The natural response of healthy human tissue to damage or attack is inflammation. The tissues of the human body may come under attack or otherwise be compromised by a variety of factors, including microbial infection, non-living foreign matter, ionizing radiation and oxidative stress. Under the right conditions, inflammation may be present in virtually all tissue types in the human body, including the major organs (heart, brain, liver, kidneys, etc) and the skin. By “oxidative stress” we mean unwanted changes in animal tissues that are caused by reactive oxygen species (pro-oxidants) present within the tissue. Oxidative stress develops through an imbalance wherein the effects of pro-oxidants dominate over the effects of anti-oxidants. Examples of pro-oxidants that accumulate in human tissues include oxygen ions, free radicals and peroxides, for example, superoxide and hydrogen peroxide. Reactive oxygen species result from normal cell metabolism, but under balanced conditions their destructive effects are checked by anti-oxidants in the organism. Examples of such anti-oxidants are superoxide dismutase and catalase.
Leukotriene B4
Helpful for developing an appreciation of the present invention is an understanding of the synthesis and role of leukotriene LTB4, a potent pro-inflammatory agent. Following an inflammatory stimulus, a first step in a complex cascade of reactions is an influx of calcium ions into certain cells. These cells may include neutrophils, eosinophils, monocytes, macrophages, mast cells, basophils and B lymphocytes. Within these cells, the influxing calcium ions and ATP bind to inactive 5-lipoxygenase and this leads to the translocation of 5-lipoxygenase out of the cytosol and into the cell membrane, where it anchors to 5-lipoxygenase activating protein (FLAP). The calcium ion influx also prompts the translocation of phospholipase A2 from the cytosol to the cell membrane, where it cleaves an arachidonic acid glycerol ester. The arachidonic acid glycerol ester is a naturally occurring phospholipid component of the cell membrane and when cleaved by phospholipase A2, it releases arachidonic acid, an unsaturated fatty acid, into the cytosol. Other mechanisms of arachidonic acid release into the cytosol include the action of certain cytokines, namely, tumor necrosis factor (TNF) and interleukin-1 (IL1). Regardless, a freed arachidonic acid molecule binds to a translocated 5-lipoxygenase molecule and is converted into 5-hydroperoxyeicosatetraenoic acid (5-HPETE). 5-HPETE is in turn acted upon by a cytosolic 5-lipoxygenase molecule to form leukotriene A4 (LTA4), which is then released into the cytosol. Some LTA4 may be secreted by the cell while some LTA4 remains in the cell and is hydrolyzed into leukotriene B4 (LTB4) under the action of LTA4 hydrolase. The secreted LTA4 may be taken up by cells that do not produce LTA4 themselves, but which are otherwise capable of converting LTA4 into LTB4. These cells may be thought of as a secondary sources of LTB4. Once produced in either primary or secondary cells, LTB4 passes through the cell membrane into the extracellular environment. The secreted LTB4 initiates a number of cellular and molecular actions that direct, as well as amplify, the inflammatory process. LTB4 has a number of functionalities that allow it to direct this stage of the inflammatory process. Broadly, LTB4 has chemotactic, chemokinetic, vasoactive, pain mediating, immuno-modulating and other properties. It also stimulates degranulation and production of superoxide within leukocytes.
Following its secretion, LTB4 attracts to the affected site, neutrophils circulating in the blood. LTB4 is the a potent chemotactic agent for neutrophils that express the appropriate chemoattractant receptors. Even topical application of LTB4 to human skin has been shown to promote the infiltration of neutrophils at the site of application; see “Production of Intraepidermal Microabscesses by Topical Application of Leukotriene B4”, R. Camp, et al., Journal of Investigative Dermatology, 1984, 82, 202-207. This and all cited references are herein, incorporated by reference, in their entirety. LTB4 binds to receptors on the neutrophil surface and this initiates the formation of structures required for motility. Having attracted neutrophils in the blood to the site of inflammation, LTB4 induces adhesion of those neutrophils to the blood vessel endothelium. LTB4 increases the permeability of the blood vessels and following adhesion to the endothelium, neutrophils pass through the endothelium and into the stromal cell environment at the site of injury or infection. The neutrophils are directed by the chemotactic activity of LTB4, which means that the neutrophils move in the direction of increasing concentration of LTB4. At this point, the primary role of neutrophils is phagocytosis. Neutrophils adhere to and then engulf unwanted organisms and debris in the extracellular spaces of the inflamed tissue. By this time, the site of inflammation is infused with inflammatory exudate, an edemaous composition of pro-inflammatory products, metabolite and debris. Neutrophils use potent enzymes and noxious microbial agents to perform phagocytosis. In another of its important functions, LTB4 influences phagocytic neutrophils to release quantities of glucuronidase and lysozyme into the extracellular environment, where they have a beneficial role in breaking down the acute inflammatory exudate and damaged tissue.
Interleukin-1β
Other than LTB4, the present invention touches on the role played by Interleukin-1β(IL-1β), a pro-inflammatory cytokine. While LTB4 may play a role in inducing adhesion of neutrophils to the endothelium, IL-1β is directly responsible for adhesion of the polymorphonuclear leukocytes (PMN) to the endothelium. IL-1β induces the expression of adhesion molecules on the surface of the endothelial cells, a necessary occurrence if neutrophils are to pass out of the blood and into the affected tissue. IL-1β is naturally present in human skin and blocking or antagonizing the role of IL-1β in adhesion, is generally anti-inflammatory.
Acute Verse Chronic Inflammation
Acute and chronic inflammation are somewhat loosely defined in the literature, but it is useful to think of four situations based on the histological and clinical presentations. As such, acute inflammation is characterized by the presence of polymorphonuclear leukocytes (mainly phagocytic neutrophils) at the site of inflammation. However, acute inflammation may further be characterized as being in a resolving or persisting state, depending on the length of time since the initial attack. Chronic inflammation is different from acute inflammation. Chronic inflammation may occur as a second stage of the inflammatory response, after the failure of the acute process to completely resolve the situation, or it may occur with an acute phase. Chronic inflammation may be caused by a persistent infection, prolonged irritation, a cellular immune response, a defective acute inflammatory response, an autoimmune disorder, lifestyle, prolonged psychological stress, etc. Chronic inflammation is characterized by the presence of mononuclear cells (mostly phagocytic macrophages, but also lymphocytes, monocytes and plasma cells) at the site of inflammation, and, like acute inflammation, may be classified as resolving or persistent. Only relatively recently has medical science identified and appreciated the role of chronic tissue inflammation in many disease etiologies. It is important to note that as inflammation progresses, both acute and chronic processes may be occurring simultaneously in close proximity to each other.
Once they have infiltrated the site of inflammation, neutrophils guided by LTB4 exert primary control over the acute inflammatory response with phagocytosis being their primary activity. However, it should also be noted that activated neutrophils are a primary source of LTB4. Thus, the potential for an amplifying feedback loop exists in which LTB4 directs the recruitment and activation of neutrophils to a site of inflammation and those neutrophils produce and release more LTB4 which recruits more neutrophils. At a later point in the acute stage, relatively small numbers of macrophages and lymphocytes also infiltrate the site to aid in removal of tissue debris and damaged cells, but the histology is still characterized by neutrophils. If the acute stage is unable to resolve the disorder and return the organism to homeostasis, a cross over from acute to chronic inflammation may occur, when neutrophils that have infiltrated the site of inflammation send out a chemical signal that reduces further neutrophil recruitment and promotes mononuclear cell influx. Activated macrophages dominate the chronic stage of inflammation. They perform many of the same functions as neutrophils, like phagocytosis, but they may also have a more system-wide effect. Also, macrophages are capable of cell division and antigen presentation to lymphocytes. Macrophages direct the chronic inflammatory response and promote the healing stage by producing a variety of cytokines (including LTB4) and growth factors. For example, TGF-β (transforming growth factor beta) is responsible for down-regulating the inflammatory function of macrophages, while also stimulating them to produce cytokines, growth factors and collagenases that support healing.
Resolving chronic inflammation requires the elimination of immune cells (macrophages and leukocytes) from the affected area. Leukocytes and macrophages tend to accumulate in a tissue compartment because of recruitment and/or local proliferation (cell division). Neutrophils, lymphocytes and macrophages are depleted from a tissue compartment by emigration and cell death. Under favorable conditions of inflammatory response, immune cells enter and exit the site of inflammation at rates that prevent their over-accumulation in the affected area. In contrast to resolving chronic inflammation, persisting chronic inflammation results when emigration and cell death do not keep up with recruitment and proliferation. Such an imbalance is a pathological condition and may generally occur when chemical signals that inhibit emigration and cell death are inappropriately produced. The mechanisms of this are not completely understood. Concerning neutrophils, one aspect seems to be that stromal cells and fibroblasts in the affected tissues may release stromal-cell-derived factor 1 (SDF-1), which is pro-retentive for neutrophils, and interferon-β (INF-β), which is pro-survival for neutrophils. Concerning activated T-cells, assorted interleukins and type I interferons (INF-α and INF-β) inhibit apoptosis and therefore, contribute to persistence of inflammation. One result of this pathological accumulation of leukocytes is the continuous release of lysosomal enzymes through exocytosis. Both neutrophils and macrophages release quantities of collagen- and elastase-destroying enzymes into the extracellular environment, where they have a beneficial role in breaking down inflammatory exudate and damaged tissue. However, these enzymes do not discriminate and may also digest healthy tissues. If the quantities of these enzymes is excessive, significant damage to healthy tissue will occur. Furthermore, the phagocytic cells also release into the environment, reactive oxygen metabolites, which may also attack healthy tissue. This attack itself, becomes an initiator of inflammatory response, extending the inflammatory state for days, months or even years. Thus in chronic, especially persisting inflammation, tissues are damaged by the causative agent as well as by the inflammatory response to that agent. In fact, in persisting inflammation, the original causative agent may have long ago been neutralized.
There is an important distinction to be made here. Some treatments are anti-inflammatory because they neutralize one or more original causative agents. For example, an invading bacteria may cause inflammation. If an effective anti-bacterial treatment neutralizes the invading bacteria, the inflammation may subside. In this sense, the anti-bacterial treatment could be called anti-inflammatory. This is different from the focus of the present invention. For example, an invading bacteria may cause inflammation. Now say, the bacteria persists for a long time such that the inflammation becomes chronic persisting. Thereafter, the invading bacteria is finally neutralized by targeted anti-bacterial treatment, but the inflammation continues because of its persisting nature. At this stage, treatment targeted at inflammation is indicated. It was not enough to remove the original causative agent. Treatment targeted at the original causative agent may have no effect on resolving the persisting inflammation. The affected person needs something that enters into the inflammatory process and interacts with the inflammatory process. The present invention is concerned with treatments targeted at inflammation. By “targeted at inflammation”, “targeted anti-inflammatory composition”, “anti-inflammatory specific” or the like, we mean a composition, method or treatment that interacts directly with the inflammatory process, other than by neutralizing one or more original causative agents, as just described.
LTB4 has been implicated in a number of persisting inflammatory disorders of the skin and internal organs, including psoriasis, eczema, erythema, acne, pruritus, cystic fibrosis, rheumatoid arthritis, asthma, allergies, colitis and others. In all of these, elevated levels of LTB4 have been observed. Such conditions are generally referred to as LTB4-mediated disorders and their study has led to the development of various LTB4 inhibitors and antagonists for treatment of chronic inflammatory disorders. Antagonists are targeted in their effect, blocking LTB4 from performing specific functions. Inhibitors, on the other hand, block the formation of LTB4 from arachidonic acid. Therefore, inhibitors potentially affect all functions that rely on LTB4 and this may have undesirable consequences. Methods of LTB4 inhibition include inhibiting 5-lipoxygenase directly as well as blocking 5-lipoxygenase-activating protein, so that 5-lipoxygenase cannot translocate into the cell membrane. Either way, the cascade leading to the formation of LTA4 is interrupted and LTB4 is not produced. In contrast, antagonists block the action of LTB4 on one or more receptors on leukocytes and/or the endothelium. Often it may be preferable to control or influence some activities of LTB4 and not others. In those cases, inhibition of LTB4 production is contraindicated while antagonism of LTB4 is indicated. As an example, during a persisting inflammatory disorder with chronically elevated LTB4 levels, it may be desirable to interrupt the inflammatory process by interrupting LTB4-mediated chemotaxis and IL-1β mediated adhesion of neutrophils. However, for neutrophils already present at the site of inflammation, LTB4 could still trigger exocytosis and the release of quantities of glucuronidase and lysozyme into the extracellular environment, where they have a beneficial role in breaking down the acute inflammatory exudate and damaged tissue. In this way, the front end of the inflammatory process may be checked, while the back end is allowed to proceed and the affected area allowed to exit the inflammatory process. As the inflammatory process subsides, the processes of healing and repair take over. In the better resolving outcomes, termed “healing”, the tissue structure remains in tact or can be regenerated by tissue cell proliferation. In the lesser resolving outcomes, termed “repair” or “organization”, the damaged tissue is replaced by scar tissue via the body's normal repair process. If either healing or repair are to succeed, the inflammatory process has to be checked and the potential for further damage reduced or eliminated. In general, the effectiveness of any treatment of conditions that are characterized by persisting elevated levels of LTB4, is evaluated by the regression or prevention of the symptoms of the condition.
Mushrooms: Immuno-Enhancement Verses Immuno-Suppression
Mushrooms belong to the Basidiomycota phylum of the fungi kingdom. Whole mushrooms and mushroom extracts have been used for centuries for a host of reported effects. Methods of use include oral ingestion, subcutaneous injection and topical application. Like any biologically active substance, the specific effects of mushrooms depend on various factors, including, the exact species of mushroom, the portion of the mushroom used, the pre-processing of the mushroom, the method of administering, the area of the body targeted for treatment, the treatment regimen and so on.
Mushroom compositions (especially edible compositions) are often touted for their immune enhancing, and energy enhancing properties. It should be remembered that immuno-enhancement is pro-inflammatory and therefore compositions of this type are contrary to the anti-inflammatory compositions of the present invention. Furthermore, many different types of products, including mushroom compositions, make anti-ageing claims. But the oft-used phrase “anti-ageing” is non-specific and may convey different, even mutually exclusive meanings, depending on the initial condition of the person undergoing an anti-ageing treatment. So, on the one hand, pro-inflammation (immuno-enhancement) may be anti-ageing while on the other hand, anti-inflammation (immuno-suppression) may be anti-ageing. Consider a person who eats a balanced diet, exercises regularly, does not smoke or consume excessive alcohol, and does not receive harmful amounts of sun exposure. As this person progresses beyond middle age, immune system function (inflammatory response) eventually weakens, despite all due care. In this case, pro-inflammatory treatments could be viewed as “anti-ageing”. Alternatively, consider a thirty year old who eats poorly, does not exercise, smokes daily, consumes more than six ounces of alcohol per day and works outdoors. At thirty, this person may have an undiminished inflammatory response, but because of constant external stimuli, that response may always be activated, i.e. chronic, leading to damage of healthy tissue. In this case, anti-inflammatory treatments should be considered anti-ageing. To summarize, “immuno-enhancement” implies pro-inflammatory, while “anti-ageing” could be pro- or anti-inflammatory.
This point was emphasized in an article entitled “Mycological Medicine” (January 2002 issue of Functional Foods & Nutraceuticals). The author noted, “As powerful immune modulators and potentiators, medicinal mushrooms are contraindicated for a number of autoimmune conditions such as systemic lupus erythematosus and collagen autoimmune disorders.” Further highlighting this point, a peer review, appearing immediately after the article, stated, “It is clear that the use of the medicinal mushroom extracts has its place in the management of certain chronic conditions, including cancer. However, the author does point out that the use of such extracts is not advocated in certain conditions, such as autoimmune states. This is a well-founded warning because these extracts enhance the functioning of the inflammatory cells, and boosting the activity of such cells is not advisable when chronic inflammation forms part of the disease pathogenesis.” Therefore, a composition, even if it claims to be anti-ageing does not necessarily have an anti-ageing effect on all persons treated with that composition. Furthermore, two compositions having one or more mushrooms in common, do not necessarily have the same general effect on all persons treated, even if both compositions claim to be anti-ageing. It depends how and on whom the compositions are used. It goes without saying, that compositions comprising extracts from taxonomically different mushrooms, do not generally behave the same way.
As noted, certain mushrooms can be used to enhance immune function. Again quoting from the article “Mycological Medicine”, “Medicinal mushrooms' powerful immune-modulating and potentiating activity help support and enhance overall immune function. Researchers also are finding that mushrooms can directly stimulate both the basic (lymphocytes, neutrophils, etc.) and secondary immune responses (immunoglobulins IgE, IgA, IgG) of the immune system. This stimulus can increase production of immune defenders such as cytokines and macrophages, which play vital roles in recognizing and removing foreign antigens, as well as releasing chemical mediators including interleukin-1.” Further quoting, “Substances that have been found to potentiate the immune system include beta-glucans, lentins, polysaccharides, polysaccharide-peptide complexes, triterpenoids, nucleosides and other secondary metabolites. Many of these bioactive substances, through their stimulatory effects on the immune system, are showing powerful antitumour, antimutagenic and anticancer activity.” The points to note are that certain mushrooms are understood to increase cytokine production and stimulate neutrophil activity, i.e. certain mushrooms are capable of pro-inflammatory activity. Also, it is believed, at least in some cases, that the polysaccharides and glucans found in mushrooms stimulate the immune system, that is, are pro-inflammatory. The author explains, “Beta-glucan binds to macrophages and other phagocytic white blood cells at certain receptors and activates their anti-infection and antitumour activity by stimulating free radical production. This, in turn, signals the phagocytic immune cells to engulf and destroy foreign bodies, be they bacteria, viruses or tumor cells.” Thus, many of the touted uses of mushroom extracts as anti-bacterial, anti-viral, anti-mutagenic, cardiovascular enhancing, etc. flow from the ability of mushrooms to address these conditions by stimulating the immune system and the inflammatory process. Finally, it must be remembered that polysaccharides and beta-glucans represent large classes of molecules. Nothing known in the field teaches or suggests that all polysaccharides or all beta-glucans are pro-inflammatory. The most that may be said is that some polysaccharides and beta-glucans are able to intervene in the inflammatory process.
Healthy skin, like all organs of the body and the body as whole, must maintain a state of homeostasis. Generally, homeostasis is disrupted by excess or deficiency, while the result is compromised skin integrity. Thus skin wrinkling and other signs of ageing skin can be a result of immuno weakness or hyper immuno activity (i.e. chronic inflammation). For example, in chronologically ageing people, the skin eventually thins and may be more easily damaged, while having a reduced ability to heal itself. As blood flow is diminished in older skin, so too is the immuno-response. Topical treatments that inhibit inflammation in the skin may be contraindicated for such people. On the other hand, eczema and psoriasis are hyper inflammatory disorders for which targeted anti-inflammatory compositions may be indicated.
Apart from the diminished capacities that accompany chronological ageing and autoimmune conditions, the human skin of any age is affected by exogenous or endogenous factors many of which are deteriorative. These factors include gravity, sun exposure, pollution, smoking, second hand smoke, pharmaceuticals, diet, trauma and others. Most of these factors (perhaps not gravity) are inflammatory to the skin and lead to deterioration of the collagen and elastin network in the surface layers of the skin. This deterioration leads to loss of skin elasticity and firmness, leading to sagging and wrinkling of the skin. Thus, even within the substantially younger population, there may occur changes in the skin which visibly manifest as wrinkles Generally, these visible manifestations are called “pre-mature ageing” of the skin.
The literature reports mushroom containing compositions that exhibit anti-inflammatory properties. WO2005/067955 claims that a topical poultice of Fomitopsis officinalis (common name: Agarikon) has long been used for anti-inflammation, and that in other forms, the mushroom is used to treat tuberculosis. The article is not specific about what types of inflammation the topical poultice may be effective in treating. Nor is it specific about how to make such poultices, even the concentration of Fomitopsis not being given. Which parts of the mushroom to use and how to prepare an extract for use in the poultice are not described. The reported components of this mushroom are beta glucans, triterpenoids, agaricin and antibiotics, but there is no explanation of how Fomitopsis officinalis intervenes, if at all, in the inflammatory process. Do the compositions mentioned neutralize the original causative agent of inflammation or are the compositions anti-inflammatory specific? It is not possible to know, because no compositions are disclosed.
Other known topical compositions of mushroom extract include the following, which are reported to treat or prevent ageing and wrinkling of the skin.
KR 2004084581 is entitled, Preservative Free Cosmetic Composition Containing The Extract of Dictyophora Indusiata To Prevent Skin Wrinkles, Wherein The Composition Displays High Antibacterial Effect And Is Thus Effective For Anti-Inflammation. Reported therein, is a preservative free cosmetic composition for the prevention of skin wrinkles containing an ethanolic extract of Dictyophora Indusiata mycelium. The extract concentration is 0.5-20% of the composition. Reportedly, the moisturizing composition displays high antibacterial effect and is thus effective for anti-inflammation. As the title itself explains, any anti-inflammatory activity of this composition comes from the composition's anti-bacterial activity. From the title and abstracts available, this can be understood to say that after the composition has neutralized invading bacteria, inflammation will subside. Nothing in the title or abstract suggest that the Dictyophora Indusiata extract directly intervenes in the inflammatory process. More specifically, nothing in the title and abstract suggest that Dictyophora Indusiata extract has any effect on LTB4 mediation of neutrophils. This is unlike compositions of the present invention which are anti-inflammatory specific and do influence the LTB4 mediation of neutrophils.
JP 11292785 describes a topical preparation containing one or more extracts of Agaricus blazei mushroom and the filtrate of the mycelium culture, combined with an active oxygen scavenger. The composition reportedly inhibits the damage of dermal fibroblast cells by ultraviolet rays; prevents the formation of peroxy lipid caused by active oxygen in the skin; is effective for the prevention and improvement of wrinkles or deterioration in skin elasticity; prevents and improves skin inflammation and roughening. The composition contains many active species other than the Agaricus mushroom, For example, active oxygen scavengers include: extracts of Hamamelis, Quercus, Aesculus, Sanguisorba, Paeonia, Ginkgo bibloba L., Betulaceae tree, parsley, carotenoid, flavonoid, tannin, superoxide dismutase, gallic acid and its salts or derivatives, hydroquinone, thioredoxin and thioredoxin reductase. JP 11228439 also describes a preparation comprising an extract of mycelia of Agaricus blazei (pref. at 0.0001-5 wt. %), at least one kind of physiologically active substance of animal origin (e.g. a placenta or spleen extract from mammal such as human or cattle, soluble eggshell membrane protein, basic or acidic fibroblast growth factor, epithelial cell growth factor, nucleic acid), 0.0001-3.0 wt. % of an anti-inflammatory agent, mucopolysaccharides (e.g. hyaluronic acid) and a 2-hydroxycarboxylic acid. Reportedly, the composition potentiates both a moisturizing effect and activates epidermal fibroblasts to treat skin aging symptoms. The reported role of Agaricus in these compositions is activation of dermal fibroblast cells, which is part of the healing and repair processes. Generally, the beta-glucans extracted from this mushroom species are reported to be used in cancer treatment where they aid in the production of interferon and interleukin (pro-inflammatory). Also, beta-glucans are known to stimulate macrophage activity (pro-inflammatory). Of course, those treatments are not generally topical. The point is that, the use of Agaricus in these references is not anti-inflammatory specific and even the presence of a separate anti-inflammatory ingredient in JP 11228439 indicates that the Agaricus is not targeted at inflammation. This is unlike compositions of the present invention wherein one or more mushroom extracts are anti-inflammatory specific and do influence the LTB4 mediation of neutrophils.
JP 63183537 reportedly describes an anti-inflammatory agent having low toxicity, containing an extract of Heterobasidiae (e.g. Auricularia auricula-judae, A. polytricha, A. mesenterica, etc.). The active ingredients of the extract are reported to be polysaccharides. The polysaccharides are xylose, mannose, glucuronic acid, etc., in the case of Auricularia auricula-judae. The extracted components of Auricularia auricula-judae are blended with various drug bases in liquid state, creamy state, etc., and used as an anti-inflammatory drug. The concentration of extract used is preferably 0.001-20.0 wt. %.
None of the foregoing references discloses, nor are the applicant's aware of prior art compositions comprising Hypsizygus ulmarius extract for use as a targeted LTB4 antagonist and/or IL-1β antagonist, in the skin. Nothing in the prior art suggests that Hypsizygus ulmarius extract could be effective for resolving persisting skin inflammation and thereby mitigating the effects of persisting inflammation, particularly visible signs of ageing, such as wrinkles. None of the foregoing discloses topical composition comprising an anti-inflammatory effective amount of Hypsizygus ulmarius extract, where the anti-inflammatory action is LTB4 antagonism and/or IL-1β antagonism.