simeprevir. In certain embodiments, HCV contacted with the herbal extracts of the invention do not develop resistance to the herbal extracts of the invention. In certain embodiments, the subject is a mammal. In other embodiments, the subject is human.

Methods of determining antiviral activity of herbal extracts

The present invention also includes methods of determining the anti-HCV activity of selected batches of herbal extracts. The method comprises preparing a water extract of a specific batch of an herb of the invention as described elsewhere herein. The water extracts are then diluted to varying concentrations and tested for anti-HCV activity in an HCV luciferase replicon cell assay for 72 hours. Using the HCV luciferase replicon assay measurements, the IC50 value for the specific batch of the herb is determined. The determined IC50 value for the specific batch of the herb is then compared to a standard IC50 value and if the determined IC50 value is lower than the standard IC50 value, then that batch is considered“active”.

In certain embodiments, batches of GWBL extract with an IC50 value less than 10 pg/mL are considered active. In certain embodiments, batches of HHP extract with an IC50 value less than 5 pg/mL are considered active. In certain embodiments, batches of BL extract with an IC50 value less than 10 pg/mL are considered active. In certain

embodiments, batches of DH extract with an IC50 value less than 50 pg/mL are considered active. In certain embodiments, batches of MYZ extract with an IC50 value less than 1 pg/mL are considered active. In certain embodiments, batches of WBL extract with an IC50 value less than 15 pg/mL are considered active.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations can be administered to the subject either prior to or after the onset of a disease or disorder contemplated in the invention. Further, several divided dosages, as well as staggered dosages can be administered daily or sequentially, or the dose can be

continuously infused, or can be a bolus injection. Further, the dosages of the therapeutic formulations can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to a patient, preferably a mammal, more preferably a human, can be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated in the invention. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a disease or disorder contemplated in the invention. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day. The pharmaceutical compositions useful for practicing the invention can be administered to deliver a dose of from 1 ng/kg/day and 100 mg/kg/day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A medical doctor, e.g ., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease or disorder contemplated in the invention.

In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In other embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable carrier. In yet other embodiments, the compound of the invention is the only biologically active agent (z.e., capable of treating HCV) in the composition. In yet other embodiments, the compound of the invention is the only biologically active agent (z.e., capable of treating HCV) in

therapeutically effective amounts in the composition.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

In certain embodiments, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account. Compounds of the invention for administration can be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

In certain embodiments, the present invention is directed to a packaged

pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second

pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder contemplated in the invention.

Formulations can be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g ., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents.

Routes of administration of any of the compositions of the invention include oral nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.

The compounds for use in the invention can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal ( e.g ., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-peritoneal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

Oral Administration

For oral application, particularly suitable are soups, teas, concentrates, tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use can be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets can be uncoated or they can be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

For oral administration, the compounds of the invention can be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g, polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethyl cellulose); fillers ( e.g ., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g, sodium starch glycollate); or wetting agents (e.g, sodium lauryl sulphate). If desired, the tablets can be coated using suitable methods and coating materials such as OP ADR Y™ film coating systems available from Colorcon, West Point, Pa. (e.g, OP ADR Y™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and

OP ADR Y™ White, 32K18400). Liquid preparation for oral administration can be in the form of solutions, syrups or suspensions. The liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g, sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g, lecithin or acacia); non-aqueous vehicles (e.g, almond oil, oily esters or ethyl alcohol); and preservatives (e.g, methyl or propyl p-hydroxy benzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a“granulation”. For example, solvent-using“wet” granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e., having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e., drug) by forming a solid dispersion or solid solution.

ET.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) melt. The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for treatment of a disease or disorder contemplated in the invention. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition can be obtained in which the active ingredient is entrapped, ensuring its delayed release.

Parenteral Administration

As used herein,“parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intra-peritoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (z.e., powder or granular) form for reconstitution with a suitable vehicle ( e.g ., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution can be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations can be prepared using a non toxic parenterally-acceptable diluent or solvent, such as water or l,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention can be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time can be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

For sustained release, the compounds can be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds. As such, the compounds useful within the methods of the invention can be administered in the form of microparticles, for example by injection, or in the form of wafers or discs by implantation.

In one embodiment of the invention, the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration. The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, about 10 minutes, or about 1 minute and any or all whole or partial increments thereof after drug administration after drug administration.

As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, about 10 minutes, or about 1 minute and any and all whole or partial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of the present invention depends on the age and weight of the patient, the current medical condition of the patient and the progression of a disease or disorder contemplated in the invention. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

A suitable dose of a compound of the present invention can be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.

The dose can be administered in a single dosage or in multiple dosages, for example from 1 to 5 or more times per day. When multiple dosages are used, the amount of each dosage can be the same or different. For example, a dose of 1 mg per day can be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

It is understood that the amount of compound dosed per day can be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose can be initiated on Monday with a first subsequent 5 mg per day dose administered on

Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the inhibitor of the invention is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a“drug holiday”). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%- 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient’s conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the disease or disorder, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long term basis upon any recurrence of symptoms and/or infection.

The compounds for use in the method of the invention can be formulated in unit dosage form. The term“unit dosage form” refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses ( e.g ., about 1 to 5 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD₅₀ and ED_50. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989);

“Oligonucleotide Synthesis” (Gait, 1984);“Animal Cell Culture” (Freshney, 1987);

“Methods in Enzymology”“Handbook of Experimental Immunology” (Weir, 1996);“Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);“Current Protocols in Molecular Biology” (Ausubel, 1987);“PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

It is to be understood that, wherever values and ranges are provided herein, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, all values and ranges encompassed by these values and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.

EXAMPLES The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Materials and Methods

Cell lines

HCV genotype lb, Conl subgenomic replicon cell line Huh-luc/neo-ET containing HCV luciferase replicon or HCV genotype 2a Conl subgenomic replicon cell line were provided by Dr. Ralf Bartenschlager from The University of Heidelberg. Huh luc/neo-ET cells were maintained in DMEM medium supplied with 10% FBS, 1 mM nonessential amino acids (Invitrogen), and 250 pg/ml G418 (Life Technologies).

Determination of anti-HCV activity by HCV luciferase replicon assay

The anti-HCV IC50 (the concentration required to reduce HCV activity to 50% of the control value) of herbal extracts were determined using an HCV luciferase replicon assay with HCV genotypes lb (conl) replicon cells free from G418 (Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science l999;285: 110-113.). Luciferase activity was measured with a luciferase assay kit (Promega) using a Farcyte Tecan luminometer. Twenty thousand (20,000) cells were seeded per well on a 48-well plate. The plates were incubated overnight at 37 °C under a 5% C0₂ atmosphere. The herbal extracts were added to the wells at various concentrations and the plates were then incubated for three days at 37 °C under a 5% C0₂ atmosphere. The media was removed and the wells were washed with phosphate-buffered saline (PBS). PBS was removed and replaced with 50 pL Lysis Buffer. The plates were rotated for 10 min. at room temperature (RT). The plates were then frozen to enhance lysis. The lysate was transferred to white plates and 50 pL luciferase was added. Luciferase activity was measure using a Farcyte Tecan luminometer. Data was plotted as a percentage of fluorescence measured versus the fluorescence of untreated control samples.

Determination of the toxicity of compounds

The IC50 (half maximal inhibitory concentration) or cytotoxicity of herbal extracts were analyzed using the methylene blue method under the same conditions as measuring IC50 (Gao W, Lam W, Zhong S, Kaczmarek C, Baker DC, Cheng YC. Novel mode of action of tylophorine analogs as antitumor compounds. Cancer Res 2004;64:678-688). In brief, cells were fixed and stained with 0.5% methylene blue in 50% ethanol, followed by extensive washing. After that, cells were solubilized in 1% Sarkosyl and cells growth was determined from the extent of absorption by spectrophotometric measurements at 595 nm.

Quantification of HCV RNA using qPCR

Selected herbal extracts obtained were examined for their impact on the expression levels of HCV RNA by using real-time PCR (qPCR). In brief, total RNA was isolated using Trizol reagent (Life Technologies). cDNA was synthesized from total RNA by using random primers according to the protocol of the Script™ cDNA synthesis kit (Bio-Rad Laboratories). qPCR assays were performed using iTaq™ SYBR® Green Supermix and the CFX96 Real- Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Gene-specific primers: forward 5’ -CGGGAGAGCC AT AGTGGTCTGCG-3’ (SEQ ID : 01 ) and reverse 5’ -

CTCGCAAGCACCCTATCAGGCAGTA-3’(SEQ ID: 02) for HCV; forward 5’- GTAACCCGTTGAACCCCATT-3’(SEQ ID: 03) and reverse 5’- CCATCC AATCGGTAGTAGCG-3’ (SEQ ID: 04) for l8s rRNA.

Western Blotting

Following herbal extract treatment, Huh-luc/neo-ET containing HCV luciferase replicon cells were lysed in 2x sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris- HC1, 2% SDS, 10% glycerol, 50 mM dithiothreitol (DTT), and 0.05% bromophenol blue) and sonicated for lOs to shear the DNA. The whole-cell extracts were then electrophoresed through 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc) with a Mini PROTEANII transfer apparatus (Bio-Rad). The membranes were blocked and probed in tris-buffered saline + tween (TBS-T) buffer (lx Tris-HCl 50mM pH7.6, NaCl l50mM, 0.2% Tween-20) containing 5% non-fat milk. Primary antibodies for NS5B, NS3, NS5A at 1 : 1000 in TBS-T were incubated with the membrane overnight at 4 C. b-actin was used as an internal control to ensure equal protein loading and detected with a monoclonal actin antibody diluted at 1 :2500 (Sigma, St. Louis, MO). After washing with TBS-T, the membranes were then further incubated with horseradish peroxidase-conjugated anti-mouse immunoglobin G (IgG) and anti-rabbit IgG (1 : 5000; Sigma), The

immunoreactive bands were visualized by enhanced chemiluminescence reagents (Perkin- Elmer Life Science Products, Boston, MA), and densitometry scanning was performed with the densitometer (Molecular Dynamics).

Solid phase extract method

Herbal water extract (1 ml, 100 mg/ml) was passed through a Discovery® DSC-18 SPE Tube bed wt. 1 g. The bound chemicals were eluted sequentially using 1 ml water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol and 100% ethanol. The different fractions were dried in vacuo to form a pellet. The dried pellet was re-dissolved to 1 ml using water.

The re-dissolved solution was regarded as a“100 mg/ml equivalent” of the original herbal water extract input for further experiments.

Liquid chromatography mass spectrometry (LC-MS)

The chromatographic separation was achieved on a ZORBAX-SBC18 column (100 mm x 2.1 mm i.d., 3.5pm, Agilent, Palo Alto, CA). Mobile phase A was water with 0.05% (v/v) formic acid and mobile phase B was acetonitrile. The liquid flow-rate was set at 0.3 mL/min, and the column temperature was maintained at 30 °C. The mobile phase consisted of linear gradients of 0.05% (v/v) formic acid (A) and Acetonitrile (B). The chromatographic system used consisted of an Agilent 1200 HPLC series, including a binary pump (Model G1312B), a vacuum degasser (Model G1379B), an autosampler (Model G1367C), and a column oven (Model G1316B). Mass of chemicals were detected by Applied Biosystems Sciex 4000 Q-trap® mass spectrometer (Applied Biosystems Sciex; Foster, CA). Data acquisition was carried out by Analyst 1.4.2® software on a DELL computer. Example 1: Preparation of herbal extracts

A number of herbal extracts were prepared from a variety of plant species. These extracts were prepared through our main methods: water extract, b-glucuronidase treated extract, honey treated extract and licorice treated extract.

Water extract

Dried herbs were ground into a powder. Herbal powder (lOOmg) was dissolved in lml water and then heated at 80°C for lh. The herbal water mixture was then cooled to room temperature. The herbal water mixture was centrifuged for 5 minutes at 13000 rpm in a desktop centrifuge. The supernatant was decanted and collected as the herbal water extract (-100 mg/ml). b-glucuronidase treated extract

For b-glucuronidase treatment, 500 mΐ herbal water extract (100 mg/ml) was added to 475 mΐ TrisHCl (100 mM, pH 6.8) and 25 mΐ purified recombinant E. colt b-glucuronidase protein (2.5 mg/ml). The mixture was incubated at 37°C for lh. After incubation, the mixture was heated at 100 °C for 5 min to inactivate the b-glucuronidase. The final mixture was collected as a b-glucuronidase treated extract (-50 mg/ml).

Honey treated extract ofMYZ

30 mL of honey solution (containing -5 g of honey) was added into 20 g of crude MYZ and then stirred and fried until dry over low heat in a pot. The honey fried MYZ was then cooled to room temperature.

Licorice treated extract ofMYZ

1.2 g of licorice was boiled with water for 20 min. The solution volume was adjusted to 12 mL, then poured into 20 g ofMYZ and then stirred and fried until dry over low heat in a pot. The licorice fried MYZ was then cooled to room temperature.

Example 2: Anti-HCV activity of selected herbal extracts

A number of herbal extracts were tested using HCV luciferase replicon assay (see materials and methods) to determine anti-HCV activity. Water extract and b-glucuronidase treated batches of a wide variety of common herbs used in traditional medicine were prepared including WBL, DH, MYZ, GWBL, HHP and BL, with concentrations ranging from 5 pg/ml to 300 pg/ml The extracts were tested for inhibition of HCV using HCV luciferase replicon cells for 72 h and the EC50 value was determined. It was found that of those tested, WBL, DH, MYZ, GWBL, HHP and BL demonstrated superior anti-HCV activity (FIG. 1).

Example 3: Anti-HCV activity of selected herbal extracts

The relative amount of HCV RNA was measured using qPCR (see materials and methods section for quantification methods) in the presence of water extract and b- glucuronidase treated extracts of MYZ, HHP, DH, WBL and BL after 24h, 48h and 72h. (FIGs. 2A and 2B).

Example 4: Anti-HCV activity of MYZ and HHP against HCVlb and HCV2a

Water extracts of MYZ and HHP were tested against HCVlb in an HCV luciferase replicon assay (see materials and methods) (FIGs. 3 A, 3C). It was found that both herbal extracts were active against the lb genotype and that the activity was strongly correlated with increasing concentration of the extract. In addition, MYZ and HHP were tested against HCV2a by measuring HCV RNA expression using qPCR at a range of concentrations (FIGs. 3B and 3D). Both extracts demonstrated no appreciable anti-HCV activity and showed no correlation between extract concentration and activity.

Water extracts of MYZ and HHP were also tested against HCVlb in an HCV luciferase replicon assay (FIGs. 4A, 5 A) over a period of 72h at a range of concentrations. The extracts were also tested against HCVlb by measuring HCV RNA expression using qPCR (FIGs. 4B, 5B) 72h after treatment. It was found that antiviral activity increased for extracts of both herbs as a function of both time and concentration. In addition, anti -viral activity was also determined by measuring the expression of selected HCV proteins 72h after treatment with extracts of MYZ and HHP (FIGs. 4C, 4D, 5C and 5D). It was found that protein expression generally decreased as a function of increasing extract concentration.

Example 5: Anti-HCV activity of MYZ and HHP fractions against HCVlb

Water extracts of MYZ and HHP were passed through a Cl 8 solid phase column and eluted with a range of ethanol solutions with different ethanol: water ratios (see material and methods for solid phase extract method). It was found that fractions of the MYZ extract that eluted with the 50% and 70% ethanol solutions yielded the strongest anti-HCV activity (FIG. 6A and 6B). These fractions contained chemicals found only in these fractions with molecular weights of 924.9 and 968.9 (FIG. 6C). Fractions of the HHP extract that eluted with the 50% and 70% ethanol solutions also yielded the strongest anti-HCV activity (FIG. 7A and 7B). These fractions contained chemicals found only in these fractions with molecular weights of 209.8, 224.1 and 294.4 in the 50% elution and 330.3 and 330.4 in the 70% elution (FIG. 7C). (These results provide evidence towards the potential active ingredients in these extracts but the identity of these compounds remains undetermined.

Example 6: Anti-HCV activity of DH batches against HCVlb

Water extracts of DH were tested against HCVlb using an HCV luciferase replicon assay (FIG. 8). It was found that some batches of DH do not demonstrate anti-HCV activity while others do. As shown in FIG. 9, different batches of each herbs may have different activity against HCV. Because of the variable activity between individual plants, methods of confirming activity prior to administration of the extract have been developed.

Batches of WBL, GWBL, DH, MYC, HHP and BL water extract were prepared using the standard procedure described in materials and methods. The water extracts (ranging from 0.2ug/ml to 300ug/ml) were tested for their inhibition of HCV using HCV luciferase replicon cells for 72 hours. For MYZ, honey processing or licorice processing are also active in against HCV using replicon luciferase assay. IC50 of each batch of herbs was then

determined. In order to be regarded as“Active” batches, the IC50 value needed to be lower than the value listed in FIG. 9. Batches of herb with IC50 less than the“Active” dose will be used for future manufacturing and batches with IC50 values higher than the“Active” dose will be discarded.

Example 7: Drug resistance testing

HCVlb replicon reported cells (1000 cells/lOcm plate) were incubated with the daclatasvir, simeprevir, MYC and HHP for three weeks (concentration are indicated in the Table 1). After the selection, clones were examined for their luciferase activity to indicate the presence of HCVlb replicon. Clones with luciferase activity are regarded as resistant clones.

Current standard of care methods of treatment of HCV can lead to the development of drug-resistant strains of the virus. For example, daclatasvir and simeprevir are two common drugs for treating HCV, and over time HCV virus clones can develop a strong resistance to these treatments.

In order to test for the development of drug resistance with the herbal extracts of the invention, batches of MYZ and HHP were prepared as described in Example 5 and tested against HCV lb. After testing, no MYZ or HHP resistant HCV clones were discovered (Table 1). All clones under the selection of MYC or HHP had no luciferase activity.

Cross-resistance between standard of care HCV treatment drugs and the herbal extracts of the invention was also investigated. MYZ and HHP were tested against daclatasvir and simeprevir resistant strains of HCV (Table 2). It was found that neither strain of drug-resistant HCV was resistant to MYZ or HHP, but each were resistant to their respective anti-HCV drug.

TABLE 1

TABLE 2: Fold change: IC50 of Resistant clones/IC50 of wt.

Example 8: Anti-HCV activity of select HHP and MYZ herbal extract batches

Various water extract batches of HHP and MYZ were tested in HCV luciferase replicon assays to determine if the activity of each batch differed from each other. The results are shown in Table 3. It was found that certain batches demonstrated much higher activity than others. The results showed that the anti-HCV qualities of different batches of HHP extract can be very different.

TABLE 3

Example 9: Inhibition of Serotonin of Selected Herbs

A high percentage of HCV patients have been found to suffer from depression. Traditional medicine has suggested that HHP, MYZ, BL, DH, WBLX and Tian Ji Huang (TJH also known as Hypericum japoni cum Thunb. exMurray or Japanese St. John's wort or all-grass of Madras Grangea) can potentially treat symptoms associated with depression. Serotonin transport is an important target for the treatment of depression. Therefore, pharmaceutical compositions having both anti-HCV activity and anti-depression activity would be greatly beneficial to HCV patients.

Different batches of HHP, MYZ, BL, DH, WBLX and TJH were tested for their activity on serotonin transport by using an uptake assay (Table 4). Appreciable serotonin transport inhibition was measured for herbal extracts of HHP and MYZ. Additionally, specific water extract batches of HHP and MYZ were tested in order to determine how serotonin inhibition might differ between batches of the same herb.

The results reported in Table 5 show that theianti-serotonin transport activity of three selected batches of HHP was much larger than that of the selected MYZ batches tested. No correlation of anti-HCV activity and anti -serotonin transport activity for the selected batches of HHP and MZY was observed (Table 3 and Table 5). In certain embodiments, both anti-HCV replicon assays and serotonin transport assays are conducted for selecting optimal batches of HHP and MYZ in order to maximize both anti- HCV and anti-depression activity. TABLE 4

TABLE 5

Other Embodiments

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A method of treating or preventing hepatitis C virus (HCV) infection in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising one or more antiviral active herbal extracts derived from the herbs selected from the group consisting of Vaccaria segetalis , Albizia julibrissin, Areca catechu and Ficus pumila Linn.

2. The method of claim 1, wherein the composition further comprises a therapeutically effective amount of one or more antiviral active herbal extracts derived from the herbs selected from the group consisting of Rheum palmatum L. and Polygala tenuifolia.

3. The method of either claim 1 or 2, wherein a therapeutically effective amount of at least one additional agent useful for treating HCV infection is administered to the subject.

4. The method of claim 3, wherein the additional agent is one selected from the group consisting of peg-interferon alfa-2a, ribavirin, elbasvir, grazoprevir, ledipasvir, sofosbuvir, paritaprevir, ritonavir, ombitasvir, dasabuvir, simeprevir, daclatasvir, dasabuvir and ledipasvir.

5. The method of either claim 1 or 2, wherein administering the composition further treats or prevents depression in the subject.

6. The method of claim 2, wherein the composition is administered to the subject orally.

7. The method of claim 6, wherein the composition is administered to the subject orally in one or more forms selected from the group consisting of a pill, tablet, capsule, soup, tea, concentrate, dragees, liquids, drops, and gelcaps.

8. The method of claim 2, wherein the therapeutically effective amount of the composition is about 0.001 mg/day to about 1,000 mg/day.

9. The method of claim 2, wherein the herbal extracts are water herbal extracts prepared in a water extract preparation method, the method comprising:

drying an amount of the herb or herbs;

grinding the dried amount of the herb or herbs into an herb powder;

adding the ground, dried amount of herb powder to an amount of water to form a mixture;

heating the mixture at a first elevated temperature for a first amount of time to form a heated mixture;

allowing the heated mixture to cool to room temperature to form a cooled mixture;

removing any undissolved solids in the cooled mixture from the cooled mixture to yield the water herbal extract.

10. The method of claim 9, wherein the amount of dried herbal powder is 100 mg and the amount of water is 1 mL.

11. The method of claim 9, wherein the first elevated temperature is about 80°C.

12. The method of claim 9, wherein the mixture is heated for about 1 hr.

13. The method of claim 9, wherein any undissolved solids are removed from the cooled mixture by centrifuging the cooled mixture and decanting the resulting solution.

14. The method of claim 9, the method further comprising:

adding an amount of the water herbal extract to an amount of a b- glucuronidase solution to form a b-glucuronidase treated solution;

incubating the b -glucuronidase treated solution to a second elevated temperature for a second amount of time to form an incubated b-glucuronidase treated solution;

heating the incubated b-glucuronidase treated solution to a third elevated temperature for a third amount of time to form a heated b -glucuronidase treated solution;

allowing the heated b-glucuronidase treated solution to cool to room temperature to form a b-glucuronidase treated extract.

15. The method of claim 14, wherein the amount of the water herbal extract is about 500 pL at a concentration of about 100 mg/mL (herb/water).

16. The method of claim 14, wherein the amount of b-glucuronidase solution comprises about 475 pL of TrisHCl (lOOmM, pH 6.8) and about 25 pL of E.coli b-glucuronidase protein (2.5 mg/mL).

17. The method of claim 14, wherein the second elevated temperature is about 37°C.

18. The method of claim 14, wherein the second amount of time is about 1 hour.

19. The method of claim 14, wherein the third elevated temperature is about l00°C.

20. The method of claim 14, wherein the third amount of time is about 5 minutes.

21. The method of claim 2, wherein the herbal extracts are honey treated herbal extracts prepared in a honey extract preparation method, the method comprising:

adding an amount of a honey solution to an amount of the herb or herbs to form a honey mixture;

stirring and heating the honey mixture until the honey mixture is dry to form a dry honey mixture;

allowing the dry honey mixture to cool to room temperature to form the honey treated herbal extract.

22. The method of claim 21, wherein the amount of honey solution is about 30 mL of about 15% (w/v) honey in water and the amount of herb or herbs is about 20 g.

23. The method of claim 2, wherein the herbal extracts are licorice treated herbal extracts prepared in a licorice extract preparation method, the method comprising:

adding an amount of a licorice solution to an amount of the herb or herbs to form a licorice mixture;

stirring and heating the licorice mixture until the licorice mixture is dry to form a dry licorice mixture; allowing the dry licorice mixture to cool to room temperature to form the licorice treated herbal extract.

24. The method of claim 23, wherein the amount of licorice solution is about 12 mL of about 10% (w/v) licorice in water and the amount of herb or herbs is about 20 g.

25. The method of claim 2, wherein the subject is a mammal.

26. The method of claim 25, wherein the mammal is a human.

27. A method of determining the anti-HCV activity of a specific batch of an herbal extract, the method comprising:

drying an amount of the herb from a specific batch of herb;

grinding the dried amount of the herb into an herb powder;

removing any undissolved solids in the cooled mixture from the cooled mixture to yield the water herbal extract;

diluting the water herbal extract to a range of concentrations; testing the water herbal extracts for inhibition of HCV in an HCV luciferase replicon cell assay for 72 h;

determining the IC50 value of the water herbal extract;

comparing the determined IC50 value to a standard IC50 value, wherein if the determined IC50 value is lower than the standard IC50 value then the specific batch of herb is considered to be active for the inhibition of HCV.

28. The method of claim 27, wherein the herb is Ficus pumila Linn and the standard IC50 value is 10 pg/mL.

29. The method of claim 27, wherein the herb is Areca catechu and the standard IC50 value is 10 m_§/ihT.

30. The method of claim 27, wherein the herb is Albizia julibrissin and the standard IC50 value is 5 m§/ihT.

31. The method of claim 27, wherein the herb is Rheum palmatum L. and the standard IC50 value is 50 m_§/ihT.

32. The method of claim 27, wherein the herb is Polygala tenuifolia and the standard IC50 value is 1 m_§/ihT.

33. The method of claim 27, wherein the herb is Vaccaria segetalis and the standard IC50 value is 15 m_§/ihT.