WO2016020724A1

WO2016020724A1 - Sphaeranthus indicus composition as il-17 inhibitor and uses thereof

Info

Publication number: WO2016020724A1
Application number: PCT/IB2014/063766
Authority: WO
Inventors: Bindu HEGDE; Asha Kulkarni-Almeida; Chandni THAKKAR; Prabha MISHRA; Sanjeev Kumar
Original assignee: Piramal Enterprises Limited
Priority date: 2014-08-07
Filing date: 2014-08-07
Publication date: 2016-02-11

Abstract

The present invention relates to herbal composition comprising an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus as interleukin-17 (IL-17) inhibitor. Further, the invention relates to the use of the said herbal composition for the treatment of disorders mediated by IL-17.

Description

SPHAERANTHUS INDICUS COMPOSITION AS IL-17 INHIBITOR

AND USES THEREOF

Field of the invention

The present invention relates to herbal composition comprising a therapeutically effective amount of an extract of flowering and fruiting heads of the plant Sphaeranthus indicus either alone or in combination with a pharmaceutically acceptable excipient, for use as interleukin-17 (IL-17) inhibitor. Further, the present invention relates to a method of treating a disease or a disorder mediated by IL-17 comprising administering to a subject in need thereof, a therapeutically effective amount of said herbal composition.

Background of the invention

Interleukin 17, also known as IL-17A, was discovered in 1993 originally as a rodent T cell cDNA transcript, cytotoxic T lymphocyte associated antigen 8 (CTLA8). Human IL-17 was subsequently identified from T cells and its biological activities on fibroblasts are reported in J. Immunology, 1995, 155(12), 5483-5486. There are five additional members of the IL-17 family, designated as IL-17B, IL-17C, IL-17D, IL-17E and IL-17F. Out of these, IL-17F is very closely related to IL-17 and can form a heterodimer with IL-17. IL-17E, also named as IL-25, is classified as Th2 cytokine. There are five receptors for IL-17 family of cytokines, i.e. IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE, of which IL-17RA and IL-17RC mediate the biological activity of IL-17. While IL-17 is produced mainly by T-cells, its receptor is expressed ubiquitously in various cell types, including myeloid cells, epithelial cells and fibroblasts. This finding indicates that IL-17 exerts various biological functions in vivo and therefore, it might be involved in the pathogenesis of a wide range on inflammatory disorders as well as infectious conditions. (Journal of Inflammation Research, 2010, 3, 33- 44).

In fact, interleukin-17 (IL-17), which is a pro-inflammatory cytokine, has received considerable attention since the discovery of a distinct CD4(+) T helper (Th) cell subset that produces it, known as the Thl7 cell subset. Thl7 cells are a distinct phenotype distinguished from the Thl and Th2 cells by the presence of intracellular IL-17 production. These cells are characterized as preferential producers of interleukin- 17 A, IL-17F, IL-21 and IL-22. Thl7 cells and their effector cytokines mediate host defensive mechanisms to various infections, especially extracellular bacterial infections and are involved in the pathogenesis of many autoimmune diseases. The effector cytokines of Thl 7 cells, therefore, mediates the crucial crosstalk between immune system and tissues and play indispensable roles in tissue immunity (Immunity, 2008, 28(4), 454-467).

As has been discussed earlier, the role of Thl7 cell subset and its signature cytokine, IL-17 in certain autoimmune diseases has been very well explored. Further, the role of Thl7 cell subset and IL-17 is explored as indicated through the study reported in J. Exp. Med., 2009, 206(7), 1457-1464. This study reports that IL-17 is elevated in several types of cancer and is also known to contribute to tumor growth. Adoptive transfer studies and analysis of the tumor microenvironment suggest that CD4+T cells are the predominant source of IL-17. Enhancement of tumor growth by IL-17 involves direct effects on tumor cells and tumor- associated stromal cells, which bear IL-17 receptors.

IL-17 and Thl7 cells play an important role in various immune-mediated diseases and hence, a compound or principle that blocks the activation of Thl7 cells may have application in the treatment of transplant rejection (Immunol. Rev. 2008, June: 223, 87-113); cystic fibrosis; atopic dermatitis; cancers such as colorectal cancer (Anticancer Research, 2006, 26, 4213-4216), ovarian cancer (Biochemical and Biophysical Research Communications, 2001, 282, 735-738), breast cancer (Breast Cancer Research, 2008, 10(6), 95), cervical cancer (Cancer Res., 1999, 54, 3698-3704), skin cancer (Cancer Res., 2010, 70(24), 10112-20), non-small cell lung cancer, melanoma and bladder cancer, multiple myeloma (Blood, 2010, 116, 3554-3563), non-Hodgkin lymphoma; multiple sclerosis; bone metastasis; uveitis; T cell mediated disorders such as acquired immune deficiency syndrome; viral diseases and asthma (Respiratory Research, 2010, 11, 78).

Currently, the treatment strategy and prognosis of autoimmune disease have greatly changed since the introduction of biologic agents such as anti- tumor necrosis factor-a (TNF- a), monoclonal antibody (mAb), anti-IL-6, anti-CD20 mAbs and soluble CTLA4-Ig which are demonstrated to be highly effective in the treatment of RA (Journal of Inflammation Research, 2010, 3, 33- 44). However, the biological-based therapy is expensive, not always effective in the treatment of diseases and may have certain drawbacks associated with its use.

Hence, there is a need for the development of improved and alternative medicaments with reduced side effects for the prevention and treatment of disorders caused by activation of Thl7 cells and increased IL-17 production.

Herbs have been known and used throughout the world for the treatment of certain disease conditions. There is evidence that products derived from plants have potential pharmacological and therapeutic effects on mammals but are devoid of deleterious side effects or have fewer side effects as compared to the synthetic based therapeutic agents. Sphaeranthus indicus is a common weed found in rice fields. It belongs to the family Asteraceae. This plant is known as mahamundi or gorakhmundi in the literature of Ayurveda. This plant, mostly available throughout India, is a branched herb with purple flowers. The use of this plant either alone or in combination with other known herbs in the treatment of certain diseases or disorders is reported (US7635494, US7344738, International Journal of Pharmacology, 2011, 7(2), 171-179 and Int. J. Ayurveda Res., 2010, 1(4), 247-253).

The present invention relates to herbal composition comprising a therapeutically effective amount of an extract of flowering and fruiting heads of the plant Sphaeranthus indicus, for use in the treatment of disorders mediated by interleukin 17 (IL-17).

Summary of the invention

The present invention relates to a method for the treatment of a disease or a disorder mediated by interleukin 17 (IL-17), comprising administering to a subject in need thereof, herbal composition comprising a therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus.

In another aspect, the present invention relates to a method for the treatment of a disease or a disorder mediated by interleukin 17 (IL-17), comprising administering to a subject in need thereof, herbal composition comprising a therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus in combination with a pharmaceutically acceptable excipient.

In another aspect, the present invention relates to a method for the treatment of a disease or a disorder mediated by IL-17, comprising administering to a subject in need thereof, herbal composition comprising therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus in combination with another therapeutically active agent.

In another aspect, the present invention relates to use of herbal composition comprising an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus as interleukin- 17 (IL-17) inhibitor.

Further, the present invention relates to the use of herbal composition comprising therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus, for the treatment of a disorder or a disease mediated by IL-17. According to a further aspect of the present invention, there is provided use of the herbal composition comprising therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus for the manufacture of a medicament for use in the treatment of a disease or a disorder mediated by IL-17.

Brief description of the drawings

Fig. 1 is graphical representation of the effect of the extract of Sphaeranthus indicus on production of IL-17 in PMA-ionomycin stimulated hPBM cells.

Fig. 2 is graphical representation of the effect of the extract of Sphaeranthus indicus on proliferation of PMA-ionomycin stimulated and unstimulated hPBM cells.

Fig. 3a depicts the effect of extract of Sphaeranthus indicus on intracellular IL-17 accumulations in donor 1.

Fig. 3b depicts the effect of extract of Sphaeranthus indicus on intracellular IL-17 accumulations in donor 2.

Fig. 4 is graphical representation of the effect of the extract of Sphaeranthus indicus on secretion of IL-17 in cytokine stimulated hPBM cells.

Fig. 5 depicts the effect of extract of Sphaeranthus indicus on intracellular and secreted IL -17 protein levels in PMA-ionomycin and cytokine stimulated hPBM cells.

Fig. 6a depicts the effect of extract of Sphaeranthus indicus on inhibition of IL-21 in hPBM cells.

Fig. 6b depicts the effect of extract of Sphaeranthus indicus on inhibition of IL-22 in hPBM cells.

Description of the invention

It should be understood that the detailed description and specific examples, while indicating embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. One skilled in the art, based upon the description herein, may utilize the present invention to its fullest extent. The following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. However, the general terms used hereinbefore and hereinafter in the context of the present invention preferably have within the context of this disclosure the following meanings, unless otherwise indicated. Thus, the definitions of the general terms as used in the context of the present invention are provided herein below.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

"An extract of the plant Sphaeranthus indicus", or "an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus" or "the extract" mentioned herein means a blend of compounds present in the flowering and fruiting heads of the plant, Sphaeranthus indicus. Such compounds are extracted from the flowering and fruiting heads of the plant using extraction procedures well known in the art (e.g., the use of solvents such as alcohols, alkyl esters, alkyl ethers, alkyl ketones, chloroform, petroleum ether, hexane and/or water). More specifically, "an extract of the plant Sphaeranthus indicus" or "extract of the flowering and fruiting heads of the plant Sphaeranthus indicus" or "the extract", mentioned herein refers to an extract of the dried flowering and fruiting heads of the plant Sphaeranthus indicus prepared using an alcohol e.g. methanol. Some of the extraction methods of the dried flowering and fruiting heads of the plant Sphaeranthus indicus are described in the PCT publication WO2007036900. In the present invention, an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus can be a standardized extract containing 3 a- hydroxy-5a,9-dimethyl-3-methylene-3a,4,5,5a,6,7,8,9b-octahydro-3H-naphtho[l,2-b]furan-2- one (7-hydroxy frullanolide (7-HF)) as a bioactive marker. The extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus containing 7-HF as the bioactive marker is described in PCT publication WO2007036900.

The term "IL-17 inhibitor" as used herein means the extract referred to herein which decreases the production of IL-17 in a subject. Alternatively, said term means that the extract can block the action or function of IL-17 in a subject.

The term "capable of inhibiting proliferation and stimulation of Thl7 cells" as used herein means capacity of the extract to decrease the proliferation and stimulation of Thl7 cells. Alternatively, said term means that the extract can block the action or function of proliferation and stimulation of Thl7 cells.

The term "capable of inhibiting IL-17 production by T cells" as used herein means capacity of the extract to decrease the production of IL-17 by T cells. Alternatively, said term means that the extract can block the action or function of the production of IL17 by T cells. The term "pharmaceutically acceptable" as used herein means the carrier, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

The term "pharmaceutically acceptable excipient" as used herein means a non-toxic, inert solid, semi-solid, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable excipients are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; malt; gelatin; talc; as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents; preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

The term " therapeutically active agent" as used herein is intended to encompass any known therapeutically active agent or investigational new drug that will produce a therapeutically beneficial pharmacological response when administered to a subject, both human and animal, in need thereof. Suitable known therapeutically active agents include, but are not limited to analgesics, immunosuppressants, anti-inflammatories, and anti-cancer agents, other than the extract of the flowering and fruiting heads of the plant Sphaeranthus indicus, which may be useful in treating the disorders mediated by IL-17. More than one therapeutically active agent may be used, if desired, in combination with the herbal composition specified herein.

The term, "therapeutically effective amount" as used herein means an amount of the active ingredient or composition, in the context of the present invention, the Sphaeranthus indicus extract sufficient to significantly induce a positive modification in the condition to be regulated or treated, but low enough to avoid side effects if any (at a reasonable benefit/risk ratio), within the scope of sound medical judgment. The therapeutically effective amount of the extract will vary with the particular condition being treated, the age and physical condition of the end user, the severity of the condition being treated/prevented, the duration of the treatment, the nature of concurrent therapy, the specific composition employed, the particular pharmaceutically acceptable carrier utilized, and like factors. As used herein, all percentages are by weight unless otherwise specified. The term "treat" or "treatment" as used herein includes prophylaxis, amelioration [i.e., reduction in the severity of the disease or accompanying symptoms], regression or curing of a disease or a disorder mediated by IL-17.

The term "herbal composition comprises an extract of the plant Sphaeranthus indicus" as used herein refers to the herbal compositions containing the extract of the flowering and fruiting heads of the plant Sphaeranthus indicus prepared using an alcohol e.g. methanol and pharmaceutically acceptable excipient. The herbal composition may contain the standardised methanolic extract as described in the PCT publication WO2007036900. One or more of the compositions described in the PCT publication WO2007036900 can be used as IL-17 inhibitor.

The term "subject" as used herein, refers to an animal, preferably a mammal, most preferably a human, who is in the need of treatment for the disorders mediated by IL-17. The term subject may be interchangeably used with the term patient in the context of the present invention.

The term "mammal" as used herein is intended to encompass humans, as well as non- human mammals which are susceptible to disorders mediated by IL-17. Non-human mammals include, but are not limited to domestic animals such as cows, pigs, horses, dogs, cats, rabbits, rats and mice, and non-domestic animals.

The term "disease(s) or disorder(s) mediated by IL-17" as used herein refers to a condition in which IL-17 has a significant role in the onset or progression of that condition. Examples of diseases or disorders mediated by IL-17 include, but are not limited to, transplant rejection, cystic fibrosis, colorectal cancer, ovarian cancer, breast cancer, cervical cancer, skin cancer, non- small cell lung cancer, melanoma, bladder cancer, multiple myeloma, non-Hodgkin lymphoma, multiple sclerosis, bone metastasis, uveitis, or T cell mediated disorders selected from acquired immune deficiency syndrome, viral diseases or asthma.

According to one aspect, the present invention relates to a method for the treatment of a disease or a disorder mediated by interleukin-17 comprising administering to a subject in need thereof, herbal composition comprising a therapeutically effective amount of an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus.

Accordingly, in an embodiment, the present invention relates to a method for the treatment of a disease or a disorder mediated by interleukin-17, comprising administering to a subject in need thereof, herbal composition comprising a therapeutically effective amount of a extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus either alone or in combination with a pharmaceutically acceptable excipient.

According to another aspect, the present invention provides a method for the treatment of a disease or a disorder mediated by IL-17, comprising administering to a subject in need thereof, herbal composition comprising a therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus in combination with a known therapeutically active agent.

The suitable known therapeutically active agents include, but are not limited to analgesics, immunosuppressants, anti-inflammatory agents and anti-cancer agents.

Analgesic is selected from the group comprising diclofenac, ketoprofen, ibuprofen and the like.

Immunosuppressant is selected from the group comprising cyclosporine, tacrolimus, sirolimus, monoclonal antibodies and the like.

Anti-inflammatory agent may include, but not limited to, steroids such as prednisolone, hydrocortisone; disease modifying anti-rheumatic drugs (DMARDs) such as methotrexate, sulfasalazine; or NSAIDS such as naproxen, diclofenac or ibuprofen.

The anti-cancer agent may include, but not limited to paclitaxel, docetaxel, doxorubicin, gemcitabine sorafenib, lapatinib, erlotinib, cisplatin or 5-fluorouracil.

In another embodiment, the present invention relates to herbal composition comprising an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus for the treatment of a disease or a disorder mediated by IL-17.

In another embodiment, the present invention relates to herbal composition comprising an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus and at least one pharmaceutically acceptable excipient for the treatment of a disease or a disorder mediated by IL-17.

In an embodiment of the present invention the herbal composition is an oral or a topical or a transdermal composition.

In an embodiment the oral composition is in the form of a tablet, capsule or syrup.

In an embodiment the topical composition is in the form of cream, gel or ointment.

In accordance with the present invention, there is provided the use of herbal composition comprising an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus as interleukin-17 (IL-17) inhibitor. In another embodiment, the present invention relates to the use of herbal composition comprising an extract of the flowering and fruiting heads of the plant alone or in combination with at least one anti-inflammatory agent for the treatment of a disease or a disorder mediated by IL-17.

Another embodiment of the present invention provides a method for inhibiting proliferation and stimulation of Thl7 cells, comprising administering to a subject in need thereof, herbal composition comprising an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus in an amount capable of inhibiting proliferation and stimulation of Thl7 cells.

In another embodiment the present invention relates to herbal composition for use in the treatment of a disease or a disorder mediated by IL-17 comprising an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus.

In another embodiment the present invention relates to herbal composition for use in the treatment of diseases or disorders mediated by IL-17 comprising a therapeutically effective amount of extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus either alone or in combination with a pharmaceutically acceptable excipient.

In another embodiment of the present invention there is provided use of herbal composition comprising an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus for the inhibition of proliferation and stimulation of Thl7 cells.

Another embodiment of the present invention provides a method for inhibiting interleukin-17 (IL-17) production by T cells, comprising administering to a subject in need thereof, herbal composition comprising an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus in an amount capable of inhibiting IL-17 production by T cells.

In another embodiment, the present invention relates to the use of herbal composition comprising therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus, for the treatment of a disorder mediated by IL-17.

In an embodiment, the extract of the flowering and fruiting heads of the plant Sphaeranthus indicus contained in the herbal composition is prepared using an alcohol such as methanol, ethanol, isopropanol, propanol and the like or mixture thereof,

In yet another embodiment, the extract of the flowering and fruiting heads of the plant Sphaeranthus indicus contained in the herbal composition is prepared using methanol.

Accordingly, in an embodiment the herbal composition contains methanolic extract of the flowering and fruiting heads of the plant Sphaeranthus indicus. In an embodiment, the extract contained in the herbal composition as described herein is obtained from dried flowering and fruiting heads of the plant Sphaeranthus indicus.

According to another embodiment the disease(s) or disorder(s) mediated by IL-17 and/or proliferation and stimulation of Thl7 cells that are treated using herbal composition comprising a therapeutically effective amount of an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus, include, but not restricted to, transplant rejection, cystic fibrosis, colorectal cancer, ovarian cancer, breast cancer, cervical cancer, skin cancer, non-small cell lung cancer, melanoma, bladder cancer, multiple myeloma, non-Hodgkin lymphoma, multiple sclerosis, bone metastasis, uveitis or a T cell mediated disorder selected from acquired immune deficiency syndrome, a viral disease or asthma.

According to an embodiment, the disease or disorder mediated by IL-17, that is treated using herbal composition comprising a therapeutically effective amount of an extract of the plant Sphaeranthus indicus, is selected from transplant rejection, cystic fibrosis, bone metastasis, multiple myeloma, multiple sclerosis or uveitis.

From among the diseases or disorders mediated by IL-17, a specific reference is made to multiple myeloma, which is the most common primary malignant neoplasm of the skeletal system. Different treatment options are available for the treatment of multiple myeloma but these are associated with severe adverse effects and affect patient compliance. It is reported that multiple myeloma results from the clonal outgrowth of malignant plasma cells and is primarily confined to the bone marrow (BM). The bone marrow is a reservoir of memory T cells with antigen specificity and capable of priming naive T cell responses. In multiple myeloma, it is known that marrow infiltrating lymphocytes (MILs) are more effectively activated and expanded, possess greater antitumor activity than peripheral blood lymphocytes (PBLs) from the same patients, and thus represent more suitable T cells for adoptive immunotherapy in this disease. Unlike marrow infiltrating lymphocytes (MILs) from normal bone marrow, myeloma MILs possess few regulatory T cells (Tregs) and demonstrate an interleukin-17 phenotype that enhances osteoclast (OC) activation. (Blood, 2010, 116(18), 3554-3563).

Another disorder mediated by IL-17 is asthma which is a common airway disorder that is characterized by chronic airway inflammation, mucus production, and airway hyper responsiveness (AHR) with airway remodeling. Airway inflammation in asthma usually involves polarization of the T lymphocyte response to T helper 2 (Th2) cells. The pathologic role of Th2 cells is mediated through the release of Th2 cytokines that are essential for immunoglobulin E (IgE) synthesis, chemokine production, airway eosinophilia, smooth muscle hyperplasia, mucus production, and AHR. The role of IL-17 in the context of allergic airway inflammation and the therapeutic potential of various strategies targeting IL-17 for asthma, particularly to regulate the IL-17 pathway for the treatment of asthma, is demonstrated and discussed in the report, Park and Lee Respiratory Research 2010, 11, 78.

Uveitis is a group of sight-threatening idiopathic intraocular inflammatory diseases including Behcet's disease (BD), birdshot retinochoroidopathy, sympathetic ophthalmia, Vogt-Koyanagi-Harada, and ocular sarcoidosis, which may be of infectious or autoimmune etiology (PNAS, May 26, 2009, 106(21), 8635-8640). For example, Behcet's disease is a systemic inflammatory disease presumably caused by an autoimmune response. The interleukins, IL-23/IL-17 pathway was demonstrated to be involved in the development and maintenance of certain inflammatory diseases (Investigative Ophthalmology & Visual Science, July 2008, vol. 49, no.7, 3058-3064). IL-2, a pleiotropic type 1 cytokine, promotes differentiation of Thl7 cells. It is discussed in the report, Proc. Natl. Acad. Sci. USA, 2011 Jun 7, 108(23), 9542-7 that IL-21 plays a key role in the development of EAU, suggesting that interfering with the action of this cytokine may have therapeutic potential for uveitis.

Multiple sclerosis (MS) is a CD4+ T cell-mediated autoimmune disease affecting the central nervous system. The widely accepted theory about pathology of MS was based on the data from experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. IL-17- producing CD4+ T cells, driven by IL-23 and referred to as Thl7 cells, play a pivotal role in the pathogenesis of EAE (Allergology International. 2008, 57, 115-120).

According to the present invention the extract of Sphaeranthus indicus blocks activation of pathogenic CD4+ Thl7cells thereby inhibiting interleukin-17 production in stimulated hPBMCs.

According to an aspect of the present invention, there is provided use of the herbal composition comprising an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus, for the manufacture of a medicament for use in the treatment of a disease or a disorder mediated by IL-17.

In an embodiment of the invention there is provided a process for preparation of herbal composition, which comprises mixing an extract of the flowering and fruiting heads of Sphaeranthus indicus with one or more pharmaceutically acceptable carrier s/excipient and formulating into appropriate therapeutic dosage forms. An embodiment of the present invention provides use of the herbal composition of the present invention for the treatment of a disease or a disorder mediated by IL-17, by administrating a therapeutically effective amount of the composition to a subject in need thereof.

In an embodiment, the disease or disorder mediated by IL-17 is multiple myeloma. In an embodiment of the present invention the herbal composition comprising an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus, is oral, topical or transdermal composition.

According to an embodiment of the present invention the herbal composition may be administered orally for example in the form of pills, tablets, coated tablets, capsules, granules, elixirs or syrup. The herbal composition for oral preparation may contain 10% to 100%, or from about 30% to 100 %, or from about 70 % to about 100% by weight of the extract of the flowering and fruiting heads of the plant Sphaeranthus indicus.

The formulation for oral administration is prepared by thoroughly blending the extract of the plant Sphaeranthus indicus into a conventional base such as sugars, starches or lubricants.

The compositions of the present invention can also be used for topical and transdermal administration. The topical compositions useful in the present invention involve formulations suitable for topical application to skin that include but are not limited to lotions, creams, gels, sticks, sprays or ointments. The herbal composition used for topical preparation may contain 1% to 50%, or from about 5% to 30 %, or from about 10 % to about 20% by weight of the extract of the flowering and fruiting heads of the plant Sphaeranthus indicus.

The topical or transdermal formulation is prepared by blending the extract of the plant Sphaeranthus indicus into a conventional base such as oils, waxes or glycols.

In an embodiment the said compositions are provided for the treatment of a disease or a disorder mediated by interleukin 17 (IL-17).

In a further embodiment, the extract of the flowering and fruiting heads of the plant Sphaeranthus indicus can be directly administered to a subject in need thereof.

Actual dosage levels of the extract of the plant Sphaeranthus indicus in the compositions of this invention may be varied, which is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration.

The dose to be administered daily is to be selected to suit the desired effect. The selected dosage level will depend upon a variety of factors including the activity of the extract of the plant Sphaeranthus indicus employed, the route of administration, the time of administration, the rate of excretion of the particular composition being employed, the duration of the treatment, use in combination with the other extracts, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The following examples illustrate but do not limit the scope of the invention. It is to be understood by those of the ordinary skill in the art that the present discussion is of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied in the exemplary construction.

Examples

The following terms/symbol/abbreviations/chemical formulae are employed in the examples:

Example 1

Preparation of extract of Sphaeranthus indicus

Dried flowering and fruiting heads of Sphaeranthus indicus (200g) were pulverized. The powdered material was extracted using methanol (2.5 L) by stirring at 60°C for 3 hrs. The extract was filtered under vacuum. This extraction process was repeated two more times. The extracts were combined and concentrated.

Yield: 23.29 g (11.65 % w/w). Example 2

In vitro assay for inhibitors of IL-17

Cytokine release assay

A modified assay of the procedure given in the reference Proc. Natl. Acad. Sci., 2009, 106 (21), 8635-8640, was used, the disclosure of which is incorporated by reference for the teaching of the assay.

Step 1

Isolation of peripheral blood mononuclear cells (PBMC)

PBMC were obtained from healthy donors by centrifugation of heparinized venous blood over Ficoll/Hypaque solution (Histopaque-1077, Sigma). Mononuclear cells in the buffy layer were collected and washed three times in sterile PBS with 2% FBS. After washing, the cells were suspended in RPMI-1640 media (Bioconcept) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS).

Step 2

Sample preparation

The extract obtained in Example 1 was dissolved in DMSO to prepare the stock solution. Eight different concentrations of the extract obtained in example 1 were prepared by serial dilution of the stock solution. The extract of Example 1 was tested at 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1 μg/ml, 0.3 μg/ml, 1 μg/ml, 3 μg/ml and 10μg/ml concentrations.

Step 3

Assay

The cells were uniformly plated in 96-well tissue culture plates (Nunc 167008) at a concentration of lxlO⁵ cells/well. After plating, the samples as prepared in step 2, were added to the cells and incubated at 37°C for 30 min. in 5% C0₂ atmosphere. The final concentration of DMSO was maintained at 0.5%. The vehicle (0.5% DMSO) was used as control. The cells were then stimulated with 25ng/ml of PMA (P1585, SIGMA) and ΙμΜ ionomycin (SIGMA, 10634). The plates were incubated in 5% C0₂ air humidified atmosphere at 37°C for 48 hrs. At the end of 48 hrs, the supernatant from the cells were collected and ELISA was performed to detect the level of IL- 17 (R&D systems, DY317E) in the supernatant. The experiment was repeated using the blood samples from two more donors. Results:

The results represented in Fig. 1 are the mean value obtained for hPBM cells (hPBMC) obtained from the blood of two donors. Primarily Fig. 1 illustrates the effect of the extract of Sphaeranthus indicus on production of IL-17 in PMA-ionomycin stimulated hPBM cells. During the preliminary screening in hPBMCs, the extract of Sphaeranthus indicus (as obtained in Example 1), after 48 hrs incubation, inhibited the release of IL-17 with IC₅₀ value of 2.4 μg/ml.

Conclusion:

The methanolic extract of the flowering and fruiting heads of Sphaeranthus indicus showed a dose-dependent inhibition of IL-17 release in hPBMCs.

Example 3

Lymphocyte proliferation assay by H thymidine uptake

A modified assay of the procedure given in the reference, J. Immunol. Methods, 1985, 82, 169, was used, the disclosure of which is incorporated by reference for the teaching of the assay.

The cells (as obtained to in step 1 of Example 2) were uniformly plated in 96-well tissue culture plates (Nunc 167008) at a concentration of lxlO⁵ cells/well. After plating, samples (as prepared in step 2 of Example 2) were added to the cells and incubated at 37°C for 30 mins. in 5% C0₂ atmosphere. The final concentration of DMSO was maintained at 0.5%. The vehicle (0.5% DMSO) was used as control. The cells were then stimulated with 25ng/ml of PMA (P1585, SIGMA) and ΙμΜ ionomycin (SIGMA, 10634). The plates were incubated in 5% C0₂ air humidified atmosphere at 37°C for 48 hrs. At the end of 24 hrs cells were pulsed with 0.5μΟΛνε11 of 3 [H] -Thymidine (BRIT, LCT3). The cells were washed twice with PBS to remove excess radioactivity of cells. Radioactivity in the incorporated cells was captured on lysis with Microscint-40, Perkin Elmer and measured by using a liquid scintillation counter (TopCount, Perkin Elmer). The anti-proliferative effect of the sample was measured using the following formula.

Control (CPM) - Treated (CPM)

% Anti-proliferation = X 100

Control (CPM) The controls containing hPBMC with PMA - ionomycin (stimulated) and hPBMC with RPMI (un- stimulated) were used for this assay.

Results:

The results of the above experiment are represented in Fig. 2. The extract of Sphaeranthus indicus (as obtained in Example 1), inhibited proliferation of PMA-ionomycin stimulated hPBMCs (IC50 = 3.0 μg/ml). The IC50 value for the unstimulated hPBMCs was found to be > 10 μg/ml.

Conclusion:

The methanolic extract of the flowering and fruiting heads of Sphaeranthus indicus inhibited the proliferation of cells without any toxic effect to naive cells [Lymphocyte proliferation assay by 3Hthymidine uptake] at the concentrations studied.

Example 4

Detection of intracellular IL-17 accumulation

A modified assay of the procedure given in the reference, Proc. Natl. Acad. Sci., 2009, 106 (21), 8635-8640, was used, the disclosure of which is incorporated by reference for the teaching of the assay.

Step 1

Isolation of hPBMCs

The hPBM cells were obtained from healthy donors. The whole blood was incubated with RosetteSep® Human CD4+T Cell Enrichment Cocktail (Stem cell technologies, 15062) for 20 mins. The incubated blood sample was layered over Ficoll/Hypaque solution and separated by centrifugation. The enriched cells were collected from the density medium: plasma interface and washed three times in sterile PBS with 2% FBS. The cells were then suspended in RPMI-1640 supplemented with lOOU/ml penicillin, 100μg/ml streptomycin and 10% fetal bovine serum (FBS).

Step 2

Sample preparation

The extract obtained in Example 1 was dissolved in DMSO to prepare the stock solution. Two concentrations of 3μg/ml and 10μg/ml, of the extract obtained in Example 1 were prepared by dilution of the stock solution. The extract of Example 1 was tested at 3 μg/ml and 10 μg/ml concentrations.

Step 3

Assay

a) The cells as referred to in step 1, were seeded in T-25 flasks at a density of lxlO⁶ cells/flask. The cells were then treated with sample (as prepared in step 2) and incubated at 37°C for 30mins in 5% C0₂ atmosphere. The final concentration of DMSO was maintained at 0.5%. The vehicle (0.5% DMSO) was used as control. b) The cells were then stimulated with 25ng/ml of PMA (P1585, SIGMA) and ΙμΜ ionomycin (SIGMA, 10634). A protein transport inhibitor, brefeldin A was added to the culture medium (BD Biosciences, 555028). The cells were incubated in 5% C0₂ air humidified atmosphere at 37°C for 12 hrs.

c) The cells were then fixed and permiabilized using BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences, 555028).

d) The cells were stained with CD4 PE (BD Biosciences, 555347) and Alexa Flour Mouse anti-Human IL-17A (BD Biosciences, 560488). Isotype antibody with matched fluorochromes was used as controls. The samples were acquired on FACS Calibur (BD Biosciences) and data were analyzed with BD CellQuest Pro Software (BD Biosciences). The result of two donors is presented in Fig. 3a and Fig. 3b.

Results:

Donor 1: The stimulation of CD4+ T cells with PMA-ionomycin in the presence of brefeldin for 12hrs resulted in 7.35% cells showing intracellular IL-17 accumulation. At 10μg/ml, the extract of Sphaeranthus indicus (as obtained in Example 1) blocked to 0.89 % of the intracellular accumulation of IL-17, resulting in 88 % inhibition of the cytokine.

Donor 2: The stimulation of CD4+ T cells with PMA-ionomycin in the presence of brefeldin for 12 hrs resulted in 2.99% cells showing intracellular IL-17 accumulation. At 3μg/ml, the extract of Sphaeranthus indicus (as obtained in Example 1) blocked to 0.2 % of the intracellular accumulation of IL-17, resulting in 93 % inhibition of the cytokine.

Conclusion:

The methanolic extract of the flowering and fruiting heads of Sphaeranthus indicus inhibited the intracellular accumulation of IL-17 and the effect exerted is significant. Example 5

Inhibition of Thl7 cells stimulated with cytokine

A modified assay of the procedure given in the reference, Proc Natl. Acad. Sci USA, 2011, May 31, 108(22):9220-5 was used, the disclosure of which is incorporated by reference for the teaching of the assay.

Step 1

Isolation of hPBMCs

The hPBMCs were obtained from healthy donors. The whole blood was incubated with RosetteSep® Human CD4+ T Cell Enrichment Cocktail (Stem cell technologies, 15062) for 20mins. The CD4+ T cells were purified from whole blood by layering over Ficoll/Hypaque solution. The cells were then suspended in RPMI-1640 supplemented with 2% FBS and 1% penicillin streptomycin solution.

Step 2

Sample preparation

Sphaeranthus indicus extract obtained in Example 1 was dissolved in DMSO to prepare the stock solution. Eight different concentrations of the extract obtained in example 1 were prepared by serial dilution of the stock solution. The extract of Example 1 was tested at 0.03μg/ml, 0.^g/ml, 0^g/ml, ^g/ml, 3μg/ml, 10μg/ml, 30μg/ml and 100μg/ml concentrations.

Step 3:

Assay:

The cells as referred to in step 1 were seeded in 96-well flat bottom plates coated with 5μg/ml of hCD3antibody at a concentration of lxlO⁵ cells/well. The cells were then treated with sample (as prepared in step 2) and incubated at 37°C for 16 hour in 5% C0₂ atmosphere. The final concentration of DMSO was maintained at 0.5%. The vehicle (0.5% DMSO) was used as control. After half an hour the cells were stimulated with 25μg/ml of hIL-6, 2^g/ml of hTGF-β and ^g/ml of hIL-Ιβ for 6 days at 37°C with 5% C0₂. At the end of 6 days, the supernatant from the cells were collected and ELISA was performed to detect the level of IL- 17 (R&D systems, DY317E) in the supernatant. Results:

The results are represented in Fig. 4. During the preliminary screening in hPBM cells stimulated with hIL-6, hTGF-β and hIL-Ιβ, the extract of Sphaeranthus indicus (as obtained in Example 1) after 6 days incubation suppressed the production of IL-17 cytokine with IC₅₀ value of 1.8+0^g/ml.

Conclusion:

The methanolic extract of the flowering and fruiting heads of Sphaeranthus indicus showed suppression of IL-17 cytokine production in hPBM cells stimulated with cytokine.

Example 6

Evaluation of IL-17 inhibition by Sphaeranthus indicus extract in CD4+ T cells stimulated with PMA-ionomycin using RT-QPCR assay

CD4+ T cells were treated with the sample (ΙμΜ and 10 μΜ) for 3 and 12 hours. Cells not treated with the said sample are referred to as untreated cells. Treated and untreated cells were stimulated with PMA-ionomycin. Stimulated untreated cells were vehicle (DMSO) control cells. RNA was isolated and utilized for RT-QPCR (real-time reverse transcription polymerase chain reaction) assay.

Gene expression was calculated by the 2^~ΔΔα method as described in METHODS, 2001, 25, 402-408 and then expressed on log2 scale. A change in 2-fold or more is considered significant in either direction. Values for the stimulated control group are expressed as fold change over the unstimulated control. Data is expressed as mean + SEM, n=3 per group.

Step 1

Sample preparation

The extract obtained in Example 1 was dissolved in DMSO to prepare the stock solution. Two different concentrations of the extract obtained in example 1 were prepared by serial dilution of the stock solution. The extract of Example 1 was tested at ΙμΜ and 10μΜ concentrations. Step 2

Isolation of hPBMCs

Step 3

Assay

The cells as referred to in step 2, were seeded in T-25 flasks at a density of lxlO⁶ cells/flask. The cells were then treated with sample (as prepared in step 1) and incubated at 37°C for 30mins in 5% C0₂ atmosphere. The final concentration of DMSO was maintained at 0.5%. The vehicle (0.5% DMSO) was used as control. The cells were then stimulated with 25μg/ml of PMA (P1585, SIGMA) and 1 μΜ ionomycin (SIGMA, 10634) and incubated in 5%C0₂ air humidified incubator at 37°C for 3 and 12 hrs. The cells not treated with said sample are referred to as untreated cells.

Step 4

Isolation of RNA:

The treated and untreated CD4+ T cells were harvested by washing with 1% Ca²⁺/Mg²⁺ free PBS directly lysed using Trizol Reagent (Invitrogen Corporation, Carlsbad, USA). The lysed cells were then homogenized by passing through a 21-gauge needle and thereafter used for RNA extraction using chloroform-ethanol precipitation protocol as described previously (Cancer research, 62, 2002, 1289-1295). 20% ice-chilled chloroform was added to the lysates and spun at 10,000 rpm for 15 minutes at 4°C. The aqueous phase was removed and an equal volume of 70% ethanol was added. This exultant was purified and concentrated using the RNeasy Mini kit (Qiagen GmbH, Hilden, Germany) as per manufacturer's directions. Further, the RNA concentrations were quantified using UV/VIS spectrophotometer (NanoDrop products, DE, USA). The RNA samples were stored in -80°C till further use. Step 5

Preparation of cDNA:

2 μg of total RNA (as isolated in step 4 above) was reverse transcribed to cDNA using cDNA synthesis kit (Invitrogen Corporation, Carlsbad, CA, USA) as per manufacturer's protocol. Ιμΐ of 50μιη Oligo dT (Cat# 18418020), Ιμΐ of 3μg/μl. Random primer (Cat# 48190), Ιμΐ of lOmM dNTP (Cat#l 8427013- 100), 2μg of RNA and nuclease-free water were mixed and to make up the reaction volume 20 μΐ in a 1.7 ml micro centrifuge tube. The mixture was mixed well and heated at 65 °C for 5 minutes on the thermomix and incubated on ice for 2-3 minutes. The contents of the tube at the bottom were collected by brief centrifugation. 8μ1 of 5X First-Strand buffer (Cat# 18080-093), 2μ1 of 0.1M DTT (Cat# 18080-093), Ιμΐ of 40 units/μΐ RNaseOUT (Cat# 10777019), Ιμΐ of 200 units/μΐ Superscript III reverse transcriptase (Cat# 18080-093), and 7μ1 of nuclease-free water were added to it and mixed by gentle pipetting. The reaction mixture was incubated at 25°C for 5 mins, followed by 50°C for 1 hour. The reaction mixture was inactivated by heating at 70°C for 15 minutes. The newly synthesized cDNA can be stored at -20°C until further use or immediately used for RT-QPCR assay.

Step 6

RT-QPCR

RT-PCR was carried out by amplifying the samples in 96-well plates using Epgradient S, Realplex4 Mastercycler System (Eppendorf, Germany) and fluorescent dye Quantifast SYBR Green PCR Master Mix (Qiagen) as per the manufacturer's protocol. For each qPCR reaction, 5μ1 of Quantifast SYBR Green PCR Master Mix (Qiagen), forward primer (0.5μΜ), reverse primer (0.5μΜ), Ιμΐ of diluted cDNA template and sufficient nuclease-free water was added to obtain a final volume of 10ul.

Amplification was done by initial denaturation at 95 °C for 5 min followed by 40 cycles of 95°C for lOsec. and 60°C for 30sec. The specificity and sensitivity of the primers and templates used in the assay were confirmed by a standard melt curve analysis. Data were analyzed using the Realplex software (version 1.5). The raw Ct values were extracted from the software using the automatic noiseband and thresholds. All gene expressions were normalized with an internal control GAPDH to check the quality of RNA.

The relative expression of each gene was calculated using relative Ct method with the formula T^AACt (Methods. 2001, 25, 402-8) (where AACt = ACt sample - ACt reference), where Ct is the number of cycles required to reach the threshold. The primer used in the RT-QPCR assay was designed using the Primer 3 software (http://frodo.wi.mit.edu/) and the primer sequence used in the RT-QPCR is presented in the following Table.

Table 1

Results:

The result of time dependent relative expression of IL-17 in samples taken from three donors is represented in below table and Fig. 5.

Table 2

Analysis of mRNA level in CD4+T cells stimulated with PMA-ionomycin showed that the methanolic extract of the flowering and fruiting heads of Sphaeranthus indicus down regulated the target gene of IL-17.

Conclusion:

The methanolic extract of the flowering and fruiting heads of Sphaeranthus indicus dose dependency down regulates the target protein gene IL-17 and the effect exerted is significant.

Example 7

Evaluation of inhibition of IL-21 and IL-22 mRNA expression by Sphaeranthus indicus extract in CD4+ T cells stimulated with PMA-ionomycin using RT-QPCR assay.

In mice, Thl7 cells selectively synthesize the inflammatory cytokines IL-17 and IL- 22 (BMC Immunology 2008, 9:47, 1471-2172). However, in humans, the production of these two cytokines has diverged into two discrete T cell subsets, Thl7 and Th22 cells, which produce IL-17 and IL-22, respectively (Hum Immunol. 2010, 71(4), 334-41). IL-21 and IL- 22 cytokines are implicated in differentiation of Thl7 cells.

The RT-QPCR study on inhibition of expression of IL-21 and IL-22 by the extract of Sphaeranthus indicus was carried out and based on the study it was found that the extract of Sphaeranthus indicus dose dependently inhibited expression of IL-21 and IL-22. These data supports the therapeutic potential of targeting IL-17 by the extract of Sphaeranthus indicus for the treatment of IL-17 mediated diseases or disorders.

Step 1

Sample preparation

The extract obtained in Example 1 was dissolved in DMSO to prepare the stock solution. Two different concentrations of the extract obtained in example 1 were prepared by serial dilution of the stock solution. The extract of Example 1 was tested at ΙμΜ and 10μΜ concentrations.

Step 2

Isolation of hPBMCs

The hPBM cells were obtained from healthy donors. The whole blood was incubated with RosetteSep® Human CD4+T Cell Enrichment Cocktail (Stem cell technologies, 15062) for 20mins. The incubated blood sample was layered over Ficoll/Hypaque solution and separated by centrifugation. The enriched cells were collected from the density medium: plasma interface and washed three times in sterile PBS with 2% FBS. The cells were then suspended in RPMI-1640 supplemented with lOOU/ml penicillin, 100μg/ml streptomycin and 10% fetal bovine serum (FBS).

Step 3

Assay

The cells as referred to in step 2, were seeded in T-25 flasks at a density of lxlO⁶ cells/flask. The cells were then treated with sample (as prepared in step 1 above) and incubated at 37°C for 30 mins in 5% C0₂ atmosphere. The final concentration of DMSO was maintained at 0.5%. The vehicle (0.5% DMSO) was used as control. The cells were then stimulated with 25μg/ml of PMA (P1585, SIGMA) and ^g/ml ionomycin (SIGMA, 10634) and incubated in 5% C0₂ air humidified incubator at 37°C for 3 hour and 12 hour. The cells not treated with the said sample are referred to as untreated cells.

Step 4

Isolation of RNA and conversion of isolated RNA into cDNA is performed according to procedure provided in Example 6 and utilized for RT-QPCR.

The relative expression of each gene was calculated using relative Ct method with the formula T^AACt (Methods. 2001, 25, 402-8) (where AACt = ACt sample - ACt reference), where Ct is the number of cycles required to reach the threshold. The primers used in the RT-QPCR assay were designed using the Primer 3 software (http://frodo.wi.mit.edu/) and the primer sequences used in the RT-QPCR are presented in the following Table 3.

Table 3

Result:

The result of time dependent inhibition of expression of IL-21 and IL-22 in samples taken from three donors at the time points i.e. at 3 hour and 12 hours is presented in below tables and in Fig. 6a and Fig. 6b respectively.

Table 4

Relative expression of IL-21 (Log₂ ) scale

Sample (ΙμΜ) Sample (10μΜ)

3 hour 12 hour 3 hour 12 hour

Donor 1 -0.26 -8.31 -3.36 -1.62

Donor 2 -1.03 -1.79 -3.68 -5.71

Donor 3 0.22 -1.66 -2.58 -6.95 Table 5

Conclusion:

It is clear from the data represented in the above tables that the Sphaeranthus indicus extract has significantly lowered expression of IL-21 and IL-22 mRNA genes.

Example 8

Evaluation of in vitro activity of Sphaeranthus indicus in multiple myeloma cell lines

A growth inhibition assay using Cell Counting Kit-8 (CCK-8) [Dojindo laboratories] was performed. Modification of the method described by the manufacturer (Dojindo laboratories) was used for this assay. Assay principle is based upon conversion of water- soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt] into a water-soluble formazan dye upon reduction in the presence of an electron mediator.

RPMI8226 and U266B 1 cell line (multiple myeloma) were purchased from ATCC (Rockville, MD, USA) and were maintained in the media as recommended in ATCC datasheet.

Step 1

Maintenance of the cell lines

The cell lines were maintained at 37°C in a humidified atmosphere of 5% C0₂ in medium supplemented with 15% fetal bovine serum [(FBS); Hyclone and 1% Penicillin/Streptomycin (Sigma) and passaged at 80-90% confluency.

Step 2

Sample preparation The extract of different batches as obtained in Example 1 was dissolved in DMSO to prepare the stock solution (20mM). Eight different concentrations of the extract obtained in example 1 were prepared by serial dilution of the stock solution. The extract of Example 1 was tested at 30μΜ, ΙΟμΜ, 3μΜ, ΙμΜ, 0.3μΜ, Ο.ΙμΜ, 0.03μΜ and Ο.ΟΙμΜ concentrations and dose response study was performed to determine the IC₅₀ value. Each concentration was evaluated in triplicate.

Step 3

Assay

A total number of 5000 cells contained in 199μ1 of the cell growth complete medium were plated overnight in a 96-well transparent tissue culture plate. To obtain three fold final dilutions of the sample, Ιμΐ of the appropriately diluted sample was added to cells in 199 μΐ medium in a 96-well plate. The control wells included cells incubated with 0.5% DMSO in medium and blank included 0.5% DMSO in medium only. The cells were incubated for 48 hrs in a C0₂ incubator at 37°C in a humidified atmosphere of 5% C0₂.

The assay was terminated by adding 15μ1 of CCK8 reagent in each well of 96 well plate and the plates were allowed to develop colour by incubating for 3-5 hrs at 37°C in a humidified atmosphere of 5% C0₂. Optical density of formazan formed was measured at the wavelength of 450 nm in a microplate reader (Spectramax). The percent inhibition was calculated in comparison with control values.

Percent growth inhibition was calculated using the following formula:

(Reading of DMSO control - Reading of treated cells)

Percent growth inhibition = X 100

Reading of DMSO control

IC₅₀ value was then calculated from graphs plotted for percent growth inhibition achieved vs concentration of the sample extract used. Results are presented in the following tables. Table 6

ICso ( g/ml)

Test RPMI8226

sample

Experiment 1 Experiment 2 Experiment 3 Experiment 4 Average+

S.D.

Extract of - - 4.1 1.1 2.6+2.1 example 1

Extract of - 8.5 1.9 - 5.2+4.7 example 1

Extract of - 12 2.5 2.1 5.5+5.6 example 1

Extract of 0.6 - 6 4 3.5+2.7 example 1

Table 7

'-' means not tested and S. D. means standard deviation Conclusion

The in-vitro study revealed that the extract of Sphaeranthus indicus showed IC₅₀ in the range of 2^g/ml to 5^g/ml in RPMI8226 and 8^g/ml to 29^g/ml in U266B 1 cell lines. This data shows that the extract of Sphaeranthus indicus is active in multiple myeloma cell lines.

Claims

We claim:

1. A herbal composition comprising a therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus for use in the treatment of a disease or a disorder mediated by interleukin-17 (IL-17).

2. The herbal composition for the use as claimed in claim 1, the extract of the flowering and fruiting heads of the plant Sphaeranthus indicus is prepared using an alcohol selected from methanol, ethanol, isopropanol or propanol.

3. The herbal composition for the use as claimed in claim 2, wherein the alcohol is methanol.

4. The herbal composition for the use as claimed in any one of the claims 1 to 3, wherein the disease or disorder mediated by interleukin-17 is selected from transplant rejection, cystic fibrosis, colorectal cancer, ovarian cancer, breast cancer, cervical cancer, skin cancer, non- small cell lung cancer, melanoma, bladder cancer, multiple myeloma, non-Hodgkin lymphoma, multiple sclerosis, bone metastasis, uveitis or a T cell mediated disorder selected from acquired immune deficiency syndrome, viral diseases or asthma.

5. The herbal composition for the use as claimed in any one of the claims 1 to 4, wherein the disease or disorder mediated by interleukin-17 is selected from transplant rejection, cystic fibrosis, bone metastasis, multiple myeloma or uveitis.

6. The herbal composition for the use as claimed in any one of the claims 1 to 5, wherein the disease or disorder mediated by interleukin-17 is multiple myeloma.

7. The herbal composition for the use as claimed in any one of the claims 1 to 6, the composition is an oral, a topical or a transdermal composition.

8. The herbal composition for the use as claimed in claim 7, wherein the oral composition is in the form of a tablet, capsule or syrup.

9. The herbal composition for the use as claimed in claim 7, wherein the topical composition is in the form of cream, gel or ointment.

10. A method for the treatment of a disease or a disorder mediated by interleukin-17 (IL- 17) comprising administering to a subject in need thereof herbal composition comprising a therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus.

11. The method as claimed in claim 10, wherein the extract of the flowering and fruiting heads of the plant Sphaeranthus indicus is prepared using an alcohol.

12. The method as claimed in claim 11, wherein the alcohol is methanol.

13. The method as claimed in any one of the claims 10 to 12, wherein the disease or disorder mediated by interleukin-17 is selected from transplant rejection, cystic fibrosis, colorectal cancer, ovarian cancer, breast cancer, cervical cancer, skin cancer, non-small cell lung cancer, melanoma, bladder cancer, multiple myeloma, non- Hodgkin lymphoma, multiple sclerosis, bone metastasis, uveitis or a T cell mediated disorder selected from acquired immune deficiency syndrome, viral diseases or asthma.

14. The method as claimed in any one of the claims 10 to 13, wherein the wherein the disease or disorder mediated by interleukin-17 is selected from transplant rejection, cystic fibrosis, bone metastasis, multiple myeloma, uveitis or T cell mediated disorders selected from acquired immune deficiency syndrome, viral diseases or asthma.

15. The method as claimed in any one of the claims 10 to 14, wherein the disease or disorder mediated by interleukin-17 is multiple myeloma.

16. A method for the treatment of diseases or disorders mediated by IL-17, comprising administering to a subject in need thereof herbal composition comprising a therapeutically effective amount of an extract of the flowering and fruiting heads of the plant Sphaeranthus indicus in combination with at least one therapeutically active agent.

17. The method as claimed in claim 16, wherein the therapeutically active agent is selected from analgesics, immunosuppressants, anti-inflammatory agents or anticancer agents.

18. The method as claimed in any one of claims 10 to 15, wherein the composition is an oral or a topical or a transdermal composition.

19. The method as claimed in claim 18, wherein the oral composition is in the form of a tablet, capsule or syrup.

20. The method as claimed in claim 18, wherein the topical composition is in the form of cream, gel or ointment.

21. A method for inhibiting proliferation and stimulation of Thl7 cells, comprising administering to a subject in need thereof, herbal composition comprising an extract of the flowering and fruiting heads of the plant, Sphaeranthus indicus in an amount capable of inhibiting proliferation and stimulation of Thl7 cells.