Bioactive phytochemicals

Abstract

The present invention relates to bioactive phytochemicals. Specifically, the present invention describes a method for producing a composition comprising (2R, 3R, 4R, 5S)-3, 4, 5-trihydroxypiperidine-2-carboxylic acid (idoBR1), the method comprising the steps of: (a) providing a plant material from a plant source including a plant of the family Cucurbitaceae; (b) fractionating the plant material to produce an extract rich in idoBR1 (idoBR1); (c) determining the following activities of the extract: (i) inhibitory activity against sialidase or TNF-alpha; or (ii) an IL-10 stimulating activity; and (d) formulating the assayed extract with a cosmetic, nutraceutical or pharmaceutically acceptable excipient or carrier to produce a cosmetic, nutraceutical or pharmaceutical composition.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980098777.7, entitled “Bioactive Phytochemicals”, filed on July 26, 2019. Technical Field

[0002] The present invention relates to a method for producing a composition comprising (2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid (idoBR1), as well as various products, compounds, compositions, medical uses and methods based on the composition, and their use in the preparation of various compositions for medicine, including the treatment of inflammation, infection, skin disorders, and in vivo inhibition of sialidase activity; and methods for isolating and purifying the composition from various plant sources.

[0003] The present invention also relates to a method for monitoring the quality of a Cucurbitaceae extract (e.g., a Cucumis extract), a method for producing a Cucurbitaceae extract, and a Cucurbitaceae extract (especially a Cucumis extract) obtainable by such a method. Background Art

[0004] Cucumber (Cucumis sativus) is a widely cultivated plant in the Cucurbitaceae family, which includes the zucchini. Cucumbers originated in India and have been cultivated in western Asia for at least 3,000 years, probably introduced to other parts of Europe by the Romans. Records of cucumber cultivation appear in France in the 9th century, in England in the 14th century, and in North America in the mid-16th century.

[0005] Cucumbers and cucumber extracts have long been known to have anti-inflammatory properties and have been used topically for various types of skin problems, including eye swelling and sunburn. Cucumbers were extremely popular in the ancient civilizations of Egypt, Greece, and Rome, where they were used not only as food but also for their skin healing properties.

[0006] Iminosugar acids (ISAs) constitute a subclass of a more widely distributed class of phytochemicals called iminosugars. Many known ISAs are phytochemicals that exist as secondary metabolites in plant tissues (where they can play a defensive role). Although iminosugars are widely distributed in plants (Watson, A. et al., 2001, Phytochemistry 56, 265), iminosugar acids are much less widely distributed and more difficult to isolate and identify (Martinez, R. et al., 2019, Amino acids 51, 991).

[0007] The iminosugar acid idoBR1 is present in older cucumber varieties but is absent in some modern commercial varieties. It has been shown to be a major component of some cucumber fruits and is the only iminosugar acid in cucumbers. It is a minor component in some squashes and gourds.

[0008] WO2013 / 054070 identified idoBR1 as an important bioactive ingredient in an anti-inflammatory herbal medicine based on cucumber extract.

[0009] Herbal food additives and medicines

[0010] There is currently a great deal of interest in the use of herbal remedies and supplements, with food manufacturers, healthcare companies and medical professionals increasingly accepting that herbal products have value and can complement established preparations and treatments. Herbal food additives and supplements are now widely available.

[0011] However, due to the complexity and inherent heterogeneity of plant materials, quality control of herbal food additives is difficult. The materials used in herbal and plant-based food additives are usually whole plants or parts or extracts thereof. Since plant materials contain many different chemical components, these materials are complex mixtures. This makes it difficult to standardize and control the quality of the material. In addition, many herbal food additives are mixtures of two or more plant-based components, and are mixtures of mixtures, thus introducing a further level of complexity. In addition, the formulas and production methods used are usually not uniform and may still not be disclosed. These factors make it difficult to ensure that two samples of a given product obtained from different sources and seemingly identical actually contain the same mixture of ingredients. This problem leads to difficulty in controlling the quality of such materials and even limits the use of certain herbal extracts by herbal practitioners.

[0012] Other problems arise from the fact that plants used in herbal practices or as food supplements / nutraceuticals are often not locally available and therefore need to be obtained from sources far from the end user. However, the supply of such plants from distant areas can be spotty and inaccurate, especially since many of these plants do not have detailed monographs including identity and quality standards. The complex mixture of ingredients found in medicinal plants varies greatly in type and concentration, depending on many factors, including the plant's source, where the plant grows, other plants or microorganisms growing near the plant, the time of year the plant is harvested, the conditions under which the material is stored and processed, and the extraction procedures used.

[0013] Therefore, there is a need for sensitive methods that can profile herbal products containing idoBR1 and thereby establish standard specifications for plant-derived products that may be associated with activity, thus allowing quality control in the production of herbal medicines, food additives, cosmetics, nutraceuticals and food supplements, and ideally quantify (structurally and / or functionally) the bioactive components.

[0014] The present inventors have now discovered that idoBR1 exhibits inhibitory activity against sialidase and TNF-α, while exhibiting IL-10 stimulatory activity. This discovery allows the development of improved methods for formulating cosmetic, nutraceutical or pharmaceutical compositions based on extracts of plant materials from botanical sources including plants of the Cucurbitaceae family, since functional assays of the relevant bioactive component idoBR1 can now be performed quickly and easily after fractionation of the plant material.

[0015] Thus, the provision of cosmetic, nutraceutical or pharmaceutical compositions that comply with standard specifications is greatly facilitated: the invention allows for rapid determination of the functional qualities of an extract. It also makes time-consuming and expensive physical characterization (e.g. by GC-MS and / or HPLC) optional. Furthermore, since the functionality of the extract has been determined, the influence of possible interferences / inhibitions of co-extracted parts can be monitored. This may be particularly important in the case of cosmetic applications in which the extract is formulated for topical application. Summary of the invention

[0016] Therefore, according to the present invention, there is provided a method for producing a composition comprising (2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid (idoBR1), the method comprising the following steps:

[0017] (a) providing plant material from a plant source comprising plants of the Cucurbitaceae family;

[0018] (b) fractionating the plant material to produce an extract enriched in idoBR1;

[0019] (c) determining the following activities of the extract: (i) inhibitory activity against sialidase or TNF-α; or (ii) IL-10 stimulating activity; and

[0020] (d) formulating the assayed extract with a cosmetically, nutraceutically or pharmaceutically acceptable excipient or carrier to produce a cosmetic, nutraceutical or pharmaceutical composition.

[0021] The plant source may include plants of the genus Cucumis or Cucurbita. A preferred species of the genus Cucumis is a plant of the species Cucumissativus (cucumber). A preferred species of the genus Cucurbita is a plant of the species Cucurbita melos or Cucurbita moschata. DETAILED DESCRIPTION

[0022] All publications, patents, patent applications, and other references mentioned herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference and was set forth in its entirety.

[0023] definition

[0024] As used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meaning that such term may have in the art:

[0025] Unless the context requires otherwise, the use of the singular herein shall be understood to include the plural, and vice versa. The term "a" or "an" used with respect to an entity shall be understood to refer to one or more of that entity. Likewise, the term "a / an", the term "one or more", and "at least one" may be used interchangeably herein.

[0026] As used herein, the term "comprise" or variations such as "include" or "comprising" should be understood to indicate the inclusion of any recited integers (e.g., features, elements, characteristics, properties, methods / method steps, or limitations) or groups of integers (e.g., features, elements, characteristics, properties, methods / method steps, or limitations), but not the exclusion of any other integers or groups of integers. Thus, as used herein, the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or methods / method steps.

[0027] The phrase "consisting essentially of" is used herein to claim one or more specified integers or steps and those integers or steps that do not materially affect the characteristics or functions of the claimed invention.

[0028] As used herein, the term "consisting of" is used to indicate that only the recited integers (e.g., features, elements, characteristics, properties, methods / method steps, or limitations) or groups of integers (e.g., features, elements, characteristics, properties, methods / method steps, or limitations) are present.

[0029] As used herein, the term "disease" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to cover any disorder, disease, abnormality, pathology, illness, condition or syndrome in which physiological function is impaired, regardless of the etiological nature (or whether the etiological basis of the disease is well established). Thus, it covers conditions caused by infection, trauma, injury, surgery, radiation ablation, poisoning or nutritional deficiency.

[0030] As used herein, the term "treatment" or "treating" refers to an intervention (e.g., administering an agent to a subject) that cures, improves or alleviates disease symptoms or eliminates one or more causes (e.g., pathologically diverse states) thereof (or alleviates the effects of one or more causes thereof). In this case, the term is used synonymously with the term "therapy".

[0031] In addition, the term "treatment" or "treating" refers to an intervention (e.g., administering an agent to a subject) that prevents or delays the onset or development of a disease or reduces (or eliminates) its incidence in a treated population. In this case, the term treatment is used synonymously with the term "prevention".

[0032] The term "subject" (which will be understood to include "individual," "animal," "patient," or "mammal" where the context permits) defines any subject in need of treatment, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, livestock, farm animals, zoo animals, sports animals, and pets. In a preferred embodiment, the subject is a human.

[0033] References herein to treatment of diabetes should be interpreted as including treatment of type 1 and type 2 diabetes itself as well as prediabetes (early diabetes) and insulin resistance. The term "prediabetes" or "early diabetes" defines the presence of elevated glucose or glycosylated hemoglobin levels in the absence of diabetes.

[0034] As used herein, the effective amount of a compound or composition defines an amount that can be administered to a subject without excessive toxicity, irritation, allergy or other problems or complications, commensurate with a reasonable benefit / risk ratio, but sufficient to provide the desired effect, such as treatment or prevention manifested by permanent or temporary improvement of the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, the mode of administration and other factors. Therefore, although it is impossible to specify an exact effective amount, those skilled in the art will be able to determine the appropriate "effective" amount in any individual case using routine experiments and background common sense. Therapeutic outcomes in this regard include eradication or alleviation of symptoms, alleviation of pain or discomfort, prolonged survival, increased mobility and other markers of clinical improvement. Therapeutic outcomes do not need to be completely cured.

[0035] The term phytochemical is used herein in a broad sense to encompass any chemical constituent of a plant, including macromolecules and small molecules. Important examples include alkaloids (e.g., iminosugars and iminosugar acids, e.g., selected from the structural classes pyrrolidine, piperidine, pyrrolizidine, indolizidine, tropane and nortropane), carbohydrate analogs, phenolic compounds, terpenoids, enzyme inhibitors, glycosides, nucleotides, amino acids, lipids and sugars.

[0036] The term "isolated" as applied to the compounds of the present invention is used herein to indicate that the compound is present in a physical environment that is different from the physical environment in which it exists in nature. For example, an isolated compound can be substantially separated (e.g., enriched or purified) relative to the complex cellular environment in which it naturally occurs. Thus, an isolated compound can take the form of an enriched fraction or extract of any of the plant sources described herein.

[0037] When the isolated material is enriched or purified, the absolute level of enrichment or purity is not critical and one skilled in the art can readily determine appropriate levels depending on the intended use of the material. Purity levels of at least 0.1% w / w, 0.2% w / w, 0.3% w / w, 0.4% w / w, 0.5% w / w, 0.6% w / w, 0.7% w / w, 0.8% w / w, 0.9% w / w, 1.0% w / w, 1.1% w / w, 1.2% w / w, 1.3% w / w, 1.4% w / w, 1.5% w / w, 1.6% w / w, 1.7% w / w, 1.8% w / w, 1.9% w / w or 2.0% w / w are preferred.

[0038] A purity level of at least 0.5-2.0% w / w, such as at least 0.8-1.5% w / w, for example at least about 1.0% w / w is particularly preferred. Where the material is isolated from a natural source, a level of 5-10% w / w may be readily obtained, if necessary, by use of appropriate enrichment techniques such as ion exchange chromatography.

[0039] In some cases, the isolated compound forms part of a composition (e.g., a crude extract containing various degrees of many other substances) or a buffer system that may, for example, contain other components. In other cases, the isolated compound may be purified to substantially homogeneity, e.g., as determined by spectrophotometry, by NMR, or by chromatography (e.g., GC-MS of trimethylsilyl derivatives).

[0040] The term herbal medicine is used herein to define a pharmaceutical composition in which at least one active ingredient (e.g., the compound) is not chemically synthesized and is a phytochemical of a plant. In most cases, this non-synthetic active ingredient is not isolated (as defined herein), but exists together with other phytochemicals associated with it in the source plant. However, in some cases, one or more plant-derived bioactive ingredients may be concentrated fractions or separated (sometimes involving high degrees of purification). However, in many cases, the herbal medicine comprises different degrees of crude extracts, infusions, or fractions of the plant, or even unprocessed whole plants (or parts thereof), but in such cases, the plant (or plant part) is usually at least dried and / or crushed. The herbal medicine can be in the form of a food supplement, a food additive, a nutraceutical, a beverage, or provided as a herbal medicine box or package in a single dose.

[0041] The term herbal food is used to define such compositions in this article, wherein at least one component is not chemically synthesized, but is a phytochemical component of a plant. In most cases, this non-synthetic component is not purified, but exists together with other phytochemicals associated with it in the source plant. However, in some cases, one or more plant-derived components may be concentrated fractions or separations (sometimes reaching high purity). However, in many cases, herbal food additives include different degrees of crude extracts, infusions or fractions of plants, or even unprocessed whole plants (or parts thereof), but in such cases, plants (or plant parts) are usually at least dried and / or crushed. Therefore, the term includes herbal foods in the form of additives and supplements used with food and beverages.

[0042] The term bioactive ingredient is used herein to define phytochemicals which are necessary or sufficient for the efficacy of the herbal medicine in which they are contained. In the context of the present invention, the bioactive ingredient comprises idoBR1.

[0043] The term nutraceutical is used herein to define a food product (or isolate thereof) that provides a physiological benefit or prevents disease.Preferred nutraceuticals of the present invention are anti-inflammatory.

[0044] The term standard specification is used herein to define the properties or phytochemical characteristics associated with the acceptable quality of a herbal, cosmetic or nutraceutical product. In this case, the term quality is used to define the overall suitability of the product for its intended use and includes the activity of ido BR1 at an appropriate concentration.

[0045] The term phytochemical profile is used herein to define a group of characteristics associated with different phytochemical constituents.

[0046] Functional assays

[0047] The extracts of the invention are assayed for the following activities: (i) inhibitory activity against sialidase or TNF-α; or (ii) IL-10 stimulating activity. Functional assays may include bioassays. Bioassays may be performed in vivo or in vitro and may include enzyme inhibition assays (e.g., sialidase inhibition). Other bioassays include receptor binding assays, cell assays (including cell replication, cell-pathogen and cell-cell interactions, and cell secretion assays), immunoassays, antimicrobial activity (e.g., bacterial and viral cell binding and / or replication) assays, and toxicity assays (e.g., LD 50 determination).

[0048] Functional characterization can also be performed indirectly through characterization that allows identification of one or more indicators of biological activity.

[0049] Exemplary techniques are described in more detail below.

[0050] Sialidase

[0051] Inhibition of sialidase (neuraminidase) activity by idoBR1 or extracts containing it can be determined by an enzymatic assay in which neuraminidase activity is measured using, for example, an enzyme from Clostridium perfringens (Sigma-Aldrich). The assay is based on an enzyme that cleaves the 2'-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid (MUNANA) substrate, resulting in the release of the fluorescent product 4-methylumbelliferone (4-MU). Thus, inhibition is based on the concentration of idoBR1 or extract required to reduce the enzyme activity by 50% (to derive the IC 50 value) to determine.

[0052] A suitable method may be as follows:

[0053] 1. Prepare the reaction mixture: sodium phosphate buffer (pH 4.5), 10 mmol MUNANA4MU-NeuAc (100 μl in buffer), 100 μl of 0.1 mg enzyme, 0.1 ml of plant extract or 100 μl of compound

[0054] 2. Incubate at 37°C for 10-30 minutes.

[0055] 3. The reaction was terminated by adding 1.25 ml of 0.25 M glycine-NaOH (pH 10.4).

[0056] 4. The released 4-methylumbelliferone (4-MU) was measured by fluorescence (emission wavelength 448 nm, excitation wavelength 365 nm).

[0057] TNF-α and IL-10

[0058] The reduced TNF-α and increased IL-10 caused by the extracts can be measured in cell culture (eg, THP-1 monocytes) or whole blood samples using ELISA.

[0059] THP-1 cells are commercially available. The cultured cells can be placed in RPMI complete medium in a microtiter plate (e.g., placed in a 96-well plate at 5 cells per well), and after incubation for 24 hours, PMA (10 ng / ml) is added to a 96-well plate to differentiate THP-1 cells and determine the impact on TNF-α and IL-10 output. The cells should be pre-treated with, for example, 200 μg / ml to 25 μg / ml of cucumber extract, followed by 2 hr LPS (100 ng / ml) stimulation. After incubation, the cell supernatant is aspirated from each well into a sterile microcentrifuge tube, and centrifuged at 1000 rpm for 2-3 minutes to precipitate any cell (if present). Then, the cell supernatant is used to evaluate the presence of TNF-α or IL-10 using ELISA. Sandwich ELISA plates coated with suitable antibodies can be widely obtained (e.g., R&D Systems, USA).

[0060] For whole blood measurements, aliquots (800 μl) of whole blood can be incubated with extracts dissolved in RPMI 1640 for a 48-hour pre-incubation period, followed by the addition of LPS (10 μg / ml) and a further incubation of 20 hours at 37° C. in a humidified (100%) atmosphere of 5% CO2 in air. At the end of the incubation period, the supernatant consisting of plasma is collected by centrifugation at 10,000 g for 30 seconds at room temperature, and TNF-α and IL-10 levels are measured using human TNF-α and IL-10 ELISA assays (kits available from, e.g., BioSource Europe SA, Belgium).

[0061] Physical characterization

[0062] The extracts of the invention may also be physically characterised (although this is not necessary). This may take the form of quantification of the phytochemical component(s) present in any given fraction or at any other stage in the process, measurement of the purity of the components, determination of molecular weight (or molecular weight distribution, or in the case of fractions containing a variety of different phytochemical components, various statistical functions of molecular weight distribution), determination of molecular formula (e) (e.g. by nuclear magnetic resonance) and various spectroscopic analyses.

[0063] Particularly useful spectral properties include:

[0064] Mass spectra (e.g., mass-to-charge ratio (m / z) values ​​and abundance), and / or

[0065] Chromatographic data (e.g. spectra, column retention times, elution curves, etc.), and / or

[0066] Photodiode array (PDA) spectroscopy (e.g. in the UV and visible range), and / or

[0067] Electrochemical detection (ED) or evaporative light scattering (ELSD) detection; and / or

[0068] Nuclear magnetic resonance (NMR) spectroscopy (including 1 H and / or 13 C NMR spectral data set).

[0069] Spectroscopic characterization can be combined with the fractionation step. For example, GC-MS and HPLC-PDA-MS-ED-ELSD can be used (as described herein) to combine fractionation with the acquisition of mass spectrometry, UV-visible spectroscopy, electrochemical response or fraction mass data and chromatographic spectral data.

[0070] Any or all of the above characteristics may be used to define a "chemical fingerprint" for any given sample (or any fraction or phytochemical constituent thereof).

[0071] Chemical characterization

[0072] The extracts of the invention may also be chemically characterised (although this is not necessary). This may take the form of measurements of, inter alia, the chemical reactivity of one or more phytochemical constituents, their solubility, stability and melting point.

[0073] Medical uses of the compounds of the present invention

[0074] Neoplasia

[0075] The compounds of the present invention are sialidase inhibitors and are therefore useful in the treatment or prevention of diseases and disorders mediated by sialidase activity and / or sialic acid.

[0076] Sialidases are involved in a variety of pathological processes including bacterial and viral infections and neoplasia, making these enzymes attractive therapeutic targets. The expression of sialidases Neu1 and Neu3 appears to be altered in diabetes (e.g., Neu1 activity discussed by Natori, Y. et al., 2013, Biol. Pharm., Bull., 36, 1027). Sialidases are also involved in atherogenesis (Sukhorukov, VN et al., 2017, Curr. Pharm. Des., 23, 4696) and osteoarthritis (Katoh, S. et al., 1999, J Immunol., 162, 5058).

[0077] Thus, the compounds of the invention are useful in the treatment or prevention of neoplastic / proliferative disorders as described in more detail below.

[0078] As used herein, the term "neoplasia" is used in a strict sense to define diseases involving abnormal proliferation of neoplastic cells. The term includes benign, precancerous and malignant neoplasias (as defined above) and is used synonymously with the term "proliferative disorder".

[0079] Neoplasia is caused by inappropriately high levels of cell division and / or low levels of apoptosis or senescence of neoplastic cells that have acquired genetic or epigenetic changes that free them from normal physiological control (i.e., the cells have been "transformed"). Neoplasia typically results in a structure called a neoplasm: an abnormal mass of tissue that grows in excess of and out of coordination with that of normal tissue and persists in the same excessive manner after the stimulus that caused the change has ceased. While most neoplasms form large tissue masses (solid tumors), some neoplasms do not form such discrete tissue masses. These include cervical intraepithelial neoplasia, anal intraepithelial neoplasia, and leukemia.

[0080] Neoplasias can be benign, potentially malignant, or malignant. Benign neoplasias include uterine fibroids and melanocytic nevi (skin moles), which are not invasive and do not transform or progress into malignant neoplasms. Potentially malignant (precancerous) neoplasms include carcinoma in situ, which are not invasive but can transform into malignant neoplasms over time.

[0081] Malignant neoplasia gives rise to neoplasms (tumors) that invade and destroy surrounding tissue, may form metastases and ultimately kill the host. The terms "malignant neoplasia" and "cancer" are used synonymously herein.

[0082] The terms "proliferative disorder" and "neoplasia" are used synonymously herein to define a class of diseases involving the pathological growth of cells in the body.

[0083] Thus, proliferative disorders include cancer, cancer metastasis, smooth muscle cell hyperplasia, systemic sclerosis, cirrhosis, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy (e.g., diabetic retinopathy), cardiac hyperplasia, benign prostatic hyperplasia, ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, hamartoma, lymphangiomatosis, sarcoidosis, and desmoid tumors. Neoplasia involving smooth muscle cell hyperplasia includes excessive proliferation of cells in the vasculature (e.g., intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion, particularly including stenosis after biological or mechanically mediated vascular injury (e.g., angioplasty)). In addition, intimal smooth muscle cell hyperplasia can include hyperplasia in smooth muscle other than the vasculature (e.g., obstruction in the bile ducts, bronchial airways, and kidneys of patients with renal interstitial fibrosis). Noncancerous proliferative disorders also include hyperproliferation of cells in the skin, such as psoriasis and its varying clinical forms, Reiter's syndrome, pityriasis rubra pilaris, and hyperproliferative variants of keratotic disorders including actinic keratosis, senile keratosis, and scleroderma.

[0084] The term "neoplasia" is also used broadly herein to define diseases involving abnormal growth and / or differentiation of cells in the body, thus including hyperplasia, metaplasia, and dysplasia.

[0085] Hyperplasia defines a condition in which normal (untransformed) cells in an organ or tissue proliferate to an abnormal degree. Therefore, it may cause an organ to be significantly enlarged, benign tumors to form, or may only be visible under a microscope. Hyperplasia is a physiological response to a specific stimulus, and the proliferating cells are still subject to normal regulatory control mechanisms (different from the tumor formation growth in which cells proliferate in an abnormal manner (which is unresponsive to normal physiological control)). Examples include congenital adrenal hyperplasia, endometrial hyperplasia, benign prostatic hyperplasia (prostatic hypertrophy), breast hyperplasia (including ductal hyperplasia), focal epithelial hyperplasia (Heck's disease), sebaceous hyperplasia, and liver hyperplasia.

[0086] Metaplasia defines a condition in which cells of one mature, differentiated type are replaced by cells of another mature, differentiated type. Examples include squamous metaplasia of the columnar epithelium of the salivary gland ducts (when stones are present), squamous metaplasia of the transitional epithelium of the bladder (again, when stones are present or associated with infection), esophageal glandular metaplasia in patients with gastric acid reflux (Barthel's esophagus), and bone metaplasia in connective tissue.

[0087] Dysplasia defines a condition characterized by abnormal maturation of cells within a tissue. This generally consists of an expansion of immature cells with a corresponding decrease in the number and location of mature cells. For example, cervical epithelial dysplasia is characterized by an increase in the population of immature cells confined to the mucosal surface. Myelodysplastic syndrome or hematopoietic dysplasia is characterized by an increase in the number of immature cells in the bone marrow and a decrease in mature, functional cells in the blood. Other examples include neurofibromatosis.

[0088] Hyperplasia, metaplasia and dysplasia are generally reversible conditions, the result of a stimulus such as damage or injury. In contrast, neoplasia is generally irreversible and is associated with cellular transformation.

[0089] The compounds of the present invention are broadly useful in the treatment of any neoplasia including proliferative disorders, benign, precancerous and malignant neoplasias, hyperplasias, metaplasias and dysplasias.

[0090] Thus, the present invention can be applied to treat proliferative disorders including, but not limited to, cancer, cancer metastasis, smooth muscle cell proliferation, systemic sclerosis, cirrhosis, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy (e.g., diabetic retinopathy), cardiac hyperplasia, benign prostatic hyperplasia, ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, hamartoma, lymphangiomatosis, sarcoidosis, and desmoid tumors. Neoplasia involving smooth muscle cell proliferation includes excessive proliferation of cells in the vasculature (e.g., intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion, particularly including stenosis following biological or mechanically mediated vascular injury (e.g., angioplasty)). In addition, intimal smooth muscle cell hyperplasia can include hyperplasia in smooth muscle other than the vasculature (e.g., obstruction in the bile ducts, bronchial airways, and kidneys of patients with renal interstitial fibrosis). Noncancerous proliferative disorders also include hyperproliferation of cells in the skin, such as psoriasis and its varying clinical forms, Reiter's syndrome, pityriasis rubra pilaris, and hyperproliferative variants of keratotic disorders including actinic keratosis, senile keratosis, and scleroderma.

[0091] Particularly preferred is the treatment of malignant neoplasia (cancer). The present invention is applicable to the treatment of any cancer, including cancers selected from the following major categories: (a) carcinoma; (b) blastoma; (c) leukemia; (d) lymphoma; (e) myeloma; (f) sarcoma and (g) mixed cancers.

[0092] Cancer refers to a malignant neoplasm of epithelial origin or a cancer of the inner or outer lining of the body. Cancer, a malignant tumor of epithelial tissue, accounts for 80% to 90% of all cancer cases. Epithelial tissue is found throughout the body. It is found in the skin and in the covering and lining of organs and internal passages such as the gastrointestinal tract. In a preferred embodiment, the cancer treated according to the present invention is selected from the following cancers: salivary gland cancer, colon cancer, rectal cancer, appendix cancer, lung cancer, thymus cancer, breast cancer, cervical cancer, bladder cancer and eye cancer.

[0093] The present invention can be applied to the treatment of all blastomas, including hepatoblastoma (e.g., Wilms' tumor, non-epithelial renal tumor, rhabdoid renal tumor, renal sarcoma and renal pPNET), medulloblastoma, pancreatic blastoma, pulmonary blastoma, pleuropulmonary blastoma, neuroblastoma (including general peripheral nerve cell tumors as well as ganglioneuroma and retinoblastoma).

[0094] The present invention can be applied to the treatment of all leukemias, myeloproliferative diseases and myelodysplastic diseases, including: lymphoid leukemias (e.g., precursor cell leukemias, mature B-cell leukemias, mature T-cell leukemias and NK-cell leukemias); acute myeloid leukemias; chronic myeloproliferative diseases; myelodysplastic syndromes and other myeloproliferative diseases. Therefore, the present invention can be applied to the treatment of various leukemias, including lymphoid leukemias, lymphocytic leukemias or lymphoblastic leukemias (malignant tumors of the lymphoid and lymphocytic blood cell series) and polycythemia vera or polycythemia vera (malignant tumors of various blood cell products but mainly red blood cells).

[0095] Lymphomas develop in the glands or nodes of the lymphatic system, which is a network of blood vessels, lymph nodes, and organs (particularly the spleen, tonsils, and thymus) that purify body fluids and produce white blood cells or lymphocytes that fight infection. Unlike leukemias, which are sometimes called "liquid cancers," lymphomas are "solid cancers." Lymphomas may also develop in specific organs, such as the stomach, breasts, or brain. These lymphomas are called extranodal lymphomas. Lymphomas are subdivided into two categories: Hodgkin lymphoma and non-Hodgkin lymphoma. The presence of Reed-Sternberg cells in Hodgkin lymphoma distinguishes Hodgkin lymphoma from non-Hodgkin lymphoma diagnostically. The present invention is applicable to the treatment of all such lymphomas and reticuloretinal neoplasms, including: (a) Hodgkin's lymphoma; (b) non-Hodgkin's lymphomas (e.g., precursor cell lymphoma, mature B-cell lymphoma, mature T-cell lymphoma, and NK-cell lymphoma; (c) Burkitt's lymphoma; and (d) other lymphoreticular neoplasms, including mantle cell lymphoma.

[0096] Thus, the present invention is applicable to the treatment of a wide range of lymphomas including, for example, tumors of the glands or nodes of the lymphatic system (including the spleen, tonsils and thymus) as well as extranodal lymphomas of the stomach, breast and brain.

[0097] Myeloma is a cancer originating from bone marrow plasma cells. Therefore, the present invention can be applied to treat hematopoietic tumors and hematological malignancies, including hematopoietic tumors and hematological malignancies of lymphoid lineage (e.g. leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B cell lymphoma (such as diffuse large B cell lymphoma), T cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma and Burkitt lymphoma), and hematopoietic tumors of myeloid lineage (e.g. acute myeloid leukemia, chronic myeloid leukemia, myeloid leukemia and imatinib-sensitive and refractory chronic myeloid leukemia, myelodysplastic syndrome, bortezomib-sensitive and refractory multiple myeloma, myeloproliferative disease or promyelocytic leukemia and thyroid follicular carcinoma).

[0098] The present invention can be applied to treat all sarcomas. Sarcomas refer to cancers that originate from supportive and connective tissues (such as bones, tendons, cartilage, muscles, and fat). The most common sarcomas generally occur in young adults and often manifest as painful lumps on bones. Sarcoma tumors are usually similar to the tissues in which they grow. Exemplary sarcomas for treatment according to the present invention include osteosarcoma (or osteogenic sarcoma), chondrosarcoma, leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelioma or mesothelioma (membrane lining of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels); liposarcoma, glioma, astrocytoma, myxosarcoma (primitive embryonic connective tissue) and mesenchymal or mixed mesodermal tumors (mixed connective tissue type). Fibrosarcomas include peripheral nerve sheath tumors and other fibrous neoplasms, such as fibroblastic and myofibroblastic tumors, nerve sheath tumors and other fibromatous neoplasms. Kaposi's sarcoma is also included. Also included are soft tissue sarcomas such as Ewing and Askin tumors of soft tissue, pPNETs of soft tissue, extrarenal rhabdomyomas; fibrohistiocytic tumors; synovial sarcomas; bony and chondromatous neoplasms of soft tissue and alveolar soft tissue sarcomas. Osteosarcoma (malignant bone tumor) includes: malignant fibrous neoplasms of bone; malignant chordomas and odontogenic malignancies. Gliomas include oligodendrogliomas, mixed and unspecified gliomas, and neuroepithelial gliomas.

[0099] The present invention can be applied to treat mixed cancers, including, for example, adenosquamous carcinomas, mixed mesodermal tumors, carcinosarcoma and teratocarcinomas. Therefore, the present invention can be applied to treat various CNS, PNS and mixed intracranial and intraspinal neoplasms, including: astrocytomas, neuroblastomas, gliomas, Schwann cell tumors, ependymomas and choroid plexus tumors (e.g., ependymomas and choroid plexus tumors); intracranial and intraspinal embryonal tumors (e.g., medulloblastomas, primitive neuroectodermal tumors (PNET), medulloepithelioma, atypical teratoid / rhabdoid tumors and other intracranial and intraspinal neoplasms (e.g., pituitary adenomas and carcinomas, sellar tumors (craniopharyngiomas), pineal parenchymal tumors, neurons and mixed neuron-glial tumors, meningiomas and general intracranial and intraspinal neoplasms).

[0100] Therefore, the present invention can be particularly applied to the treatment of: intracranial and intraspinal germ cell tumors; intracranial and intraspinal germ cell tumors; intracranial and intraspinal teratomas; intracranial and intraspinal embryonal carcinomas; intracranial and intraspinal yolk sac tumors; intracranial and intraspinal choriocarcinomas and mixed forms of intracranial and intraspinal tumors.

[0101] The present invention can also be applied to treat various germ cell tumors, trophoblastic tumors and gonadal neoplasms. Therefore, the present invention can be applied to treat malignant extracranial and extragonadal germ cell tumors, including, for example, malignant germ cell tumors of extracranial and extragonadal sites, malignant teratomas of extracranial and extragonadal sites, embryonal carcinomas of extracranial and extragonadal sites, yolk sac tumors of extracranial and extragonadal sites; choriocarcinomas of extracranial and extragonadal sites and general malignant mixed germ cell tumors of extracranial and extragonadal sites. The present invention can also be applied to treat malignant gonadal germ cell tumors, including, for example, malignant gonadal germ cell tumors, seminoma, malignant gonadal teratoma, gonadal embryonal carcinoma, gonadal yolk sac tumor, gonadal choriocarcinoma, mixed forms of malignant gonadal tumors and malignant gonadal gonadoblastoma.

[0102] Infectious Diseases

[0103] The compounds of the present invention are sialidase inhibitors and are therefore useful in treating or preventing diseases and conditions mediated by sialidase activity and / or sialic acid. Such diseases and conditions include infectious diseases (including bacterial and viral infections).

[0104] The compounds of the present invention may have anti-infection (e.g., pathogen inhibition or pathogen killing) activity against any infectious agent. Therefore, the compounds of the present invention may target a wide range of different infectious agents (i.e., have activity against a wide range of different infectious agents). Therefore, the present invention may be widely used in the treatment or prevention of any infection or infectious disease, including infectious diseases involving viruses, bacteria, fungi, protozoa, prions, or metazoan factors.

[0105] Therefore, the present invention can be widely used in the treatment or prevention of viral infection; treatment or prevention of bacterial infection; treatment or prevention of protozoan infection; treatment or prevention of fungal infection; treatment or prevention of prion infection; and / or treatment or prevention of metazoan (e.g. worm) infection or infestation. The compounds of the present invention can also be used to treat or prevent chronic, dormant or latent viral, bacterial, protozoan, fungal, prion or metazoan (e.g. worm) infection or infestation.

[0106] Viral targets include, but are not limited to, the following viruses (or classes of viruses): Retroviridae (e.g., human immunodeficiency virus, including HIV-1); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronavirus Coronoviridae (e.g. coronavirus); Rhabdoviradae (e.g. vesicular stomatitis virus, rabies virus); Filoviridae (e.g. Ebola virus); Paramyxoviridae (e.g. parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza virus); Bungaviridae (e.g. hantavirus, bunga virus, phlebovirus, and narovirus); Arenaviridae (e.g. viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) types 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses); Poxviridae (smallpox virus, vaccinia virus, poxviruses); Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., etiological agents of spongiform encephalopathies, agent), the causative agent of hepatitis D (thought to be a defective satellite of the hepatitis B virus), HCV virus (causing non-A, non-B hepatitis); Norwalk and related viruses, and astroviruses).Among the aforementioned viruses, HIV, hepatitis A, hepatitis B, hepatitis C, rabies virus, polio virus, influenza virus, meningitis virus, measles virus, mumps virus, rubella, pertussis, encephalitis virus, papilloma virus, yellow fever virus, respiratory syncytial virus, parvovirus, chikungunya virus, hemorrhagic fever virus and herpes virus, in particular varicella, cytomegalovirus and Epstein-Barr virus are particularly preferred.

[0107] • Bacterial targets include, but are not limited to, Gram-negative and Gram-positive bacteria. Examples of bacteria that can be targeted by the compounds of the present invention include, but are not limited to, Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacterium spp. (e.g., M. tuberculosis, M. leprae, M. avium, M. intracellulare, M. kansaii, and M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae, and Streptococcus pyogenes. agalactiae (Group B Streptococcus), Streptococcus viridans, Streptococcus faecalis, Streptococcus bovis, any anaerobic species of the genus Streptococcus, Streptococcus pneumoniae, Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium spp. (including C. diphtheriae), Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, aerogenes), Klebsiella spp. (including K.pneumoniae), Pasturella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus monilijormis, Treponemapallidium, Treponema pertenue, Leptospira spp., Rickettsia spp., and Actinomyces spp. (including A. israelii). Bacteria that form biofilms in vivo are specific targets of the compounds of the present invention, and these bacteria include Tannerella forsythia, Tannerella denticola, Porphyromonas gingivalis, and Gardnerella vaginalis. .

[0108] • Fungal targets include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

[0109] Protozoan targets include, but are not limited to, certain species of Plasmodium (including Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax), certain species of Toxoplasma (including T. gondii and T. cruzii), certain species of Leishmania, certain species of Cryptosporidium (including C. parvum), certain species of Cyclospora spp.) (including C. cayetanensis), Entamoeba (including E. histolytica) and Giardia spp. (including Giardia lamblia).

[0110] - Metazoan targets include parasites or pathogens, such as helminths (eg, Schistosoma spp.).

[0111] Inhibition of bacterial growth in vivo

[0112] Sialidase activity is key to the utilization of sialic acid conjugated sugars and is involved in host-pathogen interactions with bacteria. When Tannerella forsythia grows in the form of a biofilm, glycoprotein-associated sialic acid is considered to be its key in vivo nutrient source (Roy, S., 2011, Microbiology, 157, 3195). The sialidase inhibitory properties of the compounds of the present invention can also be applied to inhibit the growth of symbiotic and / or pathogenic bacteria in vivo, particularly to disrupt host-bacterial cell interactions, including inhibiting or eliminating bacterial biofilms in mammalian (e.g., human) hosts.

[0113] Thus, the compounds are useful in treating or preventing diseases and conditions mediated by or characterized by the presence of bacterial biofilms (eg, subgingival plaque biofilms and mucosal biofilms).

[0114] Such diseases include periodontal disease, bacterial vaginosis, and diseases caused by infections with Tannerella forsythia, Tannerella denticola, Porphyromonas gingivalis, and Gardnerella vaginalis (the latter species being associated with bacterial vaginosis and premature birth).

[0115] Regulation of commensal bacterial growth

[0116] The sialidase inhibitory properties of the compounds of the invention may also be applied to modulating the composition of the microbiota (and especially commensal bacteria) in a host, for example modulating the composition of commensal bacteria in a mammalian (eg human) host. Particularly preferred is the modulation of the intestinal microbiota.

[0117] Atherosclerosis

[0118] The compounds of the present invention are sialidase inhibitors and can therefore be used to treat or prevent atherosclerosis, because sialidase is involved in this process (Sukhorukov, VN et al., 2017, Curr. Pharm. Des., 23, 4696) and osteoarthritis (Katoh, S. et al., 1999, J Immunol., 162, 5058). Therefore, the compounds of the present invention can be used to treat and prevent atherosclerosis.

[0119] Inflammation

[0120] The compounds of the present invention inhibit sialidase, which is believed to be involved in the inflammatory process induced by TNF-α in osteoarthritis (Gee, K. et al., 2003, J Biol Chem. 278, 37275). In addition, the compounds of the present invention can suppress or inhibit TNF-α activity. Therefore, they can be applied to any condition in which inflammation plays a role in impaired physiological function and / or symptoms and / or pain. For example, the compounds of the present invention can be used as anti-inflammatory drugs, for example, to reduce or eliminate acute, chronic, local or systemic inflammation.

[0121] Inflammation occurs when tissue is damaged by microorganisms, trauma, chemicals, heat, cold, sunburn, or any other harmful event. Endogenous chemicals (such as bradykinin, histamine, and serotonin) are released upon injury or damage, and such chemicals activate and attract tissue macrophages and other white blood cells. In this process, chemical mediators such as TNF-α are released, causing inflammation.

[0122] Inflammatory disorders are disorders in which inflammation is persistent or chronic. In such cases, prolonged inflammation can lead to tissue destruction and result in extensive damage and eventual failure of the affected tissues and / or organs.

[0123] Therefore, the compounds of the present invention can be applied to treat non-local inflammatory disorders, such as inflammatory disorders that affect more than one organ. Such disorders include disorders caused by immune dysfunction (and therefore may have an autoimmune component). Such conditions include systemic lupus erythematosus (SLE), scleroderma, and hypersensitivity reactions.

[0124] There is increasing evidence that inflammation is associated with the development of type 2 diabetes.

[0125] The compounds of the present invention may also be used to treat local inflammatory conditions including: skin inflammation and chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, transplant rejection, vasculitis, asthma, acne, osteoarthritis, oral mucosa, gastrointestinal inflammation, eye, nose and ear inflammation, and other steroid-responsive inflammatory conditions.

[0126] In particular, the compounds of the present invention can be applied to treat inflammatory skin diseases. These inflammatory skin diseases include, for example, actinic keratosis, acne (including acne vulgaris, acne, rosacea and nodulocystic acne), allergic contact dermatitis, angioedema, bullous pemphigoid, cutaneous drug reactions, erythema multiforme, lupus erythematosus, photodermatitis, psoriatic arthritis, scleroderma and urticaria, psoriasis, dermatitis (e.g., atopic dermatitis), scleroderma, steroid-responsive skin inflammatory disorders (e.g., uremic pruritus) and skin conditions associated with exposure to sunlight, radiation, chemotherapy and environmental irritants.

[0127] The compounds of the present invention can also be applied to treat inflammatory autoimmune diseases. Such diseases may involve specific tissues or organs (such as musculoskeletal tissue, as in rheumatoid arthritis and ankylosing spondylitis), the gastrointestinal tract (such as, for example, in Crohn's disease and ulcerative colitis), the central nervous system (such as, for example, in Alzheimer's disease, multiple sclerosis, motor neuron disease, Parkinson's disease and chronic fatigue syndrome), pancreatic beta cells (such as insulin-dependent diabetes), adrenal glands (such as Addison's disease), kidneys (such as Goodpasture's syndrome, IgA nephropathy and interstitial nephritis), exocrine glands (such as Sjögren's syndrome and autoimmune pancreatitis) and skin (such as psoriasis and atopic dermatitis).

[0128] Other inflammatory disorders that may be treated according to the present invention include conditions such as osteoarthritis, periodontal disease, diabetes (including type 2 diabetes and diabetic nephropathy), chronic obstructive pulmonary disease, atherosclerosis, graft-versus-host disease, chronic pelvic inflammatory disease, endometriosis, chronic hepatitis, and tuberculosis.

[0129] Dosimetry

[0130] The compositions and compounds of the present invention may be administered topically or by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.

[0131] The amount administered may vary widely depending on the particular dosage unit employed, the duration of the treatment, the age and sex of the patient being treated, the nature and extent of the condition being treated and the particular compound selected.

[0132] In general, the effective amount of the compound used will generally be within the range of about 0.01 mg / kg to 500 mg / kg per day. The unit dose may contain from 0.05 mg to 500 mg of the compound and may be taken once or multiple times per day. As described below, the compound may be administered orally, parenterally or topically using conventional dosage unit forms with a pharmaceutical carrier.

[0133] The preferred route of administration is oral administration. In general, a suitable dosage will be in the range of 0.01 mg to 500 mg per kg body weight of the recipient per day, preferably in the range of 0.1 mg to 50 mg per kg body weight per day, and most preferably in the range of 1 mg to 5 mg per kg body weight per day.

[0134] The desired dose is preferably presented as a single dose for daily administration. However, two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day may also be used. These sub-doses may be administered in unit dosage forms, for example, in a unit dosage form containing 0.001 mg to 100 mg of active ingredient, preferably 0.01 mg to 10 mg of active ingredient, and most preferably 0.5 mg to 1.0 mg of active ingredient.

[0135] preparation

[0136] When idoBR1 is isolated from a natural source, it may be purified. However, the composition of the invention may be in the form of an herbal medicine, a food supplement, a food additive, a nutraceutical, a beverage or as a single dose in an herbal kit or package as defined above. Such herbs are preferably analyzed before use to determine whether they meet standard specifications.

[0137] The herbal medicine for use according to the present invention may be a dried plant material. Alternatively, the herbal medicine may be a processed plant material, the processing involving physical or chemical pre-processing, such as crushing, grinding, freezing, evaporation, filtering, pressing, spray drying, extrusion, supercritical solvent extraction and tincture production. In the case where the herbal medicine is administered or sold in the form of the whole plant (or part thereof), the plant material may be dried before use. Any convenient drying form may be used, including freeze drying, spray drying or air drying.

[0138] The compounds of the invention can be separated from high molecular weight components such as proteins and polysaccharides by extraction in polar solvents such as ethanol / water mixtures, e.g., ethanol / water mixtures of ≥50% v / v (e.g., up to ˜70% v / v). Other suitable techniques include various membrane techniques. These techniques include microfiltration, ultrafiltration, and nanofiltration. Alternatively or in addition, electrodialysis can also be used to concentrate charged compounds. These methods use membranes with pore sizes that only allow molecules below a certain size to pass through, or rely on the charge on the molecules to allow or not allow them to pass through the membrane. Anion and cation exchange resins can also be used to concentrate the compounds.

[0139] When the compound is separated from natural origin, the compound used according to the present invention can be purified. In the embodiment in which the compound is formulated with a pharmaceutically acceptable excipient, any suitable excipient can be used, including for example inert diluents, disintegrating agents, adhesives, lubricants, sweeteners, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate and lactose, and corn starch and alginic acid are suitable disintegrating agents. Adhesives can include starch and gelatin, and lubricants (if present) generally will be magnesium stearate, stearic acid or talcum powder.

[0140] The pharmaceutical composition may take any suitable form, and includes, for example, tablets, elixirs, capsules, solutions, suspensions, powders, granules, and aerosols.

[0141] The pharmaceutical composition may take the form of a kit of parts which may contain the composition of the invention together with instructions for use and / or the various components in unit dosage form.

[0142] Tablets for oral use may include compounds used according to the present invention, mixed with pharmaceutically acceptable excipients (such as inert diluents, disintegrants, adhesives, lubricants, sweeteners, flavorings, coloring agents and preservatives). Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate and lactose, while corn starch and alginic acid are suitable disintegrants. Adhesives may include starch and gelatin, while lubricants (if present) are generally magnesium stearate, stearic acid or talcum powder. If desired, tablets may be coated with materials such as monostearate or distearate to delay absorption in the gastrointestinal tract. Capsules for oral use include hard gelatin capsules (wherein compounds used according to the present invention are mixed with solid diluents) and soft gelatin capsules (wherein active ingredients are mixed with water or oil such as peanut oil, liquid paraffin or olive oil).

[0143] Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0144] For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compound of the present invention will generally be provided in the form of a sterile aqueous solution or suspension buffered to an appropriate pH value and isotonic. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the present invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl pyrrolidone and tragacanth, and wetting agents such as lecithin. Suitable preservatives for aqueous suspensions include ethyl p-hydroxybenzoate and n-propyl p-hydroxybenzoate.

[0145] The compounds of the present invention may also be presented as liposomal formulations.

[0146] For oral administration, one or more of the compounds may be formulated into solid or liquid products such as capsules, pills, tablets, lozenges, pastilles, melts, powders, granules, solutions, suspensions, dispersions or emulsions (the solutions, suspensions, dispersions or emulsions may be aqueous or non-aqueous). The solid unit dosage form may be a capsule, which may be a common hard-shell gelatin type or a soft-shell gelatin type containing, for example, a surfactant, a lubricant and an inert filler (such as lactose, sucrose, calcium phosphate and corn starch).

[0147] In another embodiment, the compounds of the invention are tableted with conventional tablet bases such as lactose, sucrose, and corn starch in combination with binders such as gum arabic, corn starch, or gelatin, disintegrants to aid in the breakdown and dissolution of the tablet after administration such as potato starch, alginic acid, corn starch, and guar gum, lubricants to improve the flow of tablet particles and prevent the tablet material from adhering to the surfaces of tablet dies and punches such as talc, stearic acid or magnesium stearate, calcium stearate, or zinc stearate, dyes, colorants, and flavoring agents to enhance the aesthetic qualities of the tablets and make them more acceptable to patients.

[0148] Suitable excipients for oral liquid dosage forms include diluents such as water and alcohols, for example ethanol, benzyl alcohol and polyvinyl alcohol, with or without added pharmaceutically acceptable surfactants, suspending agents or emulsifying agents.

[0149] The compounds of the invention may also be administered parenterally, ie, subcutaneously, intravenously, intramuscularly or intraperitoneally.

[0150] In such embodiments, the compound is provided as an injectable dose in a physiologically acceptable diluent together with a pharmaceutical carrier (which can be a sterile liquid or a liquid mixture). Suitable liquids include water, saline, aqueous dextrose and related sugar solutions, alcohol (such as ethanol, isopropanol or hexadecanol), diols (such as propylene glycol or polyethylene glycol), glycerol ketal (such as 2,2-dimethyl-1,3-dioxolane-4-methanol), ether (such as poly (ethylene glycol) 400), oil, fatty acid, fatty acid ester or glyceride, or acetylated fatty acid glyceride, wherein with or without addition of pharmaceutically acceptable surfactant (such as soap or detergent), suspending agent (such as pectin, carhomer, methylcellulose, hydroxypropyl methylcellulose or carboxymethylcellulose) or emulsifier and other pharmaceutical adjuvants. Suitable oils that can be used in the parenteral preparation of the present invention are oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, vaseline and mineral oil. Suitable fatty acids include oleic acid, stearic acid and isostearic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate.

[0151] Suitable soaps include fatty alkali metal salts, ammonium salts and triethanolamine salts, and suitable detergents include cationic detergents such as dimethyldialkylammonium halides, alkylpyridinium halides and alkylamine acetates; anionic detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates and sulfosuccinates; nonionic detergents such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers; and amphoteric detergents such as alkyl-β-aminopropionates and 2-alkylimidazoline quaternary ammonium salts and mixtures.

[0152] The parenteral compositions of the present invention will generally contain about 0.5% by weight to about 25% by weight of the compound used according to the present invention in the form of a solution. Preservatives and buffers may also be used. In order to minimize or eliminate the irritation at the injection site, such compositions may contain a nonionic surfactant with a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such preparations is in the range of about 5% by weight to about 15% by weight. The surfactant may be a single component with the above-mentioned HLB or may be a mixture of two or more components with the desired HLB. Examples of surfactants used in parenteral preparations are polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and high molecular weight adducts of ethylene oxide and a hydrophobic matrix formed by the condensation of propylene oxide and propylene glycol.

[0153] One or more compounds used according to the present invention can also be applied topically, and when applied topically, the carrier can suitably include a solution, an ointment or a gel matrix. The matrix can, for example, include one or more of the following substances: vaseline, lanolin, polyethylene glycol, beeswax, mineral oil, a diluent (such as water and alcohol) and an emulsifier and a stabilizer. Topical preparations can contain compounds at a concentration of about 0.1% to about 10% w / v (weight per unit volume).

[0154] When auxiliary use, one or more compounds used according to the present invention can be formulated for use with one or more other drugs. Therefore, auxiliary use can be reflected in a specific unit dose designed to be compatible (or coordinated) with one or more other drugs or in a preparation in which one or more of the compounds are mixed with one or more enzymes. Auxiliary use can also be reflected in the composition of the drug kit of the present invention in which the compound of the present invention is packaged with the enzyme (for example, as a part of a series of unit doses). Auxiliary use can also be reflected in the information and / or instructions related to the co-application of one or more of the compounds and / or enzymes.

[0155] Cosmetic preparations

[0156] The cosmetic composition of the present invention may be selected from, for example, moisturizing compositions, cleansing compositions or any composition that can provide benefits to the skin.The cosmetic composition of the present invention may comprise a cosmetically acceptable excipient or carrier, for example selected from those described below.

[0157] In one embodiment, the cosmetic composition is a cleaning composition. Suitable cleaning compositions are solid or semisolid at room temperature. Examples of useful cleaning compositions include, but are not limited to, fatty acid soaps, including glycerine soaps, synthetic detergents, and mixtures thereof. Solid cleaning compositions are widely taught in the Soap Technology of the 1990s (the contents of which are incorporated herein by reference). It is desirable that the cleaning composition be flowable.

[0158] In one embodiment of the present invention, the cleaning composition comprises glycerin soap.Examples of glycerin soaps useful in the present invention include, but are not limited to, those disclosed in U.S. Pat. Nos. 4,405,492 and 4,879,063, the disclosures of which are incorporated herein by reference.

[0159] Examples of suitable fatty acid soaps include soaps derived from hydrocarbon chain lengths of about 10 to 22 carbons (including the carboxyl carbon) and may be saturated or unsaturated. The soap may be, for example, a sodium salt, a potassium salt, an ammonium salt, a triethanolammonium salt, and mixtures thereof.

[0160] Suitable synthetic detergents include detergents known in the art for the desired purpose. Examples of detergents that can be used for personal cleaning include isethionates, sarcosinates, and glycerol ether sulfonates, which can be pure chain length variants or variants derived from commercial oils (such as coconut oil). Other suitable detergents include anionic acyl sarcosinates, methyl acyl taurates, N-acyl glutamates, alkyl sulfosuccinates, alkyl phosphates, ethoxylated alkyl phosphates, tridecyl alcohol polyether sulfates, protein condensates, mixtures of ethoxylated alkyl sulfates and alkyl amine oxides, betaines, sulfobetaines, and mixtures thereof. Alkyl ether sulfates with 1 to 12 ethoxy groups are included, especially ammonium lauryl ether sulfate and sodium lauryl ether sulfate.

[0161] The cosmetic composition may be a moisturizing composition.

[0162] Other optional components of the cosmetic compositions of the present invention include, but are not limited to, perfumes, fragrances, preservatives, colorants, dyes, anti-caking agents, and personal care ingredients, including, but not limited to, skin and hair care ingredients.

[0163] Examples of suitable personal care ingredients that can be used in the present invention include, but are not limited to, safe and effective amounts of moisturizers, sunscreen actives, skin soothers, anti-irritants, anti-inflammatory agents, emollients, conditioners, humectants, deodorants, antiperspirants, artificial tanning agents, antimicrobial agents, anti-acne agents, anti-wrinkle agents, anti-skin atrophy agents, skin firming agents, anti-itch agents, anti-fungal agents, local anesthetics, skintone evening agents, active natural ingredients, agents that minimize the appearance of unwanted hair or delay its regrowth, skin texture improving agents, and additional cleansing agents.

[0164] In one embodiment, the compounds can be used from water or alcoholic aqueous extracts by utilizing water-in-oil (w / o) emulsions such as used, for example, in dry skin treatment and emollient applications.

[0165] Emollients act by their ability to be retained on the skin surface or in the stratum corneum as lubricants, to reduce peeling and to improve skin appearance. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether siloxane copolymers, etc. Examples of suitable emollients include, but are not limited to, polypropylene glycol ("PPG")-15 stearyl ether, PPG-10 cetyl ether, steareth-10, oleth-8, PPG-4 lauryl ether, vitamin E acetate, PEG-7 glyceryl coconut ester, lanolin and combinations thereof. Vitamin E acetate, PEG-7 glyceryl coconut ester and combinations thereof are preferred.

[0166] Examples of suitable humectants include polyols. Suitable polyols include, but are not limited to, glycerol (also known as glycerol), polyalkylene glycols, alkylene polyols and derivatives thereof, including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-dibutylene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof.

[0167] Suitable skin soothing agents include, but are not limited to, panthenol, bisabolol, allantoin, aloe vera, and combinations thereof.

[0168] Suitable conditioning agents include, but are not limited to, dimethicone propyl PG-betaine, dimethicone copolyol, polyquaternium-10, guar gum, guar gum derivatives, and combinations thereof. Suitable anti-acne active ingredients include, but are not limited to, salicylic acid, sulfur, lactic acid, glycolic acid, pyruvic acid, urea, resorcinol, N-acetylcysteine, retinoic acid, benzoyl peroxide, hydroxymethyloctopirox, triclosan, azelaic acid, phenoxyethanol, phenoxypropanol, flavonoids, derivatives thereof, and combinations thereof. Salicylic acid and benzoyl peroxide are preferred.

[0169] Example

[0170] The present invention will now be described with reference to specific embodiments. These embodiments are exemplary only and are for illustration purposes only: they are not intended to limit the scope of the claimed patent or the invention described in any way. These embodiments constitute the best mode currently contemplated for practicing the present invention.

[0171] Example 1: Inhibition of sialidase by idoBR1

[0172] Introduction

[0173] Sialidases or neuraminidase are enzymes that catalyze the cleavage of terminal sialic acid in oligosaccharides and glycoconjugates. They play an important role in regulating the metabolism of molecules containing sialic acid in biological systems. They are also virulence factors for many viruses and pathogens such as Tannerella forsythia. The sialidase activity of human neutrophils is reported to play a key role in host inflammatory responses (Glanz, VY, 2019, European J.Pharmacol.842,345).

[0174] Method. The sialidase assay uses 2.8mM and 0.28mM inhibitors (or water without inhibitors) and 2.5nM sialidase (including NanH from T.forsythia) incubated in 20mM sodium phosphate buffer in the presence of 0.1mM methylumbelliferyl-N-acetylneuraminic acid (pH 7.2). The reaction is terminated at 30 seconds and 60 seconds by adding pH 10.5 60mM sodium carbonate buffer. The release of fluorescent methylumbelliferone (MU) is quantitatively measured by measuring the fluorescence emission at 450nm and the fluorescence excitation at 350nm. The sialidase activity percentage is expressed as the change in fluorescence between 30 seconds and 60 seconds compared to the reaction without inhibitor. The reaction is performed in triplicate.

[0175] result

[0176] idoBR1 inhibited sialidase without apparent dose dependence, with inhibition exceeding 30% at both concentrations used (2.8 mM [36%] and 0.28 mM [42%]). The similarity in inhibition observed at these two concentrations suggests that the inhibition is not competitive.

[0177] Example 2: Inhibition of endogenous sialidase activity measurements in THP-1 cells

[0178] Introduction

[0179] The aim of this study was to determine whether idoBR1 or a cucumber extract containing more than 1% idoBR1 (Q-actin batch B17CF001) affects the activity of sialidase in human THP-1 (monocyte-like) cell cultures. The results of this study could be a combination of decreased sialidase expression or inhibition of this enzyme by idoBR1.

[0180] method

[0181] Treatment of THP-1 cell line for sialidase activity assay

[0182] THP-1 cells were cultured in RPMI medium supplemented with mercaptoethanol and glutamine to reach 80% confluence in a culture flask, and then the cells were aspirated and centrifuged at 1500 rpm for 5 minutes. The cell pellet was then resuspended in 1 ml RPMI complete medium and counted as usual using a hemocytometer. Cells (5×106) were incubated with PMA (10 ng / ml) in separate culture dishes to differentiate THP-1 cells. To determine sialidase activity, THP-1 cells were pretreated with idoBR1 and cucumber extract-Q-actin (lot number B17CF001) at concentrations of 100 μg / ml to 12.5 μg / ml and 200 μg / ml to 25 μg / ml for 1 hour, followed by 24 hours of LPS (1 μg / ml stimulation. After incubation, cells were used to determine sialidase activity.

[0183] THP-1 cells were washed with phosphate buffered saline (PBS) and resuspended in ice-cold buffer containing 0.25M sucrose, 1mM EDTA and 0.2mM phenylmethylsulfonyl fluoride. The cell suspension was ultrasonicated on ice for 15 seconds at a low setting (6% amplitude) (VibracellTM; Sonics and Materials Inc., Newtown, CT) and then centrifuged at 25,000g for 15 minutes at 4°C. The resulting supernatant was used to determine lysosomal sialidase activity. Protein quantification of the supernatant was performed using the Bio-Rad protein assay kit as described above. To determine lysosomal sialidase activity, 200μg of total protein was mixed with 40nmol 4-methylumbelliferyl-α-N-acetyl-D-neuraminic acid (Sigma), lysosomal sialidase specific substrate, 10μmol sodium acetate buffer pH 4.6 and 200μg bovine serum albumin in a total volume of 200μl. The sialidase reaction was allowed to proceed at 37°C for 1 hour and terminated by adding 0.25M glycine NaOH pH 10.4. Released 4-methylumbelliferone was measured by fluorimetry (Synergy 2 multi-mode microplate reader) at 365nm excitation wavelength and 448nm emission wavelength. Sialidase activity was found to reach a maximum at the 16-hour cell incubation time point.

[0184] result

[0185] Inhibition of endogenous sialidase activity by IdoBR1

[0186]

[0187] Cucumber extract batch number B17CF007 (Q-Actin)

[0188] Inhibition of endogenous sialidase activity

[0189]

[0190] It was found that the sialidase activity was highest at the 16 h time point after LPS (1 μg / mL) treatment, so this incubation time was used to further evaluate the effects of idoBR1 and cucumber extract on sialidase activity in THP-1 cells.

[0191] Treat with test sample for 2 hours

[0192] The relative sialidase activity in THP-1 cells stimulated with LPS for 16 h was then measured with IdoBR1.

[0193]

[0194] Treat with test sample for 2 hours

[0195] The relative sialidase activity in THP-1 cells stimulated by 16 hours of LPS was then measured using cucumber extract batch B17CF007 (Q-Actin)

[0196]

[0197] Standard idoBR1 tested at 50 μg / ml and 100 μg / ml showed the maximum reduction in relative sialidase activity compared to the LPS control, which were 0.63 and 0.55, respectively. Q-actin at 100 μg / ml and 200 μg / ml showed the maximum reduction in relative sialidase activity compared to the control (LPS) to 0.7 and 0.62, respectively.

[0198] Example 3: ELISA for the determination of CD44-HA (hyaluronic acid) binding activity in the presence of idoBR1 Treatment of THP-1 cell lines

[0199] Introduction

[0200] CD44 has been shown to be involved in hematopoiesis, homing to mucosal lymphoid tissue, and participating in lymphocyte infiltration into inflamed tissues. The interaction of hyaluronic acid (HA) with CD44 and CD168 (RHAMM) can induce many cell behaviors, including tyrosine kinase, protein kinase C, FAK and PI3K, MAPK, NFκB and RAS, as well as the activation of cytoskeletal components required for inflammation and cancer. Although most cells express some form of CD44, not all cells constitutively bind HA (Kryworuchko, M. et al., 1999, Cellular Immunol., 194, 54; Nandi et al., 2000, J.Biol.Chem., 275, 14939). Functionally active HA adhesion to CD44 is produced by inducing sialidase through MAPK activation. Studies conducted to understand the role of MAPK in LPS-induced inflammatory responses have shown that MAPK p42 / 44-mediated TNF-α production and subsequent TNF-α-mediated p38 activation lead to HA-adhesive CD44 production caused by sialidase activity (Gee, K. et al., 2003, J Biol Chem. 278, 37275).

[0201] method

[0202] THP-1 cells were cultured in RPMI medium supplemented with mercaptoethanol and glutamine to reach 80% confluence in the culture flask, and then the cells were aspirated and centrifuged at 1500 rpm for 5 minutes. The cell pellet was then resuspended in 1 ml of RPMI complete medium and counted as usual using a hemocytometer. Cells (5×106) were incubated with Phorbol 12-myristate 13-acetate (PMA) (10 ng / ml) in a separate culture dish to induce THP-1 cell differentiation. In order to determine the CD44-HA binding activity, THP-1 cells were pretreated with idoBR1 or cucumber extract-Q-actin (lot number B17CF001) at concentrations of 100 μg / ml to 12.5 μg / ml and 200 μg / ml to 25 μg / ml for 1 hour, followed by 24 hours of LPS (1 μg / ml) stimulation. After incubation, cell lysates were taken for further analysis.

[0203] Anti-CD44 monoclonal antibody (Invitrogen, 2 μg) was coated in 50 mM carbonate / bicarbonate buffer (pH 9.6) in each well of a 96-well plate and then incubated overnight at 4°C. Unbound antibodies were removed using PBS (PBS-T washing solution) containing 0.05% Tween 20. Wells were blocked with 1% BSA and incubated at 37°C for 1 hour. Wells were thoroughly washed three times with PBS-T washing solution by adding 200 μl PBS-T washing solution to the wells. 50 μl cell lysate was added to the wells and incubated at 37°C for 1 hour. Then, wells were washed three times by soaking the wells for 30 seconds by adding 200 μl PBS-T before each wash. Biotinylated-hyaluronic acid (HA) antibody was added, followed by streptavidin-HRP to form immune complexes, and incubated at 37°C for 60 minutes. Aspirate the solution and wash the wells three times with 200 μl of wash solution by soaking the wells for 30 seconds. Add 50 μl of Chromogen A and Chromogen B to each well. Incubate the plate at 37°C in the dark for 15 minutes. Stop the reaction by adding 50 μl of stop solution and read the absorbance at 450 nm.

[0204] result

[0205] Inhibition of CD44-HA binding by IdoBR1

[0206]

[0207] Inhibition of CD44-HA binding by cucumber extract batch B17CF007 (Q-Actin)

[0208]

[0209]

[0210] CD44-bound HA was found to be 110.45 ng / ml in LPS-stimulated (1 μg / ml) THP-1 cells. Ido-BR1 at 100 μg / ml showed the greatest reduction in CD44-HA levels in LPS-induced THP-1 cell inflammatory response compared to LPS control (26.62%). Cucumber extract-Q-actin at 200 μg / ml showed the greatest reduction in CD44-HA levels in LPS-induced THP-1 cell inflammatory response compared to LPS control (30.60%).

[0211] Example 4: Reduction of TNF-α production in human blood by idoBR1 and Q-actin cucumber extracts

[0212] Introduction

[0213] TNF-α is a cytokine produced by monocytes (macrophages) and T lymphocytes, a key element in the cascade of inflammatory responses, and has many pleiotropic effects as the main orchestrator of disease states (Beutler, B. et al., 1989, Annual Review of Immunology, 7, 625). The biological effects of TNF-α depend on its concentration and site of production: at low concentrations, TNF-α can produce the desired homeostasis and defense functions, but at high concentrations, TNF-α can synergize with other cytokines, especially interleukin-1 (IL-1), systemically or in certain tissues, thereby exacerbating many inflammatory responses. The purpose of this study was to evaluate the anti-inflammatory activity of idoBR1 or cucumber extracts with idoBR1 in terms of their ability to regulate TNF-α levels in human whole blood.

[0214] method

[0215] Blood and buffy coat fractions were provided by Scottish National Blood Transfusion Service (SNBTS), Glasgow, UK (SNBTS). Ficoll Histopaque (1.077 g / l), lipopolysaccharide (from Salmonella abortus equi) were purchased from Sigma-Aldrich Co. Ltd. (UK). PGE was from Cayman Chemical Co. (Ann Arbor, MI). Human TNF-α antibodies for TNF-α ELISA assays were from Invitrogen / Life Sciences Europe. All drugs were dissolved in RPMI 1640 medium from Gibco BRL, UK.

[0216] Blood was used without any further processing after donation. It was kindly provided by the Scottish National Blood Transfusion Service from normal healthy donors, defined by ensuring that they were negative for HIV, hepatitis B and C, CMV and parasitic diseases such as malaria (as tested by the National Blood Transfusion Service). Our laboratory also confirmed that they were free of acute inflammatory disease at the time of blood collection by measuring basal levels of TNF-α (always <50pg / ml).

[0217] Cell stimulation and TNF-α measurement

[0218] Aliquots (800 μl) of whole blood were incubated with compounds dissolved in RPMI 1640 (as indicated in the results) for the appropriate preincubation period, followed by the addition of LPS and continued incubation for an additional 20 h at 37° C. in a humidified (100%) atmosphere of air with 5% CO 2. At the end of the incubation period, supernatants of plasma or culture medium were collected by centrifugation at 10,000 g for 30 s at room temperature and TNF-α levels were measured using the Human TNF-α ELISA System (BioSource Europe SA, Belgium, provided by Invitrogen).

[0219] result

[0220] The potent activity of cucumber extract and idoBR1 against LPS-induced TNF-α in human blood is listed in the following two tables. The results show that cucumber extract (pilot Q-actin) containing 0.09% idoBR1 can reduce TNF-α, while idoBR1 is effective at well below 10 μM, confirming that idoBR1 alone can contribute to the anti-inflammatory effects of cucumber extract. Q-actin contains 10-100 times more idoBR1 than the pilot extract used here. Q-actin extract containing 10 times less idoBR1 has a 10-fold lower effect on TNF-α (data not shown).

[0221] The second study showed that idoBR1 was even more active in human blood in the presence of preincubation (significant at 0.01 μM). The IC 50, 182nM for blood, and 27nM for inhibition of TNF-α production by human monocytic cell line THP-1 cells. As measured by trypan blue absorption or MTT dye conversion, idoBR1 did not significantly change the viability of THP-1 cells. The inhibitory effect of idoBR1 (at 10μM) was comparable to that of the same pretreatment with dexamethasone (50μM), with inhibition rates of >50% and >65%, respectively. Mifepristone (a glucocorticoid receptor antagonist) alone with LPS greatly increased TNF-α production by THP-1 cells, however, in the presence of dexamethasone, it reversed the inhibitory effect of dexamethasone, but not that of idoBR1. The data clearly show that idoBR1 can inhibit the production of TNF-α in human blood. Therefore, it seems likely to be a potent anti-inflammatory agent. It also seems likely to work through a new mechanism that is different from the steroid receptor pathway.

[0222] The table shows the effect of various idoBR1 concentrations on TNF-α production in LPS-stimulated human blood. Whole blood was pre-incubated with varying concentrations of idoBR1 for 48 hours, then LPS (10 μg / ml) was added and incubated for an additional 20 hours. After incubation at 37°C (5% CO2, 100% humidity), plasma was collected from the blood by centrifugation, and TNF-α levels in the plasma samples were measured by ELISA.

[0223]

[0224] Values ​​represent mean ± sd of n = 3. * indicates P < 0.05 compared with LPS alone (null idoBR1).

[0225] The table gives the effect of various concentrations of cucumber extract (0.09% idoBR1) on TNF-α production in LPS-stimulated human blood. Whole blood was pre-incubated with varying concentrations of cucumber Q-Actin extract for 48 hours, then LPS (10 μg / ml) was added and incubated for another 20 hours. After incubation at 37°C (5% CO2, 100% humidity), plasma was collected from the blood by centrifugation, and TNF-α levels in plasma samples were measured by ELISA. Values ​​represent mean ± sd of n = 3. * indicates P < 0.05 compared to LPS alone.

[0226]

[0227] Example 5: Effects of idoBR1 and cucumber extract on the expression of cytokines IL-10, IL-12 and Effects of IL-1β

[0228] Introduction

[0229] IL-10 is an important negative regulator of anti-inflammatory cytokines and pro-inflammatory cytokines. Various cell types including T cells, B cells and monocytes / macrophages secrete IL-10 under different immune activation conditions (Moore, K. et al., 1993, Annu. Rev. Immunol., 11, 165). In vitro studies have shown that IL-10 inhibits the release and function of IL-1β, IL-6, TNF-α, granulocyte macrophage colony stimulating factor and IL-12 (Casatella, M. et al., 1993, J. Exp. Med., 178, 2207; de Waal Malefyt, R. et al., 1991, J. Exp. Med., 174, 1209; Fiorentino, D. et al., 1991, J. Immunol., 147, 3815), thereby revealing the normal endogenous feedback mechanism of immune response and inflammation control (Asadullah, K. et al., 1998, J. Clin. Invest. 101, 783; Joosten, L. et al., 1997, Arthritis Rheum., 40, 249). Studies have shown that IL-10 exerts its inhibitory effect on IL-12p40 and p35 and TNF-a gene expression mainly at the transcriptional level (Aste-Amezaga, M. et al., 1998, J. Immunol., 160, 5936). Among the proinflammatory cytokines involved in the pathogenesis of various autoimmune diseases, IL-12 is the main stimulator of IFN-γ production and the development of T helper (Th) 1 autoimmune response (Paunovic, V. et al., 2008, Rheumatology, 47, 771). It has been shown that IL-12 synergizes with various cytokines and induces the production of IFN-γ and proinflammatory cytokines. Monocytes / macrophages produce IL-1β and TNF-α, which mediate inflammation after infection or by stimulating LPS. It can induce inflammatory responses and decomposition independently and in combination with other mediators. The biological activation of cells by IL-1β is mediated by interaction with the membrane receptor IL-1R1 (IL-1RI, CD121a), which can also bind to another IL-1 group, IL-1α.

[0230] method

[0231] ELISA assay on THP-1 monocytes

[0232] Sample preparation for ELISA assay

[0233] The cells in the 80% fused culture flask were aspirated and centrifuged at 1500rpm for 5 minutes. The cell pellet was then resuspended in 1ml RPMI complete medium and inoculated into each well of a 96-well microtiter plate at 1×105 cells / well. After 24 hours of incubation, PMA (10ng / ml) was added to the 96-well plate to differentiate the THP-1 cells to determine TNF-α production. THP-1 cells were pretreated with idoBR1 or cucumber extract-Q-actin (lot number B17CF001) at a concentration of 200μg / ml to 25μg / ml for 1 hour, serially diluted twice, and then stimulated with LPS (100ng / ml) for 2 hours. After incubation, the cell supernatant of each well was aspirated into a sterile microcentrifuge tube and centrifuged at 1000rpm for 2-3 minutes. The cell supernatant was then used to evaluate the presence of cytokines using ELISA.

[0234] Sandwich ELISA assay

[0235] ELISA plates (R&D Systems, USA) coated with IL-12, IL-1β or IL-10 antibodies were used for the following studies. After mixing, 50 μL of Assay DiluentRD1F was added to each well. 200 μL of sample (idoBR1 or extract) or control was added to each well and covered with tape. After incubation for 2 hours at room temperature, each well was aspirated and washed 4 times with wash buffer (400 μl). After the last wash, any remaining wash buffer was removed by aspiration or decantation. The plate was inverted and blotted with a clean paper towel. 200 μl of the appropriate human conjugate was added to each well, then covered with new tape and incubated at room temperature for 1 hour. The aspiration / washing was then repeated. Then, 200 μL of substrate solution was added to each well and further incubated for 20 minutes at room temperature in the dark. 50 μL of stop solution was added to each well. The color in the well changed from blue to yellow. OD was measured at 450 nm within 30 minutes.

[0236] result

[0237]

[0238]

[0239] Reduction of IL-12 at different IdoBR1 concentrations

[0240]

[0241]

[0242] Reduction of IL-12 at different concentrations of cucumber extract (Batch No. B17CF007)

[0243]

[0244] Reduction of IL-1β under different IdoBR1 concentrations

[0245]

[0246] Reduction of IL-1β at different concentrations of cucumber extract (Batch No. B17CF007)

[0247]

[0248] The anti-inflammatory marker IL-10 increased by 3.04-fold and 3.65-fold with idoBR1 and cucumber extract, respectively. The IL-10 results suggest an anti-inflammatory effect. IdoBR1 at 100 μg / mL showed a 24.53% decrease in IL-12 levels in the LPS-induced inflammatory response of THP-1 cells compared to the LPS control. Q-actin, a cucumber extract at 200 μg / mL, showed a 25.88% decrease in IL-12 levels in the LPS-induced inflammatory response of THP-1 cells compared to the LPS control. idoBR1 at 100 μg / mL reduced IL-1β levels in the LPS-induced inflammatory response of THP-1 cells by 24.07% compared to the LPS control. Q-actin, a cucumber extract at 200 μg / mL, showed the greatest reduction (22.53%) in IL-1β levels in the LPS-induced inflammatory response of THP-1 cells.

[0249] Example 6: Fitness exercise and Q-actin together increase IL-10 measured in human blood

[0250] Introduction

[0251] We have demonstrated the modulation of the anti-inflammatory cytokine IL-10 in THP-1 cells by idoBR1 and cucumber extract (Q-actin). Cytokine modulation was tested here in humans taking Q-actin and given an intense exercise regimen that naturally induces an inflammatory response in the muscles.

[0252] method

[0253] IL-10 was measured in blood samples during an exercise recovery experiment using 7 placebo subjects and 10 Q-actin subjects. Subjects took 10 mg Q-actin or placebo (both in capsule form) twice daily for 4 days starting on day 0 and performed intense exercise on days 1, 2, 3, and 4 (recovery days). Blood samples were collected before and after exercise on days 1, 2, 3, and at the end of recovery day 4. IL-10 was measured by ELISA assay.

[0254] result

[0255] Once exercise began, subjects who had received Q-actin showed a trend toward a significantly greater increase in IL-10, confirming the results obtained in LPS-stimulated THP-1 cells.

[0256] The table shows the IL-10 response of blood from subjects who underwent a vigorous exercise regimen (plus and minus Q-actin, cucumber extract containing >1% idoBR1)

[0257]

[0258] D1-Pre-exercise = blood collected before exercise

[0259] D1-1H = blood collected 1 hour after exercise

[0260] Example 7: MAPK signaling effects of idoBR1 and cucumber extracts containing idoBR1

[0261] Introduction

[0262] The MAPK signaling cascade plays an important role in the initiation of inflammatory responses. The induction of inflammatory cytokine genes requires the activation of MAPK, and the stimulation of the extracellular regulated protein kinase / mitogen-activated protein kinase (ERK / MAPK) pathway is crucial for downstream inflammatory responses (Kaminska, B., 2005, Biochim. Biophys. Acta, 1754, 253; Buchholz, K. et al., 2007, Infection and Immunity, 75, 5924). The MAPK pathway is also required for the expression of inflammatory mediator genes including COX-2, iNOS, IL-1β and TNF-α. It is reported that ERK and / or p38MAPK are involved in the upregulation of IL-1β (Baldassare, J. et al., 1999, J. Immunol., 162, 5367).

[0263] method

[0264] THP-1 cells were cultured in RPMI medium supplemented with mercaptoethanol and glutamine to reach 80% confluence in the culture flask, then they were aspirated and centrifuged at 1500 rpm for 5 minutes. The cell pellet was then resuspended in 1 ml of RPMI complete medium and counted as usual using a hemocytometer. Cells (5×106) were incubated with PMA (10 ng / ml) in separate culture dishes to differentiate THP-1 cells. To determine protein expression p38 and p42 / 44, THP-1 cells were pretreated with idoBR1 (100 μg / ml and 50 μg / ml) and cucumber extract (Q-actin lot number B17CF001) (200 μg / ml and 100 μg / ml) for 1 hour, followed by 2 hours of LPS (1 μg / ml) stimulation. After incubation, cells were harvested and whole protein was isolated.

[0265] Western Blot Procedure Cell pellets were lysed and protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad). Total cell protein was subjected to 8% polyacrylamide SDS gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Thermoscientific). The membrane was probed with mouse anti-phospho-p38 mAb (Thermoscientific) or mouse anti-phospho-p42 / 44 mAb (Thermoscientific) followed by a horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody (Thermoscientific). All immunoblots were visualized by ECL (Amersham Biosciences). The test sample Q-Actin tested at 100 μg / ml and 200 μg / ml showed a relative decrease of 0.92 and 0.83 in phosphorylated p38 expression compared to the LPS control, respectively. In case of 50μg / ml and 100μg / ml of idoBR1, it showed a decrease in phosphorylated p38 expression of 0.88 and 0.80, respectively, compared to the LPS control. Q-actin tested at 100μg / ml and 200μg / ml showed a relative decrease in phosphorylated ERK 42 / 44 expression of 0.81 and 0.78, respectively, compared to the LPS control. IdoBR1 at 50μg / ml and 100μg / ml showed a decrease in phosphorylated ERK 42 / 44 expression of 0.80 and 0.76, respectively, compared to the LPS control.

[0266] The table shows the relative expression of p42 / 44

[0267]

[0268] The table gives the relative expression of p38 / .

[0269]

[0270] Thus, idoBR1 and cucumber extract (Q-actin) containing idoBR1 were shown to reduce the MAPK signaling cascade, which plays an important role in inflammatory responses.

[0271] Example 8: Oral availability and in vivo stability of idoBR1

[0272] Introduction

[0273] The aim of this study was to investigate the oral availability of idoBR1 from edible cucumbers / gherkins by measuring the amount of idoBR1 in urine. This study not only suggests oral availability and possible systemic activity of idoBR1, but also supports the ability of this compound to pass through cell membranes unchanged, thus supporting local availability.

[0274] method

[0275] Parisian pickles (seeds purchased from Lidl 2013) were organically grown and three were eaten at noon by a male and a female volunteer. The weight of fresh cucumbers eaten was 260 g in each case, and a similar weight of 30 g was taken from all the cucumbers eaten and kept for analysis. The volunteers did not eat cucurbitaceous food 15 hours before the experiment. A pre-consumption urine sample was collected within 3 hours as t=0, and then a 9-hour sample was collected for females and a 15-hour sample was collected for males. The 30 g cucumber sample was homogenized in 50% ethanol (water), filtered after extraction for 15 hours, and the idoBR1 fraction was bound to the cation exchange resin IR120 in the H+ form. After washing the column with water, the material replaced with 2M ammonia solution (52.3 mg) was dried and analyzed by GC-MS after trimethylsilylation using Pierce TriSil. Then, 0.2 mg of castanospermine was added to the remaining 51 mg of material for comparative quantitative purposes. All urine samples were processed similarly using the cation exchange resin, but the material displaced with ammonia solution was run a second time through the same cation exchange resin (now in the ammonium form) to reduce the strong base (which would bind to IR120 in the ammonium form) and only the unretained material was retained. The urine idoBR1 fraction was dried and diluted to 20 ml in water. 500 ul of each was sampled and 0.025 mg of castanospermine was added.

[0276] result

[0277] GCMS analysis of cucumbers This was performed on a Perkin Elmer Turbomass Gold GCMS. The spectrum of the main peak at 10.33 minutes matched the GCMS spectrum of authentic idoBR1 (900288 PhytoQuest Ltd, UK). The relative response factor between authentic BR1 (900125, PhytoQuest Ltd) and castanospermine was calculated to be 1:2. Assuming the same response factor, the amount of idoBR1 in the 30 g sample was estimated to be 1.5 mg, which means that the volunteers consumed approximately 260 / 30×1.5 mg=13 mg idoBR1.

[0278] Urine results

[0279] Urine samples collected before eating cucumbers did not show a significant peak at the retention time of idoBR1 (10.33min). After 15 hours, the male showed an excretion of about 2.4mg of idoBR1 compared to the castanospermine reference peak area, but more accurate measurements of intake and excretion are needed to achieve the final mass balance. The female had excreted about 2.1mg idoBR1. This study confirms that idoBR1 is orally available and can be measured in urine, meaning that it can enter the bloodstream through oral intake and at least a large portion is excreted intact in the urine. This suggests that the compound can pass membranes in the digestive tract and is significant in the urine. No obvious conjugation was observed in the urine analysis.

[0280] The remaining idoBR1 may stay in the body for longer. Therefore, it appears to be a potent anti-inflammatory agent, and possibly a long-lasting one.

[0281] Example 9: Effects of idoBR1 on microglia

[0282] Introduction

[0283] Microglia are resident macrophages of the central nervous system (CNS). These cells are the main form of active immune defense in the CNS. In neurodegenerative disorders (such as Alzheimer's disease and Parkinson's disease), microglia are activated for a long time and promote the release of proinflammatory cytokines, thereby further destroying normal CNS activities. People have considerable interest in checking the extent to which bioactive food ingredients reduce oxidative stress and / or reduce the effects of inflammation by reducing proinflammatory gene expression.

[0284] method

[0285] idoBR1 was used in cell cultures of the murine microglial cell line BV-2 in the presence or absence of suboptimal LPS at 0, 20, 40 and 80 μg / ml. TNF-α and nitrite production were measured 24 hours later. Interestingly, idoBR1 from Q-actin was found to be effective in reducing TNF-α and nitrite production by stimulated microglia.

[0286]

[0287] *Significance calculated in the case of ±LPS and for test samples compared to LPS+OidoBR1 (n=>3).

[0288] Cells were pretreated with compounds for 30 min and then restimulated with LPS for 24 h. Data for all experiments are presented as mean ± SEM of at least 3 experiments. Values ​​were compared using one-way ANOVA followed by post hoc Student Newman-Keuls test. Data were analyzed using GraphPad Prism software. *p<0.05; **p<0.01; ***p<0.001 Equivalent solution

[0289] The foregoing description describes the present preferred embodiments of the present invention in detail. It is expected that those skilled in the art will make many modifications and variations in their practice after considering these descriptions. These modifications and variations are intended to be encompassed in the claims attached hereto.

Claims

1. Use of a pharmaceutical composition comprising idoBR1 and a pharmaceutically acceptable excipient or carrier in the preparation of a medicament for treating viral infection or bacterial infection.

2. The use as claimed in claim 1, wherein the drug is used to treat bacterial infection.

3. The use according to claim 2, wherein the drug is used to inhibit the growth of commensal and / or pathogenic bacteria in vivo.

4. The use according to claim 2, wherein the drug is used to disrupt host-bacterial cell interactions and / or inhibit or eliminate the formation of bacterial biofilms in mammalian hosts.

5. The use according to claim 4, wherein the mammal is a human.

6. Use according to any one of claims 3 to 5, wherein the medicament is for the treatment or prevention of diseases and disorders mediated by or characterised by the presence of bacterial biofilms.

7. The use according to claim 6, wherein the bacterial biofilm is selected from subgingival plaque biofilm and mucosal biofilm.

8. Use as claimed in claim 2, wherein the medicine is used to treat periodontal disease, bacterial vaginosis and / or a method for treating a disease caused by infection with Tannerella forsythia, Tannerella denticola, Porphyromonas gingivali or Gardnerella vaginalis.

9. Use of a composition comprising idoBR1 in the preparation of a medicament for regulating the growth of commensal bacteria in a mammalian host.

10. The use according to claim 9, wherein the drug is used to regulate the composition of commensal bacteria in a mammalian host.

11. The use according to claim 10, wherein the mammalian host is a human host.

12. Use of a pharmaceutical composition comprising idoBR1 and a pharmaceutically acceptable excipient or carrier in the preparation of a medicament for treating atherosclerosis.

13. The use according to claim 12, wherein the atheroma formation is atherosclerosis.

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