Patent Application
20150359834
The present invention relates to formulations and methods for the modulation of airway contraction.
Asthma, a growing health concern worldwide, particularly in industrialized countries, is a complex disease associated with an elevation of IgE production, abnormally high levels of Th2 cytokines, and airway inflammation. The immediate allergic asthmatic response is the release of histamine and leukotrienes from IgE-activated mast cells that leads to bronchoconstriction and airway hyperreactivity (AHR). Increased airway smooth muscle (ASM) contraction and reduced ASM relaxation in chronic asthma contributes to the excessive airway narrowing and AHR characteristic of asthmatic attacks.
Despite a better understanding of the pathophysiology of asthma, there are still considerable gaps in knowledge as well as a need for new classes of drugs. Current standard asthma management is primarily directed towards suppressing airway inflammation with inhaled corticosteroids (ICSs) and relieving bronchoconstriction with bronchodilators. This standard therapy controls symptoms in most, but not all asthmatic patients while on treatment. Severe asthma is difficult to control. ICSs are generally safe, however, their prolonged use, especially at high doses has been accompanied by concern about both systemic and local side effects. These include osteoporosis, hypothalamic-pituitary-adrenal axis suppression, immune suppression resulting in increased susceptibility to infections such as esophageal candidiasis and frequent upper respiratory tract infections in children, development of cataracts in elderly patients, mood changes, and pharyngitis. Long-term treatment with β2-adrenoceptor (β2-AR) agonists that directly relax ASM contraction, can result in bronchodilator resistance, cardiovascular diseases and arrhythmias. Therefore, development of new alternative or complementary therapies for this disease is becoming increasingly important.
In recent years, interest and use of complementary and alternative medicine (CAM) has been increasing. Traditional Chinese medicine (TCM) has a long history of human use in China and other Asian countries for treating and preventing diseases including asthma, and is part of accepted medical practice in these countries. TCM is also beginning to play a role in the US health care system. There is also increasing scientific understanding of the mechanisms of actions of certain TCMs for allergy and asthma. Kao et al. (2001) showed that the TCM formula xiao-qing-long-tang (XQLT) inhibited bronchoconstriction and airway eosinophil infiltration in ovalbumin-sensitized guinea pigs, and suppressed airway smooth muscle contraction (Kao et al., 2001).
ASHMI™ (Anti-asthma Herbal Medicine Intervention), an aqueous extract of Ganoderma lucidum (Fr.) P. Karst. (Ling Zhi), Sophora flavescens Aiton (Ku Shen) and Glycyrrhiza uralensis Fisch. ex DC (Gan Cao), has been shown to prevent allergic asthma airway hyperreactivity in mice and inhibit acetylcholine (ACh) induced airway smooth muscle (ASM) contraction in tracheal rings from allergic asthmatic mice.
We previously demonstrated that ASHMI™, an aqueous (aq.) extract of G. lucidum, S. flavescens and G. uralensis, eliminated both the immediate airway response and late-phase AHR, and significantly reduced pulmonary inflammation and airway remodeling in a murine allergic asthma model (Busse et al., 2010; Li et al., 2000; Zhang et al., 2010). In a clinical trial, ASHMI™ treatment reduced the use of β2-AR agonists, while significantly improving objective lung function and reducing symptom scores of asthmatics to a similar extent as conventional treatment, and showed a high safety profile (Kelly-Pieper K et al., 2009; Wen et al., 2005). We further found that, in addition to its anti-inflammatory effect, ASHMI™ inhibited ex vivo acetylcholine-induced ASM contractility in tracheal rings of allergic asthmatic mice without activating β2-ARs (Zhang et al., 2010). However, the individual constituent ingredients and compounds responsible for inhibiting ASM contraction have not been identified.
There is therefore a need in the medical arts for an identification and refinement of the bioactive constituents of the ASTHMI™ formula to reduce airway smooth muscle contractility.
In an embodiment of the present invention the herb S. Flavescens is used to reduce activity in contractile tissue, particularly airway smooth muscle.
In an embodiment, the invention comprises aqueous extracts the herb S. Flavescens and methods of use thereof to reduce activity in contractile tissue, particularly airway smooth muscle.
In an embodiment the invention comprises, a flavinoid-rich fraction of an extraction of S. Flavescens and methods of use thereof to reduce activity in contractile tissue, particularly airway smooth muscle.
In an embodiment the invention comprises, the compounds trifolirhizin and 3′,7-dihydroxy-4′-methoxy-isoflavone, either alone or in combination, and methods of use thereof to reduce activity in contractile tissue, particularly airway smooth muscle.
In an embodiment the invention comprises, the compounds Matrine, Oxymatrine and Sophocarpine either alone or in combination, and methods of use thereof to reduce activity in contractile tissue, particularly airway smooth muscle.
In an embodiment the invention comprises an alkaloid rich fraction of an extract of S. Flavescens and methods of use thereof to reduce activity in contractile tissue, particularly airway smooth muscle.
In an embodiment the invention comprises a nutritional supplement comprising S. Flavascens or extracts thereof.
In an embodiment the invention comprises methods for isolating the bioactive constituents of S. Flavescens that have an effect on airway smooth muscle contractility.
Supplemental
Supplemental
Table 1. 1H and 13C NMR spectra data of trifolirhizin.
Table 2. 1H NMR spectra data of 3′,7-dihydroxy-4′-methoxy-isoflavone.
Table 3-1 Fractions generated by Liquid-Liquid Extraction of ASHMI or its component herbs. Solvents used were Dichloromethane, Ethyl Acetate and Methane
Dried root of Sophora flavescens (Fabaceae), known in Chinese as Ku-Shen, is widely used in traditional Chinese Medicine as a “cooling” drug for the treatment of gastrointestinal hemorrhage, skin diseases, and pyretic stranguria. The herb is an antipyretic, anthelminitic and diuretic agent. Phytochemical studies have revealed the presence of quinolizidine alkaloids and prenylated flavonoids, both of which show a wide spectrum of pharmacological activities.
The present invention provides a S. Flavescens extract that is effective in reducing airway smooth muscle (hereinafter “ASM”) contraction. In an embodiment the extract may comprise an aqueous extract of S. Flavescens. The extract may further comprise flavinoid enhanced fractions of S. Flavescens. The extract may further comprise the compound trifolirhizin. In an embodiment of the invention the compound trifolirhizin may be derived from S. Flavescens other natural sources or produced synthetically. In an embodiment the invention may comprise the compound 3′,7-dihydroxy-4′-methoxy-isoflavone, derived from S. Flavascens, or other natural sources or produced synthetically.
In another embodiment, the extract may comprise an alkaloid extraction of S. Flavescens. The alkaloid extraction may further comprise the compounds Matrine, Oxymatrine and Sophocarpine. In an embodiment of the invention, the compounds Matrine, Oxymatrine and Sophocarpine may be derived from natural sources such as S. Flavescens or produced synthetically.
In an embodiment, a method for reducing the contractility of mammalian airway smooth muscle is described. The method comprises the steps of administering an effective amount of an S. Flavascens extract to a mammal.
In another embodiment, the invention is a method for modulating airway contractility by administering to a subject, in need thereof, any of the following compounds, either alone or in combination, along with an acceptable carrier or excipient: trifolirhizin, 3′,7-dihydroxy-4′-methoxy-isoflavone, matrine, oxymatrine and sophocarpine
Extracts of S. flavascens may be obtained through any appropriate methods known to those skilled in the art. For example, aq. extracts of ASHMI™ and the individual herbal medicines, G. lucidum, S. flavescens and G. uralensis were provided by Sino-lion (Zhong Shi) Pharmaceutical Company (Weifang, China). Each raw herb was soaked in water for 30 min, and then boiled for 2 hr. The decoction was then concentrated under reduced pressure (P=0.08 Mpa), and thermal spray dried. Based on the ratio of individual raw herbs G. Lucidum, S. flavescens and G. uralensis in the ASHMI™ formula (62.5%, 28.1% and 9% respectively), and the yield of extracts of G. lucidum, S. flavescens, and G. uralenisis (6%, 20% and 17% respectively), ASHMI™ used in this study was a combination of the 3 herbal extracts in a ratio of 35%, 45%, 20% of G. lucidum, S. flavescens and G. uralenisis respectively. HPLC grade methylene chloride, ethyl acetate, acetonitrile with 0.1% formic acid, potassium chloride, and aluminum hydroxide (Alum, Imject®) were purchased from Fisher Scientific (Pittsburgh, Pa.). Albumin (OVA, Grade VI), Acetylcholine chloride (ACh, 99%), ICI 118,551 hydrochloride (>98% TLC), dexamethasone, and fenoterol hydrobromide were purchased from Sigma Aldrich (St Louis, Mo.).
The present invention also provides a dietary supplement comprising any of the S. Flavescens extracts of the invention. The S. Flavescens extracts or individual compounds extracted therefrom may be formulated into dietary supplements, including capsules, tablets, powders, solutions, gels, suspensions, creams, pastes, gels, suppositories, transdermal patches, and the like. These dietary supplements in, for instance, powder or solution form, may be added to nutraceuticals, foods and/or beverages to form functional nutraceutical, food, and/or beverage products. The dietary supplements may be formulated as powders, for example, for mixing with consumable liquids such as milk, juice, water or consumable gels or syrups for mixing into other dietary liquids or foods. The dietary supplements of this invention may be formulated with other foods or liquids to provide pre-measured supplemental foods, such as single serving bars. Typical food products that may incorporate the S. Flavescens extract of the present invention include dairy foods such as yogurt, cereals, breads, snack food products, fruit juices and other soft drinks Flavorings, binders, protein, complex carbohydrates, vitamins, minerals and the like may be added as needed. Preferably, the S. Flavescens extract is formulated for oral administration.
The present invention also provides a dietary supplement comprising of the S. Flavescens extracts described herein. The dietary supplement when administered to a mammal, including humans, may reduce Airway Smooth Muscle contractility, and therefore be a useful treatment for asthma. A successful course of treating, for instance, individuals with asthma, is characterized, inter alia, by causing the Airway Smooth Muscle of those individuals to relax and/or reduce contractility.
The dietary supplements of the present invention are intended for daily administration or as needed. The magnitude of a prophylactic or therapeutic dose of the dietary supplement in asthmatic individuals will vary with the severity of the condition being treated and the route of administration. The dose, and perhaps the dose frequency, will also vary according to the age, body weight, and response of the individual.
The dietary supplements of the present invention may be formulated in a conventional manner (i.e. by dry mixing, dry or wet granulation, direct compression), in admixture with pharmaceutically acceptable carriers, excipients, vitamins, minerals and/or other nutrients. Representative carriers and excipients include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like, in the case of oral solid preparations (such as powders, capsules, and tablets).
Any suitable route of administration may be employed to administer the dietary supplements of the invention to an individual. Suitable routes include, for example, oral, rectal, parenteral, intravenous, topical, transdermal, subcutaneous, and intramuscular. Although any suitable route of administration may be employed for providing the patient with an effective amount of the extract according to the methods of the present invention, oral administration is preferred, including solid dosage forms such as tablets, capsules, or powders. It is also preferred that the extract is formulated for use in functional nutraceutical, food, or beverage products.
An embodiment of the invention is a method for treating disorders of brochoconstriction or airway contractility, as in, for example, asthma, in a subject in need thereof which comprises administering to the subject a therapeutically effective amount of any of the extracts or compounds, or compositions described herein.
Also included in the invention is the use of any of the extracts described herein for the preparation of a medicament for treating disorders of bronchoconstriction or airway contractility, as in, for example, asthma in a subject in need thereof. The term “treating” as used herein refers to a method for improving, halting, retarding or palliating disorders of bronchoconstriction or airway contractility, as in, for example, asthma in the subject in need thereof. All such methods of treatment are intended to be within the scope of the present invention.
In accordance with the methods of the present invention, the individual components of the compositions described herein can also be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly.
The term “subject” as used herein, refers to an animal (preferably, a mammal; most preferably, a human) who has been the object of treatment, observation, or experiment.
The term “therapeutically effective amount” as used herein, means that amount of active compound, or extract, or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human, that is being sought by a researcher, veterinarian, medical doctor, or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
To prepare the compositions of this invention, one or more extracts of S. Flavascens, or compounds purified therefrom, as the active ingredient(s), is intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending of the form of preparation desired for administration (e.g. oral or parenteral). Suitable pharmaceutically acceptable carriers are well known in the art. Descriptions of some of these pharmaceutically acceptable carriers may be found in The Handbook of Pharmaceutical Excipients, published by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain.
Methods of formulating compositions have been described in numerous publications such as Pharmaceutical Dosage Forms: Tablets, Second Edition, Revised and Expanded, Volumes 1-3, edited by Lieberman et al; Pharmaceutical Dosage Forms: Parenteral Medications, Volumes 1-2, edited by Avis et al; and Pharmaceutical Dosage Forms: Disperse Systems, Volumes 1-2, edited by Lieberman et al; published by Marcel Dekker, Inc.
In preparing a composition of the present invention in liquid dosage form for oral, topical, inhalation/insufflation and parenteral administration, any of the usual pharmaceutical media or excipients may be employed. Thus, for liquid dosage forms, such as suspensions (i.e., colloids, emulsions and dispersions) and solutions, suitable carriers and additives include but are not limited to pharmaceutically acceptable wetting agents, dispersants, flocculation agents, thickeners, pH control agents (i.e., buffers), osmotic agents, coloring agents, flavors, fragrances, preservatives (i.e., to control microbial growth, etc.) and a liquid vehicle may be employed. Not all of the components listed above will be required for each liquid dosage form.
In solid oral preparations such as, for example, powders, granules, capsules, caplets, gelcaps, pills and tablets (each including immediate release, timed release and sustained release formulations), suitable carriers and additives include but are not limited to diluents, granulating agents, lubricants, binders, glidants, disintegrating agents, and the like. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated, gelatin coated, film coated or enteric coated by standard techniques.
Preferably these compositions are in unit dosage forms from such as tablets, pills, capsules, powders, granules, lozenges, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, autoinjector devices or suppositories for administration by oral, intranasal, sublingual, intraocular, transdermal, parenteral, rectal, vaginal, inhalation or insufflation means. Alternatively, the composition may be presented in a form suitable for once-weekly or once-monthly administration; for example, an insoluble salt of the active compound, such as the decanoate salt, may be adapted to provide a depot preparation for intramuscular injection.
For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g., conventional tabletting ingredients such as diluents, binders, adhesives, disintegrants, lubricants, antiadherents, and glidants. Suitable diluents include, but are not limited to, starch (i.e., corn, wheat, or potato starch, which may be hydrolized), lactose (granulated, spray dried or anhydrous), sucrose, sucrose-based diluents (confectioner's sugar; sucrose plus about 7 to 10 weight percent invert sugar; sucrose plus about 3 weight percent modified dextrins; sucrose plus invert sugar, about 4 weight percent invert sugar, about 0.1 to 0.2 weight percent cornstarch and magnesium stearate), dextrose, inositol, mannitol, sorbitol, microcrystalline cellulose (i.e., AVICEL™ microcrystalline cellulose available from FMC Corp.), dicalcium phosphate, calcium sulfate dihydrate, calcium lactate trihydrate, and the like. Suitable binders and adhesives include, but are not limited to acacia gum, guar gum, tragacanth gum, sucrose, gelatin, glucose, starch, and cellulosics (i.e. methylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, and the like), water soluble or dispersible binders (i.e., alginic acid and salts thereof, magnesium aluminum silicate, hydroxyethylcellulose (i.e. TYLOSE™ available from Hoechst Celanese), polyethylene glycol, polysaccharide acids, bentonites, polyvinylpyrrolidone, polymethacrylates and pregelatinized starch), and the like. Suitable disintegrants include, but are not limited to, starches (corn, potato, etc.), sodium starch glycolates, pregelatinized starches, clays (magnesium aluminum silicate), celluloses (such as crosslinked sodium carboxymethylcellulose and microcrystalline cellulose), alginates, pregelatinized starches (i.e., corn starch, etc.), gums (i.e., agar, guar, locust bean, karaya, pectin, and tragacanth gum), cross-linked polyvinylpyrrolidone, and the like. Suitable lubricants and antiadherents include, but are not limited to, stearates (magnesium, calcium and sodium), stearic acid, talc waxes, stearowet, boric acid, sodium chloride, DL-leucine, carbowax 4000, carbowax 6000, sodium oleate, sodium benzoate, sodium acetate, sodium lauryl sulfate, magnesium lauryl sulfate, and the like. Suitable gildants include, but are not limited to, talc, cornstarch, silica (i.e., CAB-O-SIL™ silica available from Cabot, SYLOID™ silica available from W. R. Grace/Davison, and AEROSIL™ silica available from Degussa), and the like. Sweeteners and flavorants may be added to chewable solid dosage forms to improve the palatability of the oral dosage form. Additionally, colorants and coatings may be added or applied to the solid dosage form for ease of identification of the drug or for aesthetic purposes. These carriers are formulated with the pharmaceutical active to provide an accurate, appropriate dose of the pharmaceutical active with a therapeutic release profile.
Generally these carriers are mixed with the pharmaceutical active to form a solid preformulation composition containing a homogeneous mixture of the pharmaceutical active of the present invention, or a pharmaceutically acceptable salt thereof. Generally the preformulation will be formed by one of three common methods: (a) wet granulation, (b) dry granulation, and (c) dry blending. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from about 0.01 mg to about 500 mg of the active ingredient of the present invention. The tablets or pills containing the novel compositions may also be formulated in multilayer tablets or pills to provide a sustained or provide dual-release products. For example, a dual release tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric materials such as shellac, cellulose acetate, cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, methacrylate and ethylacrylate copolymers, and the like. Sustained release tablets may also be made by film coating or wet granulation using slightly soluble or insoluble substances in solution (which for a wet granulation acts as the binding agents) or low melting solids a molten form (which in a wet granulation may incorporate the active ingredient). These materials include natural and synthetic polymers waxes, hydrogenated oils, fatty acids and alcohols (i.e., beeswax, carnauba wax, cetyl alcohol, cetylstearyl alcohol, and the like), esters of fatty acids metallic soaps, and other acceptable materials that can be used to granulate, coat, entrap or otherwise limit the solubility of an active ingredient to achieve a prolonged or sustained release product.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include, but are not limited to aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable suspending agents for aqueous suspensions, include synthetic and natural gums such as, acacia, agar, alginate (i.e., propylene alginate, sodium alginate, and the like), guar, karaya, locust bean, pectin, tragacanth, and xanthan gum, cellulosics such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose and hydroxypropyl methylcellulose, and combinations thereof, synthetic polymers such as polyvinyl pyrrolidone, carbomer (i.e., carboxypolymethylene), and polyethylene glycol; clays such as bentonite, hectorite, attapulgite or sepiolite; and other pharmaceutically acceptable suspending agents such as lecithin, gelatin, or the like. Suitable surfactants include but are not limited to sodium docusate, sodium lauryl sulfate, polysorbate, octoxynol-9, nonoxynol-10, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polyoxamer 188, polyoxamer 235, and combinations thereof. Suitable deflocculating or dispersing agent include pharmaceutical grade lecithins. Suitable flocculating agent include but are not limited to simple neutral electrolytes (i.e., sodium chloride, potassium, chloride, and the like), highly charged insoluble polymers and polyelectrolyte species, water soluble divalent or trivalent ions (i.e., calcium salts, alums or sulfates, citrates and phosphates (which can be used jointly in formulations as pH buffers and flocculating agents). Suitable preservatives include but are not limited to parabens (i.e., methyl, ethyl, n-propyl and n-butyl), sorbic acid, thimerosal, quaternary ammonium salts, benzyl alcohol, benzoic acid, chlorhexidine gluconate, phenylethanol, and the like. There are many liquid vehicles that may be used in liquid pharmaceutical dosage forms, however, the liquid vehicle that is used in a particular dosage form must be compatible with the suspending agent(s). For example, nonpolar liquid vehicles such as fatty esters and oils liquid vehicles are best used with suspending agents such as low HLB (Hydrophile-Lipophile Balance) surfactants, stearalkonium hectorite, water insoluble resins, water insoluble film forming polymers, and the like. Conversely, polar liquids such as water, alcohols, polyols and glycols are best used with suspending agents such as higher HLB surfactants, clays silicates, gums, water soluble cellulosics, water soluble polymers, and the like. For parenteral administration, sterile suspensions and solutions are desired. Liquid forms useful for parenteral administration include sterile solutions, emulsions and suspensions. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.
Furthermore, extracts and/or compounds of the present invention can be administered in an intranasal dosage form via topical use of suitable intranasal vehicles or via transdermal skin patches, the composition of which are well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the administration of a therapeutic dose will, of course, be continuous rather than intermittent throughout the dosage regimen.
Extracts and/or compounds of the present invention can also be administered in a form suitable for intranasal or inhalation therapy. For such therapy, extracts and/or compounds of the present invention are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped or as an aerosol spray from a pressurized container or a nebulizer (such as, a metered dose inhaler, a dry powder inhaler or other conventional or non-conventional modes or devices for inhalation delivery) using a suitable propellant (such as, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container or nebulizer may contain a solution or suspension of the active compound. Capsules and cartridges (such as, those made from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
Extracts and/or compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, multilamellar vesicles and the like. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, phosphatidylcholines, and the like.
Extracts and/or compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include, but are not limited to polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxy-ethylaspartamidephenol, and polyethyl eneoxidepolylysine substituted with palmitoyl residue. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, to homopolymers and copolymers (which means polymers containing two or more chemically distinguishable repeating units) of lactide (which includes lactic acid d-, l- and meso lactide), glycolide (including glycolic acid), ε-caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one (including its dimer 1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels and blends thereof.
The therapeutically effective amount of extracts and/or compounds or composition thereof may be from about 0.001 mg/kg/dose to about 300 mg/kg/dose. Preferably, the therapeutically effective amount may be from about 0.001 mg/kg/dose to about 100 mg/kg/dose. More preferably, the therapeutically effective amount may be from about 0.001 mg/kg/dose to about 50 mg/kg/dose. Most preferably, the therapeutically effective amount may be from about 0.001 mg/kg/dose to about 30 mg/kg/dose. Therefore, the therapeutically effective amount of the active ingredient contained per dosage unit (e.g., tablet, capsule, powder, injection, suppository, teaspoonful, and the like) as described herein will be in the range of from about 1 mg/day to about 21,000 mg/day for a subject, for example, having an average weight of 70 kg. For oral administration, the compositions are preferably provided in the form of tablets containing, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular compound used, the mode of administration, the strength of the preparation, and the advancement of the disease condition. In addition, factors associated with the particular subject being treated, including subject age, weight, diet and time of administration, will result in the need to adjust the dose to an appropriate therapeutic level. Advantageously, compounds of the present invention may be administered in a single daily dose or the total daily dosage may be administered in divided doses of two, three or four times daily.
Preparation of Flavonoid Rich Fraction from S. flavescens by Methylene Chloride Extraction
Enrichment for flavinoids may be accomplished through any appropriate methods known to those skilled in the art. For example, we first used methylene chloride (MC) extraction to generate less-polar compounds (
A 100 mg/ml MC-extract solution was prepared by dissolving MC extract powder into a mixture of water and acetonitrile (v:v=1:1) containing 0.1% formic acid and loaded into a preparative HPLC system. Separation was performed on a C18 reverse phase preparative column (XBridge™ Prep C18 5 μm OBD™, 19×150 mm, Waters, Milford, Mass.) with distilled water as mobile phase A and acetonitrile containing 0.1% formic acid as mobile phase B at a constant flow rate of 20 ml/min and detected under photodiode array (PDA) detector at wavelength 254 nm. The initial gradient was 30% B for 20 min; increased to 50% in 4 min; increased to 100% B in 2 min; maintained at 100% for another 2 min. Five sub-fractions were collected according to a time based collection method (MC-F1˜5).
Further separation of MC-F1 sub-fractions was performed using preparative HPLC. 19 subfractions were collected, and each fraction was analyzed using analytical HPLC. Those fractions containing individual peaks were dried under vacuum, and their structures determined using NMR and LC-MS. The fractions containing multiple-peaks were saved for further separation.
Isolation and Identification of 3′,7-dihydroxy-4′-methoxy-isoflavone and Trifolirhizin from MC-F1:
Isolation of individual compounds contained in S. Flavescens may be accomplished through any method known to those having skill in the art. For example, the MC-F1 fraction was further fractionated by preparative HPLC and 19 subfractions were collected. Based on analytical HPLC chromatograms subfractions 8 and 11 each contained a single peak, designated MC-F1-8S and MC-F1-11S (purity>90%). The remaining fractions containing either mixtures (not pure) or flow through solvent were combined for further investigation. The structures of these compounds (
LC-MS and NMR Method
Mass spectra data were collected on a micromass LCT premier TOF mass spectrometer (Waters Corporation, Milford, Mass.) coupled with a Waters Alliance 2695 HPLC system in a positive electrospray mode (ESI+). 1H and 13C NMR spectra were obtained on a JOEL instrument (at 300 MHz for 1H NMR and 75 MHz for 13C NMR) using DMSO-d6 as the solvent.
Quantification of the biologically active compounds present in S. flavescens was performed by HPLC. The analyses were carried out with a Waters Alliance 2695 separation module (Waters) equipped with PDA detector. The separation was performed on a Zorbax-C18 (150×4.6 mm) column with mobile phase A (0.1% formic acid) and B (CH3CN) at a flow rate of 1.0 ml/min. The linear gradient from 2% to 46% B over 75 min was used for separation. Samples were dissolved in mobile phase mixture (v:v=1:1). UV wavelength was monitored from 210 nm to 400 nm. Five different concentrations of standard compound (5, 8, 10, 12, and 15 μg/ml) were prepared and analyzed with HPLC and the Empower software (Waters). The calibration curve was generated by using peak area obtained at 254 nm of standard at different concentrations. The R2 was 0.9995.
S. flavescens Inhibited Acetylcholine-Induced ASM Contraction in Tracheal Rings from Asthmatic Mice
To determine which herbal constituents in ASHMI™ have a direct inhibitory effect on ASM contraction, an asthmatic mouse model was induced by ovalbumin (OVA) sensitization and challenge as described previously (Zhang et al., 2010). Tracheal rings from asthmatic mice were treated with an aq. extract of ASHMI™ (100 μg/ml), or individual herb aq. extracts, G. lucidum (35 μg/ml), S. flavescens (45 μg/ml) and G. uralenisis (20 μg/ml), for 30 min prior to ex vivo acetylcholine provocation. The ASHMI™ concentration was based on our previous publication (Zhang et al., 2010). Concentrations of individual herb extracts were equivalent to their individual concentrations in ASHMI™. ASHMI™ treatment significantly reduced acetylcholine-induced ASM contraction in tracheal rings over a range of 10-6 M-10-4 M acetylcholine (
Identification of the S. flavescens Flavonoid Fraction and Subfraction that Inhibit Acetylcholine-Induced ASM Contraction
We first generated a methylene chloride (MC) extract from an aq. extract of S. flavescens to obtain a flavonoid rich extract (designated MC extract,
Isolation and Identification of 3′,7-dihydroxy-4′-methoxy-isoflavone and Trifolirhizin from MC-F1
The MC-F1 fraction was further fractionated by preparative HPLC and 19 subfractions were collected. Based on analytical HPLC chromatograms subfractions 8 and 11 each contained a single peak, designated MC-F1-8S and MC-F1-11S (purity>90%). The remaining fractions containing either mixtures (not pure) or flow through solvent were combined for further investigation. The structures of these compounds (
3′,7-dihydroxy-4′-methoxy-isoflavone (6.0 μg/ml) had no effect on ASM contraction to acetylcholine (
Studies of pharmacological actions of trifolirhizin are limited. It has been shown to inhibit inflammatory responses (Zhou et al., 2009), and melanin synthesis by melanoma cells (Yu et al., 1999). However, it has not previously been evaluated for a potential effect on ASM. Our study is the first demonstration that trifolirhizin inhibits ASM contraction. A previous open label study reported that S. flavescens aq. extract produced remarkable clinical improvement of asthma symptoms, reduction of b2 agonist use, dose of inhaled corticosteroid, and improvement of PEF in refractory asthmatics (Hoang et al., 2007). Hoang et. al. suggested that the two major matrine-type alkaloids, matrine and oxymatrine, may be the primary active components in the herbal mixture responsible for the anti-asthma effect (Hoang et al., 2007). However, S. flavescens contains not only matrine-type alkaloids, it also contains active flavonoids which may contribute other anti-asthmatic effects (Jin et al., 2010).
In another open label study S. flavescens aq. extract was given by inhalation. Wheezing and chest pain diminished, and in some cases disappeared in 10 minutes, and this effect lasted for up to 36 hours (Zhang et al., 2010). Our current study is the first to identify the S. flavescens flavonoid trifolirhizin as an active compound that contributes to S. flavescens inhibition of ASM contraction. Mechanisms of ASM modulation are complex, but mainly involve G protein-coupled receptors (GPCRs) controlling intracellular calcium and the responsiveness of the actin myosin complex. Gs-coupled receptors, including β2-ARs and prostaglandin E2 receptors, primarily mediate relaxation as a consequence of cAMP signaling. Gq and Gi-coupled receptors include muscarinic M3, histamine H1 and leukotriene CysLT1 mediate contraction (Zhang et al., 2007). Activation of Gq-coupled receptors results in activation of phospholipase C, increased intracellular Ca+2 and ASM contraction. Activation of receptors coupled to Gi (such as adenosine A1/A3, and muscarinic M2) leads to inhibition of adenylyl cyclase, and activation of phospholipase C. Aberrant GPCR signaling in ASM may contribute to elevated ASM tone in asthma. Our previous study showed that ASHMI inhibition of ASM contraction is β2-AR independent (Zhang et al., 2010) and that was confirmed for trifolirhizin in the present study where pretreatment with the β2-AR antagonist, ICI 118,551 did not block the effect of trifolirhizin's pretreatment on impairment of acetylcholine in sensitized murine tracheal rings (
The primary therapy for acute constriction of airway smooth muscle is β2-AR agonists which are inhaled for the acute relaxation of hyperresponsive airway smooth muscle. We performed studies with trifolirhizin to determine whether it could relax pre-contracted airway smooth muscle and how the magnitude of relaxation compared to a classic β2-AR agonist fenoterol. In order to investigate the relaxation effect of trifolirhizin on ASM, we tested this isolated compound on acetylcholine pre-contracted sensitized murine tracheal rings. Trifolirhizin relaxed the pre-contracted tracheal rings stimulated by 10-5 M acetylcholine in a dose-dependent manner. The maximal relaxation was 85% of the initial contractile tone at a trifolirhzin concentration of 20 μg/ml (
The amount of bioactive flavonoid trifolirhizin present in the S. flavescens was quantified by HPLC. Pure trifolirhizin at five different concentrations 5.0 to 15.0 μg/ml were analyzed on HPLC. The calibration curves were plotted using the peak areas of each condition and the equation of the peak area and the concentration was generated. The trifolirhizin concentration in the aqueous extract of S. flavescens was approximately 6.7 μg/100 mg. Previous in vitro use of trifolirhizin involved concentrations ranging from 10-20 μM to 100-200 μM in the cultures of mouse J774A.1 macrophages (McGraw et al., 2003), or 50 μM in the cultures of melanoma B16 cells. The trifolirhizin concentration used in our ex vivo study was 6.0 μg/ml (13.4 μM), which is in the lower range of concentrations shown to be active in other systems. Although effective concentrations for ASM were comparable to those previously reported, they reflect higher concentrations than would occur in ASHMI™. Whether this reflects the presence of other ASM-relaxant compounds in ASHMI™, or potentiation of trifolirhizin by other ASHMI™ components, is yet unknown.
ASHMI-Mediated Suppression of Tracheal Contractility is Reproduced by S. flavascens but not G. lucidum or G. uralensis
Murine tracheal rings from OVA-allergic mice were pretreated for 30 minutes with either 100 ug/ml of ASHMI, 35 ug/ml of G. lucidum or 20 ug/ml of G. uralensis. Tracheal ring contraction to Ach was markedly suppressed by ASHMI as expected (P<0.01 to P<0.001 vs None). Of the three herbs tested, only S. flavescens reduced Ach-induced contractility. At 45 μg/ml, S. flavescens was able to reproduce the degree of suppression observed in rings treated with ASHMI. (P<0.01 to P<0.001 vs None). Extracts of G. lucidum or G. uralensis did not alter tracheal ring contractility to the doses of Ach tested.
Dichloromethane Soluble Fractions of S. flavescens Potently Inhibit Tracheal Ring Contractility to Ach
We tested solvent fractions of S. flavescens (SF1-7) for their potential to inhibit tracheal contraction to Ach. As shown in
To verify if indeed the Fraction 1 and 3 (dichloromethane soluble) components were functional in whole ASHMI fractions we also evaluated fractions of ASHMI generated using similar methods used to fractionate S. flavescens. We found that ASHMI fraction 3 (AS3) as observed for SF fraction 3 dramatically inhibited tracheal contractility (
Having observed that Fraction 3 of S. flavescens was a potent inhibitor of Ach-stimulated tracheal contraction we tested major compounds present in this fraction in myography experiments. According to investigations using mass spectrometric techniques, the major compounds identified in SF Fraction 3 were the matrine type alkaloids-Matrine, Oxymatrine and Sophocarpine. The structures of these compounds are shown in
Data from multiple experiments are shown in
Results described in this study shed light on the identity of compounds participating in the direct effects of ASHMI on airway contractility. It appears that anti-contractile responses of ASHMI are restricted to those contained in S. flavescens. Furthermore, only the dichloromethane soluble fractions of both ASHMI and S. flavescens demonstrated inhibitory activity. The basic Dichloromethane soluble fraction (Fraction 3) was of special interest as Fraction 3 of both ASHMI and S. flavescens were the effective at inhibiting contraction. Communications with Dr. LaVerne Brown and Dr. Nan Yang have indicated that Fraction 3 of ASHMI is mostly comprised of compounds from S. flavescens supporting the observation that AS3 and SF3 were similar in their ability to suppress tracheal contractility to Ach. Interestingly while SF1 showed very potent inhibitory effects, the corresponding fraction of ASHMI (AS1) was not as effective. This may likely be attributed to the fact that S. flavescens compounds of fraction 1 display altered extractability or function when fractionated as a part of whole ASHMI. SF1 compounds are being actively investigated by my lab colleague Banghao Liang.
Further focusing on SF3, its major compounds Matrine, Sophocarpine and Oxymatrine were tested by tracheal ring myography and results indicate that these matrine type alkaloids have considerable inhibitory activity on tracheal contractility. This is the first known demonstration of anti-contractile properties of these compounds in the airway. Experiments suggest that, similar to ASHMI, suppressive effects of these alkaloids are also inhibited by Indomethacin.
Matrine, Oxymatrine and Sophocarpine are well known quinolizidine alkaloids found in many Sophora species 3-5. Matrine and Oxymatrine in particular have been investigated in human and animal studies in China. Most studies have focused on the anti-cancer and anti-viral properties of these compounds. Mechanistic studies are largely restricted to anti-cancer research and include regulation of cancer-relevant proteins such as ERK, Bcl-2 family of proteins, telomerase and mitochondrial caspases. Of note, a few publications have noted that matrine and oxymatrine protect from cardiac arrhythmia in rat models 0.13 The mechanism of protection was suggested to be related to beneficial modulation of Ca2+ transience in myocytes. More recent publications suggest that Matrine has anti-inflammatory properties by virtue of inhibition of NFkB signaling. It is yet undetermined if mechanisms proposed in the above mentioned studies have relevance to observation in our experiments. The doses of Matrine or Oxymatrine used for experiments in cancer cell lines are often 25-50 fold higher than the dose of 10 μg/ml used in our study and the effects are usually observed over treatment of several hours if not days. Studies related to anti-inflammatory properties also use similarly high doses. An added difficulty of understanding the research literature about matrine compounds is the lack of availability of English translations of many publications. Currently, there is little research on matrine alkaloids outside of China.
A dried aqueous extract of SF (200 g) was dissolved in water (4 L) and loaded on macroporous resin column (Amberlite* XAD-7 HP, Acros Organics, New Jersey, USA), and sequentially eluted with distilled water, 20% ethanol, 50% ethanol, and 95% ethanol, sequentially. The 4 fractions were concentrated under reduced pressure, to yield SFF1, SFF2, SFF3 and SFF4.
GU-F2 (20 g) was subjected on silica gel column chromatography, eluted with dichloromethane-ethyl acetate-methanol (5:4:0), dichloromethane-ethyl acetate-methanol (5:4:0.5), dichloromethane-ethyl acetate-methanol (5:4:1), dichloromethane-ethyl acetate-methanol (5:4:2), dichloromethane-ethyl acetate-methanol (5:4:4), with 0.5% ammonium hydroxide (30%), sequentially. Elute were collected each 300 ml, on the basis of TLC profiles, fractions were combined with one purified compound or 2-3 major compounds. SFF2-15 and SFF2-17 were labeled as PC-2A and PC2B, sequentially. PC2 contains single peak.
Balb/c mice were sensitized by intraperitoneal (i.p.) injection of 200 μg OVA plus 2 mg of aluminum hydroxide (alum) in 400 μl of PBS on days 0 and 7. Mice were challenged intratracheally (i.t.) with 100 μg of OVA in 50 μl of PBS weekly for 2 weeks (days 14 and 21). Mice were challenged i.t. once daily with 100 μg of OVA (50 μl) on 2 consecutive days (days 28 and 29) 48 hr before sacrifice and tissue harvesting.
Forty-eight hrs after the last i.t. OVA challenge, mice were sacrificed and tracheas were excised and cut into 3 tracheal rings as previously described. Each tracheal ring was attached to a pair of mounting pins in the organ chamber of a Myograph System Model 610M system (AD Instruments, Denver, Colo.) and bathed in a chamber containing physiological salt solution (PSS (In g/L—NaCl: 6.95, KCl: 0.35, MgSO4.7H2O:0.28, KH2PO4: 0.1561, CaCl2.2H2O: 0.36, NaHCO3: 2.1, EDTA: 0.01, Glucose: 1.09, pH=7.4). All chambers were maintained at 37° C. and were continuously bubbled with 100% 02. The change of tracheal ring tension was recorded by Power Lab Chart recording software (AD Instruments). After incubation at 37° C. for 15 min at baseline tension, passive tension applied to the tracheal ring was adjusted to 1.00 mN. Following stabilization of passive tension, tracheal rings were contracted using 60 mM KCl twice, to assess ASM contractility. Tracheal rings were washed with PSS buffer 3 times and allowed to rest at 1.00 mN for 30 min after each stimulation. The individual compounds as indicated in Supplement
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
All references cited herein, including all patents, published patent applications, and published scientific articles, are incorporated by reference in their entireties for all purposes.
This application is the National Stage of International Application No. PCT/US14/12306 filed on Jan. 21, 2014 which claimed priority from provisional application No. 61/754,831 filed on Jan. 21, 2013.
The research leading to the present invention was supported, in part, by National Institutes of Health grant P01 AT002647 to XM Li.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/12306 | 1/21/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61754831 | Jan 2013 | US |
