EP3873507A1

EP3873507A1 - A multi-component composition for use in resolving inflammation

Info

Publication number: EP3873507A1
Application number: EP19801496.1A
Authority: EP
Inventors: Natascha KRÖMMELBEIN; Bernd Seilheimer
Original assignee: Biologische Heilmittel Heel GmbH
Current assignee: Biologische Heilmittel Heel GmbH
Priority date: 2018-10-30
Filing date: 2019-10-29
Publication date: 2021-09-08
Also published as: CO2021005806A2; WO2020089205A1

Abstract

The present invention relates to a composition for use in resolving inflammation in a subject in need thereof, wherein said composition comprises the following ingredients: Achillea millefolium, Aconitum napellus, Arnica montana, Atropa bella-donna, Bellis perennis, Calendula officinalis, Echinacea purpurea, Echinacea, Hamamelis virginiana, Hepar sulfuris, Hypericum perforatum, Matricaria recutita, Mercurius solubilis Hahnemanni, and Symphytum officinale. The invention also relates to a pharmaceutical composition comprising said composition and an anti-inflammatory drug. Moreover, the present invention also contemplates a kit for use in resolving inflammation in a subjectin need thereof comprising the (i) said composition and (ii) an anti-inflammatory drug.

Description

A multi-component composition for use in resolving inflammation

The present invention relates to a composition for use in resolving inflammation in a subject in need thereof, wherein said composition comprises the following ingredients: Achillea millefolium, Aconitum napellus, Arnica montana, Atropa bella-donna, Bellis perennis, Calendula officinalis, Echinacea purpurea, Echinacea, Hamamelis virginiana, Hepar sulfuris, Hypericum perforatum, Matricaria recutita, Mercurius solubilis Hahnemanni, and Symphytum officinale. The invention also relates to a pharmaceutical composition comprising said composition and an anti-inflammatory drug. Moreover, the present invention also contemplates a kit for use in resolving inflammation in a subject in need thereof comprising the (i) said composition and (ii) an anti-inflammatory drug.

Since the acute inflammatory response is protective, evolved to permit repair of injured tissues and eliminate invading organisms, it is ideally self-limited and leads to complete resolution of leukocyte infiltrates and clearance of cellular debris enabling return to homeostasis (Serhan, 2014).

Countless times each day, the acute inflammatory response protects from invading microbes, injuries, and insults from within, as in surgery-induced tissue injury. These challenges go unnoticed because they are self-limited and naturally resolve without progressing to chronic inflammation. Peripheral blood markers of inflammation are present in many common diseases, including inflammatory bowel disease, cardiovascular disease, neurodegenerative disease, and cancer. While acute inflammation is protective, excessive swarming of neutrophils amplifies collateral tissue damage and inflammation (Serhan and Levy, 2018).

For many years, resolution of inflammation was considered a passive phenomenon associated with the removal of inflammatory stimuli, end of chemoattractant synthesis, dilution of chemokines, and prevention of further leukocyte recruitment. Meanwhile, however, several studies indicate that endogenous inhibitors of leukocyte trafficking act as a counteractive mechanism against chemokine and adhesion molecule promoters of cell recruitment. Moreover, there are indications that the resolution of inflammation is an active process brought about by the biosynthesis of active mediators, the so-called specialized pro-resolving mediators (SPMs). In this context, homeostasis is recovered after the induction of SPMs that act on specific cellular receptors to regulate leukocyte trafficking and blunt production of inflammatory mediators, while also promoting clearance of dead cells and tissue repair (Sansbury and Spite, 2016).

The cardinal signs of resolution entail the limitation or cessation of blood-bome cell extravasation, the counter regulation of chemokines and cytokines, the switching off of signaling pathways associated with leukocyte survival, the induction of leukocyte apoptosis and their subsequent removal through efferocytosis by macrophages, the reprogramming of macrophages from classically activated to alternatively activated cells, the return of non-apoptotic cells to the vasculature or lymph, and finally the initiation of healing processes. Failure of one or more steps in the resolution of inflammation may be involved in the pathogenesis of several human chronic inflammatory diseases, such as impaired wound or tissue healing or fibrosis. Additionally, chronic, unresolved inflammation may be involved in the development of autoimmunity (Sugimoto et al., 2016).

Various drugs have been reported to be modulators of inflammation. However, these drugs, typically, act antagonistically with respect to the pro-inflammatory environment (Serhan, 2014). The most prominent anti-inflammatory agents are non-steroidal anti-inflammatory drugs such as selective or non-selective cyclooxygenase 2 inhibitors. Moreover, homeopathic drug compositions such as Traumeel® (Biologische Heilmittel Heel GmbH, Germany) have been reported to exhibit anti-inflammatory activities in acute inflammation (Schneider, 2011).

So far, there are only a limit number of pro-resolving agents under investigation in clinical trials. Resolvin El and a synthetic analog RX- 100045 are tested in numerous inflammatory diseases (Lee, 2012). An LXA₄ based compound is investigated for topical treatment of eczema (Wu et al, 2013). AP214, a melanocortin agonist, elicits pro-resolving effects in experimental systems and kidney injury (Montero -Melendez et al, 2011).

There is a need for compositions that can resolve inflammation in a subject to therapeutically address those pathophysiological conditions were the resolution of inflammation is impaired.

The technical problem underlying the present invention can be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

The present invention relates to a composition for use in resolving inflammation in a subject in need thereof, wherein said composition comprises the following ingredients: Achillea millefolium, Aconitum napellus, Arnica montana, Atropa bella-donna, Bellis perennis, Calendula officinalis, Echinacea purpurea, Echinacea, Hamamelis virginiana, Hepar sulfuris, Hypericum perforatum, Matricaria recutita, Mercurius solubilis Hahnemanni, and Symphytum officinale.

The term "composition" as used herein refers to a mixture of extracts from, inter alia, biological sources such as plants which are further defined elsewhere herein. Preferably, the said composition can comprise further other ingredients and, more preferably, comprises a pharmaceutically acceptable carrier and/or diluent.

Preferably, such further ingredients can be stabilizing agents, wetting agents, pharmaceutical carriers, additional pharmaceutically active agents, release controlling agents and the like. Preferred diluents encompass water, alcohols, physiological saline solutions, buffers, such as phosphate buffered saline solutions, syrup, oil, water, emulsions, various types of wetting agents, and the like. Preferably envisaged in accordance with the present invention is a composition which further comprises at least one pharmaceutically acceptable carrier and/or diluent. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may include a solid, a gel, or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, European Pharmacopeia, Homeopathic Pharmacopeia of the USA or HAB. The pharmaceutically acceptable diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

The composition shall be adapted for use in resolving inflammation. Accordingly, it will be understood that dependent on the desired mode of administration the composition shall be formulated for a systemic or topical application. Preferably, the composition envisaged herein is formulated for a systemic or local application. Preferably, oral application, e.g. in the form of tablets, solution or drinking ampules, is envisaged or application via injection. However, depending on the nature and the mode of action, the composition may be administered by other routes as well including dermal, intra-muscular, subcutaneous, oral, intravenous or topical administration. The composition can be, preferably, formulated for a bolus administration or can be made for continuous applications as set forth elsewhere herein in detail. Preferably, the composition according to the present invention is formulated as a medicament as set forth elsewhere herein in detail.

The term“inflammation” as used herein refers to a pathophysiological response of a subject against harmful stimulation by, e.g., pathogens, cellular damage or irritants. Typical signs of inflammation are heat, pain, redness or other skin reactions, swelling and impaired tissue function. Inflammation may be acute inflammation or chronic inflammation. Preferably, inflammation referred to in accordance with the present invention is chronic inflammation. Inflammation and, in particular chronic inflammation, may be, preferably, associated with a disease or disorder selected from the group consisting of: rheumatoid arthritis, osteoarthritis, peritonitis, irritable bowel syndrome, COPD, systemic lupus erythematosus, multiple sclerosis, psoriasis, diabetes mellitus type 1, musculoskeletal injuries, muscle inflammation, and joint inflammation, such as ankle sprain.

The term "resolving inflammation" as used herein refers to an active process which results in restoration of tissue integrity and function in a tissue being affected by inflammation. More specifically, inflammation is a pathophysiological process in response to infection or tissue damage. The process, typically, starts when tissue-resident cells of the innate immune system detect infection or damage within the tissue. Secretion of chemical signals such as chemokines and cytokines lead to the recruitment of circulating neutrophils to the site of damage or infection. Lipid mediator class switching occurs as neutrophils congregate in pus or purulent exudates. Lipoxins (LXs) stimulate non-phlogistic monocyte recruitment. LXs, resolvins (Rvs), and other specialized pro-resolving mediators (SPMs) are produced in pus to limit or stop further neutrophil tissue infiltration. SPMs, Rvs, maresins (MaRs), and protectins, each stimulate efferocytosis of apoptotic neutrophils and cellular debris by macrophages. Resolving macrophages and apoptotic neutrophils also produce SPMs. Edema also brings circulating n-3 polyunsaturated fatty acids (PUFAs) into exudates for temporal conversion to SPMs by exudate cells. MaRs and specific Rvs enhance wound healing and tissue regeneration (Serhan, 2017). Afterwards, tissue homeostasis can be restored. There are various inflammatory diseases where the resolution of inflammation is apparently impaired resulting, e.g., in chronic inflammation.

Under resolving inflammation in accordance to the present invention it is to be understood that the active process of resolution of inflammation is triggered and that such chronic inflammation processes can be resolved. Typical characteristic changes of resolution of inflammation are synthesis of so-called specialized pro-resolving mediators (SPMs) and recruitment of macrophages, in particular, of the resolving phenotype. Thus, resolving inflammation as referred to herein, preferably, involves reduction of neutrophil recruitment to the site of inflammation. Also preferably, resolving inflammation involves inducing the synthesis of at least one SPM and, more preferably, at least one SPM which is selected from the group consisting of: LxA₄, LxB₄, RVD₂, RvDs, Mar-2, and NPDi. Such SPMs are, preferably, significantly increased after 4 hours upon administration of the composition to a subject compared to a control subject. Moreover, resolving inflammation involves, preferably, the recruitment of macrophages. More preferably, said macrophages are significantly increased at the site of inflammation after 24 hours upon administration of the composition to a subject compared to a control subject. Preferably, resolving inflammation as referred to herein may refer to any significant improvement of the resolution such as the initiation of the synthesis of SPMs and/or the recruitment of macrophages. However, more preferably, resolving inflammation means curing inflammation such that tissue homeostasis and function is completely restored. It will be understood that a resolution of the inflammation may not occur in 100% of the subjects to which the composition has been administered. The term, however, requires that the resolution occurs in a statistically significant portion of the subjects (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.05, 0.01, 0.005, or 0.0001.

The term“subject” as used herein refers to a mammal and preferably a human. Said subject shall be, preferably, in need of inflammation resolution, i.e. it shall exhibit an acute or chronic inflammation. More preferably, the subject has chronic inflammation and/or further suffers from a disease associated therewith and, preferably, disease or disorder selected from the group consisting of: rheumatoid arthritis, osteoarthritis, peritonitis, irritable bowel syndrome, COPD, systemic lupus erythematosus, multiple sclerosis, psoriasis, diabetes mellitus type 1 , musculoskeletal injuries, muscle inflammation, and joint inflammation, such as ankle sprain.

The plants referred to in accordance with the present invention, i.e. Achillea millefolium (milfoil), Aconitum napellus (monkshood), Arnica montana (mountain arnica), Atropa bella donna (deadly nightshade), Bellis perennis (daisy), Calendula officinalis (calendula), Echinacea purpurea (purple cone flower), Echinacea (narrow-leaved cone flower), Hamamelis virginiana (witch hazel), Hypericum perforatum (St. John's wort), Matricaria recutita (chamomile), and Symphytum officinale (comfrey), are well known to the person skilled in the art and botanically characterized. Accordingly, the said plants as a starting material for preparing the composition according to the present invention can be identified, cultured and/or harvested without further ado. For example, the plants can be obtained by growing developing seedlings in the green house under standard conditions of humidity, temperature, and illumination into plants, culturing said plants for a period of time sufficient to allow for production of secondary plant metabolites and harvesting the plants or parts thereof required for the extraction process defined elsewhere herein. Furthermore, the said plants as a starting material for preparing the composition according to the present invention can, preferably, also be obtained from wild harvesting. In this case, the plants are identified using micro- and macroscopic testing methods.

Other components of the composition are also well-known chemicals in homeopathic applications, such as Hepar sulfuris (calcium sulphide) and Mercurius solubilis Hahnemanni (mercurico amidonitrate). Again, these components, in particular as homeopathic ingredients, are well known to the person skilled in the art.

The composition according to the present invention comprises the said components in the following homeopathic dilutions:

Achillea millefolium D3,

Aconitum napellus D2,

Arnica montana D2,

Atropa bella-donna D2,

Bellis perennis D2,

Calendula officinalis D2,

Echinacea purpurea D2,

Echinacea D2,

Hamamelis virginiana Dl,

Hepar sulfuris D6,

Hypericum perforatum D2,

Matricaria recutita D3,

Mercurius solubilis Hahnemanni D6, and

Symphytum officinale D6.

The term“Achillea millefolium D3“ as used herein refers to a dilution of Achillea millefolium mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. D-Dilutions of the mother tincture can be made by applying the following dilution scheme: 3 parts of mother tincture are diluted in 7 parts ethanol (70% V/V) to obtain a Dl dilution. This dilution is further processed as described below.

The term“Aconitum napellus D2“ as used herein refers to a dilution of Aconitum napellus mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 2 parts of mother tincture are diluted in 8 parts ethanol (50% V/V ) to obtain a Dl dilution. This dilution is further processed as described below. The term“Arnica montana D2“ as used herein refers to a dilution of Arnica montana mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture (corresponding to the Dl dilution) to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 1 part of the Dl dilution is subsequently diluted in 9 parts ethanol (90% V/V) in order to obtain a D2 dilution. This dilution is further processed as described below.

The term“Atropa bella-donna D2“ as used herein refers to a dilution of Atropa bella-donna mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 2 parts of mother tincture are diluted in 8 parts ethanol (50% V/V ) to obtain a Dl dilution. This dilution is further processed as described below.

The term“Bellis perennis D2“ as used herein refers to a dilution of Bellis perennis mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 2 parts of mother tincture are diluted in 8 parts ethanol (50% V/V) to obtain a Dl dilution. This dilution is further processed as described below.

The term“Calendula officinalis D2“ as used herein refers to a dilution of Calendula officinalis mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 3 parts of mother tincture are diluted in 7 parts ethanol (70% V/V) to obtain a Dl dilution. This dilution is further processed as described below.

The term“Echinacea purpurea D2“ as used herein refers to a dilution of Echinacea purpurea mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 3 parts of mother tincture are diluted in 7 parts ethanol (70% V/V) to obtain a Dl dilution. This dilution is further processed as described below.

The term“Echinacea D2“ as used herein refers to a dilution of Echinacea mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 3 parts of mother tincture are diluted in 7 parts ethanol (70% V/V) to obtain a Dl dilution. This dilution is further processed as described below.

The term“Hamamelis virginiana Dl“ as used herein refers to a dilution of Hamamelis virginiana mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. The mother tincture is further processed as described below.

The term“Hepar sulfuris D6“ as used herein refers to a dilution of Hepar sulfuris which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned material to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 1 part of the material is triturated with 9 parts of lactose monohydrate in order to obtain the trituration Dl . 1 part of the trituration Dl is triturated with 9 parts of lactose monohydrate in order to obtain the trituration D2. The D2 trituration is further triturated (1 :10) as specified before in order to obtain triturations D3 and D4. The trituration D4 is further processed as described below.

The term“Hypericum perforatum D2“ as used herein refers to a dilution of Hypericum perforatum mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 3 parts of mother tincture are diluted in 7 parts ethanol (70% V/V) to obtain a Dl dilution. This dilution is further processed as described below.

The term“Matricaria recutita D3“ as used herein refers to a dilution of Matricaria recutita mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 3 parts of mother tincture are diluted in 7 parts ethanol (70% V/V ) to obtain a Dl dilution. This dilution is further processed as described below.

The term“Mercurius solubilis Hahnemanni D6“ as used herein refers to a dilution of Mercurius solubilis Hahnemanni which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned material to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 1 part of the material is triturated with 9 parts of lactose monohydrate in order to obtain the trituration Dl. 1 part of the trituration Dl is triturated with 9 parts of lactose monohydrate in order to obtain the trituration D2. The D2 trituration is further triturated (1 : 10) as specified before in order to obtain triturations D3 and D4. The trituration D4 is further processed as described below.

The term“Symphytum officinale D6“ as used herein refers to a dilution of Symphytum officinale mother tincture which has been prepared according to the current versions of the homeopathic pharmacopeias and, in particular, the German homeopathic pharmacopeia (HAB), preferably, of 2017 or the European Pharmacopeia (Ph. Eur.), preferably, 9.5. Preferably, the aforementioned mother tincture to be used for the composition according to the invention is characterized by the analytical parameters set forth in the respective monograph. 1 part of the Dl dilution is subsequently diluted in 9 parts ethanol (70% V/V) in order to obtain a D2 dilution. 1 part of the D2 dilution is further diluted in 9 parts ethanol (70% V/V) in order to obtain D3, 1 part of the D3 dilution is then diluted in 9 parts ethanol (50% V/V) in order to obtain a D4 dilution. This dilution is further processed as described below.

Achillea millefolium dilution Dl, Matricaria recutita dilution Dl and Symphytum officinale dilution D4 are mixed together and the mixture is potentised twice 1 : 10 with water for injections in order to obtain Achillea millefolium dilution D3, Matricaria recutita dilution D3 and Symphytum officinale dilution D6. Aconitum napellus dilution Dl, Atropa bella-donna dilution Dl, Bellis perennis dilution Dl, Calendula officinalis dilution Dl, Echinacea dilution Dl, Echinacea purpurea dilution Dl, Hypericum perforatum dilution Dl are mixed together and the mixture is potentised 1 :10 with water for injections in order to obtain Aconitum napellus dilution D2, Atropa bella-donna dilution D2, Bellis perennis dilution D2, Calendula officinalis dilution D2, Echinacea dilution D2, Echinacea purpurea dilution D2, Hypericum perforatum dilution D2.

Hepar sulfuris trituration D4 and Mercurius solubilis Hahnemanni trituration D4 are potentised twice 1 :10 with water for injections in order to obtain Hepar sulfuris dilution D6 and Mercurius solubilis Hahnemanni dilution D6, respectively.

Hamamelis virginiana mother tincture is potentised 1 :10 with water for injections in order to obtain Hamamelis virginiana dilution Dl . These finally envisaged dilutions are subsequently mixed with each other, with the Arnica montana dilution D2 and with water for injections and sodium chloride to obtain the composition to be used in accordance with the present invention.

Preferably, a composition envisaged for use in accordance with the present invention is the commercially available drug composition Traumeel® as drops, tablets, ointment, injection solution or gel (Biologische Heilmittel Heel GmbH, Germany).

In the following, the components and amounts present in the composition for use according to the invention are listed for tablets, ointment, drops, gel and injection solution (ampoules):

Typically, the composition for use according to the present invention is to be administered in an amount sufficient for resolving inflammation. It will be understood that the dosage to be applied may depend on several factors well known to the clinician including gender, age, body weight and general health condition. Moreover, the dosage will depend on the kind of administration. Typical, a topical administration may require a different dosage from systemic administration. Similarly, the mode of administration will also affect the therapeutically effective dosage. For example, oral administration may require another dosage than intraperitoneal injection. Preferably, the composition according to the present invention shall be administered as a solution. More preferably, the dosage provided to a subject shall correspond to the amount provided by at least about 1.5 ml composition (injection solution) per kg body weight for 6 days in mice.

If the composition for use according to the present invention is formulated as a solution, a 2.2 ml ampoule shall be administered per day OOmI/kg body weight). Such an ampoule, preferably, may be administered between one to three times a week. However, the dosage may be, preferably, increased up to 5 or up to 10 times said dosage. In acute cases, the ampoule shall be preferably administered daily.

Preferably, the aforementioned dosage can be provided by a single administration (bolus) or may be achieved by more than one administration steps within the day. Moreover, it is also envisaged that the dosage is, preferably, provided by using continuous release devices which provide for a continuous administration of the dosage over the day.

How the composition can be formulated in a proper manner in order to provide the aforementioned daily dosage is well known in the art. Typical formulations may be tablets, capsules, solutions such as tinctures for dermal or liquids for oral applications, injection solutions, gels or ointments.

The composition according to the present invention may be used together with anti-inflammatory drugs or together with other resolving drugs for inflammation. Anti-inflammatory drugs may be non-steroidal anti-inflammatory drugs (NSAIDs). Such NSAIDs may be selective or non- selective inhibitors of cyclooxygenase 2 (COX-2) that are commonly used for anti-inflammatory therapy. Typical NSAIDs are Salicylates and, preferably, Aspirin, Diflunisal, Salicylic acid and other salicylates, Salsalate, Propionic acid derivatives and, preferably, Ibuprofen, Dexibuprofen, Naproxen, Fenoprofen, Ketoprofen, Dexketoprofen, Flurbiprofen, Oxaprozin, Loxoprofen, Acetic acid derivatives and, preferably, Indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Aceclofenac, Nabumetone, Enolic acid (Oxicam) derivatives and, preferably, Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lomoxicam, Phenylbutazone, Anthranilic acid derivatives (Fenamates) and, preferably, Mefenamic acid, Meclofenamic acid, Flufenamic acid, Tolfenamic acid, and selective COX-2 inhibitors (Coxibs), preferably, Celecoxib, Parecoxib, and Etoricoxib. Besides NSAIDs, other homeopathic drugs or compositions may also be applied together with the composition for use in accordance with the present invention. The aforementioned anti-inflammatory drugs or other resolving drugs for inflammation may be administered together with the composition according to the invention or sequentially before or after administration of said composition. If the composition is to be administered together with the said anti-inflammatory drugs or other resolving drugs for inflammation, this may be achieved by administering a single composition consisting of the composition for use in accordance with the present invention and said anti-inflammatory drugs or other resolving drugs for inflammation or they may be administered as separate compositions.

Advantageously, it has been found in accordance with the present invention that a composition comprising the aforementioned ingredients when administered to mice suffering from zymosan- induced peritonitis was able to resolve efficiently inflammation. The composition positively modified the kinetics of neutrophil recruitment and shortened the resolution index by almost 6 hours compared to a control. Moreover, macrophages were significantly increased in comparison to the control group at the site of inflammation having a positive impact on inflammation resolution after 24 hours as well. Finally, it was observed that synthesis of various SPMs was significantly increased after 4 hours in the treatment group compared to the control. Accordingly, the composition for use according to the invention was identified to exhibit strong inflammation resolving properties in a model organism. Thanks to the present invention it will be possible to effectively resolve inflammation and, in particular, chronic inflammation in various pathological contexts.

In general, the present invention also relates to a method of resolving inflammation in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a composition comprising the following ingredients: Achillea millefolium, Aconitum napellus, Arnica montana, Atropa bella-donna, Bellis perennis, Calendula officinalis, Echinacea purpurea, Echinacea, Hamamelis virginiana, Hepar sulfuris, Hypericum perforatum, Matricaria recutita, Mercurius solubilis Hahnemanni, and Symphytum officinale. Typically, a subject being in need of resolution of inflammation is a subject as specified elsewhere herein, and, in particular a subject suffering from chronic inflammation or a disease associated therewith as mentioned herein.

In the following, particular preferred embodiments of the composition for use in accordance with the present invention are specified:

In one preferred embodiment of the composition for use, said resolving inflammation involves reduction of neutrophil recruitment to the site of inflammation. In another preferred embodiment of the composition for use, said resolving inflammation involves inducing the synthesis of at least one specialized pro-resolving mediator (SPM). More preferably, said at least one SPM is selected from the group consisting of: LxA₄, LxB₄, RvD₂, RvDs, Mar-2, and NPDi. More preferably, said at least one SPM is significantly increased after 4 hours upon administration of the composition to a subject compared to a control subject.

In a preferred embodiment of the composition for use, said resolving inflammation involves recruitment of macrophages. More preferably, said macrophages are significantly increased at the site of inflammation after 24 hours upon administration of the composition to a subject compared to a control subject.

In yet a preferred embodiment of the composition for use, said ingredients of the composition are used in the following homeopathic dilutions:

Achillea millefolium D3,

Aconitum napellus D2,

Arnica montana D2,

Atropa bella-donna D2,

Bellis perennis D2,

Calendula officinalis D2,

Echinacea purpurea D2,

Echinacea D2,

Hamamelis virginiana Dl,

Hepar sulfuris D6,

Hypericum perforatum D2,

Matricaria recutita D3,

Mercurius solubilis Hahnemanni D6, and

Symphytum officinale D6.

More preferably, said composition is to be administered in an amount corresponding to the amount provided by at least about 1.5 ml composition per kg body weight for 6 days in mice.

In a preferred embodiment of the composition for use, said inflammation is chronic inflammation. More preferably, said inflammation is associated with a disease or disorder selected from the group consisting of: rheumatoid arthritis, osteoarthritis, peritonitis, irritable bowel syndrome, COPD, systemic lupus erythematosus, multiple sclerosis, psoriasis, diabetes mellitus type 1, musculoskeletal injuries, muscle inflammation, and joint inflammation, such as ankle sprain.

In a preferred embodiment of the composition for use, said composition is to be administered to the subject together with an anti-inflammatory drug. In yet a preferred embodiment of the composition for use, said composition is to be administered to the subject after treatment of the said subject with an anti-inflammatory drug.

More preferably, said anti-inflammatory drug is a non-steroidal anti-inflammatory drug (NS AID) and, most preferably, is selected from the group consisting of: Salicylates and, preferably, Aspirin, Diflunisal, Salicylic acid and other salicylates, Salsalate, Propionic acid derivatives and, preferably, Ibuprofen, Dexibuprofen, Naproxen, Fenoprofen, Ketoprofen, Dexketoprofen, Flurbiprofen, Oxaprozin, Loxoprofen, Acetic acid derivatives and, preferably, Indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Aceclofenac, Nabumetone, Enolic acid (Oxicam) derivatives and, preferably, Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lomoxicam, Phenylbutazone, Anthranilic acid derivatives (Fenamates) and, preferably, Mefenamic acid, Meclofenamic acid, Flufenamic acid, Tolfenamic acid, and selective COX-2 inhibitors (Coxibs), preferably, Celecoxib, Parecoxib, and Etoricoxib.

In a preferred embodiment of the composition for use, said subject is a human.

The invention also relates to a pharmaceutical composition comprising the composition described herein above and an anti-inflammatory drug, preferably, as described herein above.

The present invention also contemplates a kit for use in resolving inflammation in a subject in need thereof comprising the (i) composition described above and (ii) an anti-inflammatory drug, preferably, as described above.

All references cited throughout this description are herewith incorporated by reference with respect to their disclosure contents in their entireties and with respect to the disclosure content to which it is specifically referenced.

FIGURES

Figure 1: Neutrophil trafficking during zymosan-induced peritonitis. HE-100 and vehicle (1.5 ml/kg) were administered i.p. once daily for 6 days. Following a time lapse of 12-13 hours after the last injection peritonitis was induced by zymosan (0.1 mg/mouse). At 0, 4, 8 and 24 hours, mice (6-8 animals/group/time point) were euthanized, peritoneal exudates were collected and analyzed by flow cytometry. HE- 100 positively modifies kinetics of neutrophil recruitment and shortens the resolution index (Ri) by almost 6 hours. Ri for an investigated compound is defined as the time gain to reduce the neutrophil number to 50 % compared to the control condition: Ri = T50 vehicle-T5o compound.

Figure 2: Macrophage trafficking during zymosan- induced peritonitis. HE-100 and vehicle (1.5 ml/kg) were administered i.p. once daily for 6 days. Following a time lapse of 12-13 hours after the last injection peritonitis was induced by zymosan (0.1 mg/mouse). At 0, 4, 8 and 24 hours, mice were euthanized, peritoneal exudates were collected and analyzed by flow cytometry. More macrophages were detected in the treatment group at 24 hours. Macrophages are involved in the clearance of debris at the inflamed site (efferocytosis). Increase in their numbers is believed to have a positive impact on resolution. *p- value < 0.05.

Figure 3: Synthesis of SPMs 4 hours after zymosan- induced peritonitis. HE- 100 and vehicle (1.5 ml/kg) were administered i.p. once daily for 6 days. Following a time lapse of 12-13 hours after the last injection peritonitis was induced by zymosan (0.1 mg/mouse). At 0, 4, 8 and 24 hours, mice were euthanized, peritoneal exudates were collected and analyzed by LC-MS/MS. Increased synthesis of SPMs were detected in the treatment group at 4 hours. Increase in their numbers is believed to have a positive impact on resolution. *p- value < 0.05.

EXAMPLES

The invention shall now be illustrated by the following Examples which shall, whatsoever, not be construed as limiting the scope of the invention in any way.

Example 1: HE-100 and vehicle (1.5 ml/kg) were administered i.p. once daily for 6 days. Mice have been weighed daily to adapt the volume of compounds. Following a time lapse of 12-13 hours after the last injection peritonitis was induced by zymosan (0.1 mg/mouse). At 0, 4, 8 or 24 hours, mice were euthanized (6-8 animals/ group/ time point). The inflammatory exudate was obtained by washing the peritoneal cavity with PBS. Cells were immediately counted using a cell counter Scepter (Merck Millipore) and labelled for subpopulation identification and quantification by cytometry (Macs quant analyzer - Miltenyi). Exudates were centrifuged and suspended to obtain a suspension at lxlO⁶ cells/ml. The cells were then labelled with the following antibody (Miltenyi biotech): CD45-vioblue (Miltenyi); F4/80-FITC (Miltenyi); GR1- PE (Biolegend). Among CD45 positive cells, neutrophils were sorted as follow: GRl⁺, F4/80 .

Example 2: HE-100 and vehicle (1.5 ml/kg) were administered i.p. once daily for 6 days. Mice have been weighed daily to adapt the volume of compounds. Following a time lapse of 12-13 hours after the last injection peritonitis was induced by zymosan (0.1 mg/mouse). At 0, 4, 8 or 24 hours, mice were euthanized (6-8 animals/ group/ time point). The inflammatory exudate was obtained by washing the peritoneal cavity with PBS. Cells were immediately counted using a cell counter Scepter (Merck Millipore) and labelled for subpopulation identification and quantification by cytometry (Macs quant analyzer - Miltenyi). Exudates were centrifuged and suspended to obtain a suspension at lxlO⁶ cells/ml. The cells were then labelled with the following antibody (Miltenyi biotech): CD45-vioblue (Miltenyi); F4/80-FITC (Miltenyi); GR1- PE (Bio legend). Among CD45 positive cells, resident macrophages were sorted as follow: GRF, F4/80^+,hlgh; infiltrating monocytes were sorted as GR1 , F4/80⁺. Example 3: The extraction protocol and FC/MS/MS analysis were performed as described (Fe Faouder et al, 20l3)and adapted from Ambiotis Standard Operating Procedure. Dedicated samples of peritoneal lavages were extracted using oasis HFB 96 wells solid phase extraction (Waters). FC-MS/MS analysis was performed on U-HPFC system (Agilent FC1290 Infinity) coupled to Agilent 6490 triple quadrupole MS (Agilent Technologies) equipped with electro- spray ionization operating in negative mode. Reverse-phase U-HPFC were performed with Kinetex Biphenyl column (2.1 mm*50 mm* 1.7 pm) (Phenomenex) maintained at 50 °C. For monitoring and quantification of bioactive lipids, analyses were run in multiple reaction monitoring (MRM) detection mode. Identification was conducted using pure authentic standards. Peak detection, integration, and quantitative analysis were done by use of MassHunter Quantitative Analysis Software (Agilent Technologies). Bioactive lipids that were quantified in samples are:

FXA₄ for Arachidonic acid derived mediators.

RVD₂, RvDs and NPDi for Docosahexaenoic acid derived mediators.

CITED LITERATURE

Le Faouder, P. et al. (2013)‘LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites’, Journal of

Chromatography B , 932, pp. 123-133. doi: l0.l0l6/j.jchromb.20l3.06.0l4.

Lee, C. H. (2012)‘Resolvins as new fascinating drug candidates for inflammatory diseases’, Archives of Pharmacal Research, 35(1), pp. 3-7. doi: l0.l007/sl2272-0l2-0l2l-z.

Montero-Melendez, T. et al. (2011)‘The melanocortin agonist AP214 exerts anti-inflammatory and proresolving properties’, American Journal of Pathology. Elsevier Inc., 179(1), pp. 259-269. doi: 10.1016/j.ajpath.20l 1.03.042.

Sansbury, B. E. and Spite, M. (2016)‘Resolution of acute inflammation and the role of resolvins in immunity, thrombosis, and vascular biology’, Circulation Research, 119(1), pp. 113-130. doi: 10.1161/CIRCRESAHA.116.307308.

Schneider, C. (2011)‘Traumeel: an emerging option to nonsteroidal anti-inflammatory drugs in the management of acute musculoskeletal injuries’, International Journal of General Medicine,

4, pp. 225-234. doi: l0.2l47/ijgm.sl6709.

Serhan, C. N. (2014)‘Pro-resolving lipid mediators are leads for resolution physiology’, Nature, 510(7503), pp. 92-101. doi: l0.l038/naturel3479.

Serhan, C. N. (2017)‘Treating inflammation and infection in the 21 st century: New hints from decoding resolution mediators and mechanisms’, FASEB Journal, 31(4), pp. 1273-1288. doi:

10.1096/fj .201601222R.

Serhan, C. N. and Levy, B. D. (2018)‘Resolvins in inflammation: Emergence of the pro resolving superfamily of mediators’, Journal of Clinical Investigation, 128(7), pp. 2657-2669. doi: 10.1172/JCI97943.

Sugimoto, M. A. et al. (2016)‘Resolution of inflammation: What controls its onset?’, Frontiers in Immunology, 7(APR). doi: 10.3389/fimmu.20l6.00160.

Wu, S.-H. et al. (2013)‘Efficacy and safety of l5(R/S)-methyl-lipoxin A 4 in topical treatment of infantile eczema’ , British Journal of Dermatology, 168(1), pp. 172-178. doi: 10.111 l/j.1365- 2133.2012.11177.X.

Claims

1. A composition for use in resolving inflammation in a subject in need thereof, wherein said composition comprises the following ingredients: Achillea millefolium, Aconitum napellus, Arnica montana, Atropa bella-donna, Bellis perennis, Calendula officinalis, Echinacea purpurea, Echinacea, Hamamelis virginiana, Hepar sulfuris, Hypericum perforatum, Matricaria recutita, Mercurius solubilis Hahnemanni, and Symphytum officinale.

2. The composition for use of claim 1, wherein said resolving inflammation involves reduction of neutrophil recruitment to the site of inflammation.

3. The composition for use of claim 1 or 2, wherein said resolving inflammation involves inducing the synthesis of at least one specialized pro-resolving mediator (SPM).

4. The composition for use of claim 3, wherein said at least one SPM is selected from the group consisting of: LxA₄, LxB₄, RvD₂, RvDs, Mar-2, and NPDi.

5. The composition for use of claims 3 or 4, wherein said at least one SPM is significantly increased after 4 hours upon administration of the composition to a subject compared to a control subject.

6. The composition for use of any one of claims 1 to 5, wherein said resolving inflammation involves recruitment of macrophages.

7. The composition for use of claim 6, wherein said macrophages are significantly increased at the site of inflammation after 24 hours upon administration of the composition to a subject compared to a control subject.

8. The composition for use of any one of claims 1 to 7, wherein said ingredients of the composition are used in the following homeopathic dilutions:

Achillea millefolium D3,

Aconitum napellus D2,

Arnica montana D2,

Atropa bella-donna D2,

Bellis perennis D2,

Calendula officinalis D2, Echinacea purpurea D2,

Echinacea D2,

Hamamelis virginiana Dl,

Hepar sulfuris D6,

Hypericum perforatum D2,

Matricaria recutita D3,

Mercurius solubilis Hahnemanni D6, and

Symphytum officinale D6.

9. The composition for use of claim 8, wherein said composition is to be administered in an amount corresponding to the amount provided by at least about 1.5 ml composition per kg body weight for 6 days in mice.

10. The composition for use of any one of claims 1 to 9, wherein said inflammation is chronic inflammation.

11. The composition for use of any one of claims 1 to 10, wherein said inflammation is associated with a disease or disorder selected from the group consisting of: rheumatoid arthritis, osteoarthritis, peritonitis, irritable bowel syndrome, COPD, systemic lupus erythematosus, multiple sclerosis, psoriasis, diabetes mellitus type 1, musculoskeletal injuries, muscle inflammation, and joint inflammation, such as ankle sprain.

12. The composition for use of any one of claims 1 to 11, wherein said composition is to be administered to the subject together with an anti-inflammatory drug.

13. The composition for use of any one of claims 1 to 11, wherein said composition is to be administered to the subject after treatment of the said subject with an anti-inflammatory drug.

14. The composition for use of claim 12 or 13, wherein said anti-inflammatory drug is a non steroidal anti-inflammatory drug (NS AID).

15. The composition for use of claim 14, wherein said NSAID is selected from the group consisting of: Salicylates and, preferably, Aspirin, Diflunisal, Salicylic acid and other salicylates, Salsalate, Propionic acid derivatives and, preferably, Ibuprofen, Dexibuprofen, Naproxen, Fenoprofen, Ketoprofen, Dexketoprofen, Flurbiprofen, Oxaprozin, Loxoprofen, Acetic acid derivatives and, preferably, Indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Aceclofenac, Nabumetone, Enolic acid (Oxicam) derivatives and, preferably, Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lomoxicam, Phenylbutazone, Anthranilic acid derivatives (Fenamates) and, preferably, Mefenamic acid, Meclofenamic acid, Flufenamic acid, Tolfenamic acid, and selective COX-2 inhibitors (Coxibs), preferably, Celecoxib, Parecoxib, and Etoricoxib.

16. The composition for use of any one of claims 1 to 15, wherein said subject is a human.

17. A pharmaceutical composition comprising the composition described in any one of claims 1 to 16 and an anti-inflammatory drug, preferably, as described in any one of claims 11 to 16.

18. A kit for use in resolving inflammation in a subject in need thereof comprising the (i) composition described in any one of claims 1 to 17 and (ii) an anti-inflammatory drug, preferably, as described in any one of claims 11 to 17.