WO2009078022A2

WO2009078022A2 - Synergistic combinations of growth factors with herbal compositions for the enhancement of tissue repair

Info

Publication number: WO2009078022A2
Application number: PCT/IL2008/001636
Authority: WO
Inventors: Gabriel Nussbaum; William Zev Levine
Original assignee: Izun Pharmaceuticals Corporation
Priority date: 2007-12-17
Filing date: 2008-12-17
Publication date: 2009-06-25
Also published as: WO2009078022A3

Abstract

The present invention provides synergistic combinations of growth factors and an extract of the plant species Sambucus nigra. In another aspect, the present invention is also directed to methods of treatment comprising the administration of a synergistic combination of one or more growth factors together with an extract of Sambucus nigra to a mammalian subject in need of such treatment.

Description

Synergistic combinations of growth factors with herbal compositions for the enhancement of tissue repair

Field of the invention

The present invention relates to compositions for use in enhancing tissue repair. More specifically, the present invention discloses compositions comprising synergistic combinations of one or more growth factors together with an extract of Sambucus nigra or a group of extracts where one of them is Sambucus nigra.

Background of the invention

Wound healing is the process by which an organism reconstitutes damaged tissue, and restores its function. Mucosal and dermal wounds heal through different stages, including hemostasis, inflammation, proliferation, and remodeling of the collagen matrix. The healing process is complex and involves temporal and spatial coordination of cellular and extracellular events involving epithelium and connective tissue. Many cell types participate in the healing process. Chief among them are fibroblasts, whose migration, proliferation, and collagen deposition restore tissue strength, integrity, and structure. The complex process of wound healing is orchestrated by a myriad of growth factors .

Growth factors are naturally occurring bioactive proteins that regulate the proliferation and differentiation of responsive cells. Based on amino acid sequence homology, growth factors can be grouped into families. Growth factors function as signals between cells and are essential for the regulation of many cellular processes such as blood vessel differentiation and tissue regeneration. Following tissue damage cells secrete growth factors in order to stimulate the repair process. For example, following skin wounding growth factors within the provisional wound matrix stimulate fibroblasts to proliferate and synthesize new collagen and other components of the extracellular matrix. In turn, growth factors anchor themselves to matrix components, allowing the extracellular matrix to serve as a growth factor reservoir.

On example of such a growth factor is transforming growth factor beta (TGFb) . TGFb is a multifunctional protein that controls proliferation, differentiation, and functions such as synthesis of cytokines and extracellular matrix components in many cell types. TGFb is synthesized at the site of tissues derived from mesenchyme such as bone, cartilage, and connective tissue and is present in those tissues both during development and during tissue repair. The therapeutic benefit of TGFb has been shown in numerous studies of different types of wound healing (inflammatory, traumatic, chemical, etc.), and tissue repair following fracture or degenerative conditions. These, and other related medical conditions and diseases, are major causes of hospitalization, morbidity and mortality.

The ECM is composed of various types of glycoproteins, proteoglycans and polysaccharides. The ECM is crucial for supporting and anchoring cells and sequestering the growth factors that act upon them. Intercellular communication is therefore very much dependent upon an intact and healthy ECM. Collagens are among the most abundant glycoproteins in the ECM and confer strength and integrity to the connective tissue .

The present inventors have shown that herbal compositions containing extracts of Sambucus nigra and Centella asiatica, alone or mixed, or in combination with Echinacea purpurea are effective in the treatment of diseases characterized by degeneration of collagen or other extracellular matrix components. Examples of such diseases include degenerative bone or joint diseases such as those caused by arthritis (osteoarthritis or rheumatoid arthritis), ocular conditions such as keratoconjunctivitis, skin conditions such as age related changes, and wound healing of all types.

Degenerative conditions affecting the joints are a major cause of morbidity. Breakdown of the integrity of the synovial fluid and connective tissue elements in the joints are a major component of these conditions regardless of their etiology. Therapeutic agents that protect synovial fluid and connective tissue elements are therefore expected to be of benefit for the treatment of these conditions .

Similarly, the eyeball between the retina and the lens is bathed in a clear gelatinous substance known as the vitreous humor, of which collagen and hyaluronic acid are important components. Agents that enhance these factors would be of benefit for acute and chronic conditions affecting the integrity of the eye.

Skin changes with age, such as wrinkles, are a major cosmetic concern. Given its ability to strengthen and give W 2

-4-

fullness to the connective tissue, agents that build or protect ECM components, in particular collagen and hyaluronic acid, are expected to benefit in the management of these changes.

The clinical use of recombinant growth factors such as recombinant TGFb has been limited by high cost, and short duration of action in vivo. Therefore methods to enhance the effect of such factors are necessary in order to harness their therapeutic potential. Previous attempts to obtain this enhancement include the use of peptides that activate latent TGFb (US 6,384,189) and the use of gene therapy approaches to achieve sustained growth factor expression in vivo, as described in several patents.

A need exists for a simple, safe and cost-effective method for enhancing the therapeutic activity of growth factors in vivo.

One aim of the present invention is to meet this need by- providing synergistic combinations of one or more growth factors with one or more herbal extracts or fractions thereof.

A further aim is to provide methods for treating disease in mammalian subjects (particularly humans) by means of administering the aforementioned synergistic combinations.

Further aims and objectives will become apparent as the description proceeds. Suromary of the invention

The inventors have now unexpectedly found that compositions comprising growth factors (including, but not limited to TGFb, platelet-derived growth factor (PDGF), bone morphogenic proteins (BMPs), fibroblast growth factor (FGF) , keratinocyte growth factor (KGF) , granulocyte colony-stimulating factor (G-CSF) , Macrophage colony- stimulating factor (M-CSF) , granulocyte-macrophage colony- stimulating factor (GM-CSF) and Epidermal growth factor (EGF) ) together with an extract of Sambucus nigra alone, or, in the alternative, together with a mixture of extracts wherein one of the extracts is Sambucus nigra, synergistically potentiate the actions of said growth factors. Combinations of S. nigra (or herbal mixtures containing S. nigra) with growth factors can therefore enhance the cellular response to those growth factors and thereby improve the reparative response.

The aforementioned compositions (which will be described in more detail hereinbelow) are effective in the treatment of diseases wherein enhanced growth factor activity is desirable. Examples of such diseases include but are not limited to wound healing of all types, degenerative, inflammatory, or traumatic bone and joint diseases such as those caused by arthritis (osteoarthritis or rheumatoid arthritis) , ocular conditions such as keratoconjunctivitis, skin conditions such as those caused by inflammation or degenerative changes such as age related changes, for example wrinkles.

The present invention is thus primarily directed to compositions comprising a synergistic combination of one or more growth factors together with an extract of a Sambucus species (preferably Sambucus nigra) alone, or, in the alternative, together with a mixture containing an extract of Sambucus nigra together with additional herbal extracts. A particularly preferred mixture of extracts for use in the present invention comprises extracts of Sambucus nigra, and one or both of Centella asiatica and Echinacea species. Preferably, the growth factor present in the combination is a recombinant growth factor. However, in certain cases, other non-recombinant forms (e.g. growth factors purified from tissues, body fluids and other sources of natural products) may also be used.

As mentioned above, the synergistic combinations of the present invention comprise one or more growth factors together with an extract of a Sambucus species. In a particularly preferred embodiment, the Sambucus species is S. nigra. In some embodiments, the synergistic combination may contain other herbal extracts. In one preferred embodiment, the herbal extract components of the claimed combination comprise S. nigra, Centella asiatica and Echinacea species. The present inventors have found synergistic interactions between Sambucus extracts and several different growth factors of different chemical classes and having markedly different biological activities. In view of the wide range of structurally and functionally unrelated growth factors that have been found by the inventors to interact synergistically with S. nigra, it is believed that said synergistic interaction is a general property of S. nigra, and is therefore to be found in combinations of said plant extract with a very large number of different growth factors, regardless of their chemical structure, target cell type or biological activity.

In particular, the present invention is directed to compositions comprising combinations of a Sambucus extract together with a growth factor selected from the group consisting of platelet derived growth factor (PDGF) , fibroblast growth factors (e.g. basic FGF), bone morphogenic proteins (e.g. BMP-2, BMP-4, BMP-7), transforming growth factor beta (TGF-beta) , keratinocyte growth factor (KGF) , granulocyte colony-stimulating factor (G-CSF) , Macrophage colony-stimulating factor (M-CSF) , granulocyte-macrophage colony-stimulating factor (GM-CSF) and Epidermal growth factor (EGF) . However, it is to be recognized that the present invention is not to be limited to combinations containing these named growth factors. Rather, combinations of Sambucus extracts with other growth factors are also included within the scope of the present invention.

In one preferred embodiment of the invention, the aforementioned combination of one or more growth factors with herbal components is realized in a single composition. In other preferred embodiments, the growth factor (s) and the herbal components are prepared as separate compositions which are suitable for being administered separately.

In another aspect, the present invention is directed to a method for treating diseases or conditions characterized by reduced levels of collagen and/or other extracellular matrix components in the affected tissues, wherein said method comprises administering a synergistic combination of one or more growth factors together with an extract of Sambucus nigra or fractions thereof with or without additional herbal extracts to a mammalian subject in need of such treatment. In one preferred embodiment, the mammalian subject is a human subject.

In another aspect, the present invention is directed to a method for treating diseases characterized by degeneration of collagen and/or other extracellular matrix components, wherein said method comprises administering a synergistic combination of one or more growth factors together with an extract of Sambucus nigra or fractions thereof with or without additional herbal extracts to a mammalian subject in need of such treatment. In one preferred embodiment, the mammalian subject is a human subject.

In the aforementioned methods of treatment, the disease or condition to be treated is selected from the group consisting of: wound healing, degenerative, inflammatory, or traumatic bone and joint diseases such as those caused by arthritis (osteoarthritis or rheumatoid arthritis), ocular conditions such as keratoconjunctivitis, skin conditions including age related changes such as wrinkles, and mucosal conditions such as oral mucositis.

In one preferred embodiment of the method of the invention, the growth factor used is selected from the group consisting of PDGF, FGF, BMP-2, BMP-4, BMP-7, TGF-beta, KGF, G-CSF, M-CSF, GM-CSF and EGF. In one particularly preferred embodiment, the growth factor is TGFb. In another preferred embodiment, the growth factor is platelet-derived growth factor (PDGF) . In addition to the Sambucus nigra herbal extract, the combination used in the method of the present invention may also comprise extracts of additional plants. Such additional plants may include any of the plants commonly used in herbal remedies. Two preferred additional plants are plants of the Centella genus and/or the Echinacea genus .

Thus, in some preferred embodiments of the above-disclosed methods, said methods comprise the administration of one or more growth factors together with an herbal composition comprising an extract of Sambucus nigra and a mixture of extracts of plants selected from the group consisting of Centella asiatica and one or more Echinacea species. In a particularly preferred embodiment, the method involves the use of a composition containing one or more growth factors and a mixture of the aforementioned three plant extracts, wherein said mixture is the mixture of Sambucus nigra, Centella asiatica and Echinacea purpurea that is described in co-owned international patent application no. PCT/IL02/00402 (published as WO 02/094300) , the disclosure of which is incorporated herein in its entirety.

In another preferred embodiment, the method involves the administration of a composition containing one or more growth factors together with Sambucus nigra as the sole herbal ingredient.

In one preferred embodiment of the above-disclosed methods of the present invention, the growth factor (s) and the herbal extract (s) are administered in a single composition. In another preferred embodiment, the growth factor (s) and the herbal extract (s) are administered separately. In the latter case, both components may be administered by the same route (e.g. both topically or both systemically) or by different routes (e.g. one component topically and the other component systemically) . In addition, the two separately-administered components may be administered either at the same time or consecutively (in either order) .

In another aspect, the present invention is directed to the use of the synergistic combinations and compositions described hereinabove in the preparation of medicaments. In addition to the aforementioned herbal extracts and growth factors, said medicaments may also contain other active and non-active ingredients (e.g. excipients) . In one preferred embodiment, these medicaments are suitable for use in treating diseases or conditions characterized by reduced levels of collagen and/or other extracellular matrix components in the affected tissues. In another preferred embodiment, the medicaments are suitable for use in treating diseases characterized by degeneration of collagen and/or other extracellular matrix components .

All the above and other characteristics and advantages of the present invention will be further understood from the following illustrative and non-limitative examples of preferred embodiments thereof. Brief description of the drawings

Pig. 1 graphically depicts the synergistic enhancement of TGF-beta-induced IL-Il production by fibroblasts due to the presence of either S. nigra alone or a mixture of S. nigra, Centella asiatica and Echinacea purpurea.

Fig. 2 graphically depicts the synergistic enhancement of PDGF-stimulated IL-Il production by fibroblasts due to the presence of a S. nigra extract.

Fig. 3 graphically depicts the strong synergistic interaction between S. nigra and PDGF in a fibroblast proliferation model.

Fig. 4 graphically depicts the strong synergistic interaction between S. nigra and fibroblast growth factor in a fibroblast proliferation model.

Fig. 5 graphically depicts the synergistic interaction between an S. nigra extract and fibroblast growth factor in a wound contracture model .

Fig. 6 demonstrates the synergistic interaction between an S. nigra extract and BMP-7 in the regulation of the fibronectin gene (FNl) .

Fig. 7 illustrates the synergistic interaction between an S. nigra extract and BMP-2 in the regulation of the SPOCKl and TIMP3 genes. Fig. 8 graphically depicts the synergistic interaction between an S. nigra extract and BMP-2 in the regulation of various ECM-related genes.

Pig. 9 further illustrates the synergistic interaction between an S. nigra extract and BMP-2 in the regulation of various ECM-related genes .

Pig. 10 further illustrates the synergistic interaction between an S. nigra extract and BMP-2 in the regulation of various ECM-related genes .

Fig. 11 illustrates the synergistic interaction between an S. nigra extract and BMP-4 in the regulation of various ECM-related genes.

Fig. 12 further depicts the synergistic interaction between an S. nigra extract and BMP-4 in the regulation of various ECM-related genes.

Fig. 13 further illustrates the synergistic interaction between an S. nigra extract and BMP-4 in the regulation of various ECM-related genes.

Fig. 14 further illustrates the synergistic interaction between an S. nigra extract and BMP-4 in the regulation of various ECM-related genes.

Fig. 15 graphically illustrates the synergistic interaction between an S. nigra extract and BMP-7 in the regulation of genes involved in osteogenesis. Fig. 16 graphically illustrates the synergistic interaction between an S. nigra extract and b-FGF in the regulation of genes involved in angiogenesis .

Pig. 17 further illustrates the synergistic interaction between an S. nigra extract and b-FGF in the regulation of genes involved in angiogenesis.

Fig. 18 further illustrates the synergistic interaction between an S. nigra extract and b-FGF in the regulation of genes involved in angiogenesis.

Fig. 19 graphically illustrates the synergistic interaction between an S. nigra extract and KGF in the stimulation of keratinocyte migration.

Detailed description of the preferred embodiments

In the context of the method of treatment of the present invention, the compositions comprising TGFb or other growth factor and individual plant extracts or combinations thereof (as described above) may be administered to the subject being treated by any suitable route. In one preferred embodiment, the compositions are generally given by local or topical administration, in order to reduce the possibility of side effects. However, in other embodiments of the method of the present invention, the compositions may also be administered by a systemic route, including but not limited to the peroral, intravenous, intramuscular, subcutaneous, intradermal, intra-articular and intra-disc routes . As described above, the growth factor (s) and the herbal extracts may either be formulated as a single composition, or alternatively, may be prepared as separate compositions. In the latter case, the growth factor composition and the (separate) herbal composition may be administered either simultaneously or sequentially. Furthermore, in such cases, both compositions may be administered either via the same route (e.g. topical) or by two different routes (e.g. the herbal composition locally and the growth factor composition systemically, or vice versa) .

Examples of suitable dosage forms for use in the presently disclosed method include (but are not limited to) liquids, gels, sprays, creams, ointments, pastes, lotions, solutions, mouthwashes, lozenges, candies, chewing gums and sustained release formulations. Examples of suitable formulations of this type are to be found in co-owned international patent application no. PCT/IL02/00402 (published as WO 02/094300) . The compositions may also be formulated for administration by injection (via various routes including intradermal, subcutaneous, submucosal, intramuscular, etc.). In one preferred embodiment the compositions may be incorporated into transmucosal or transdermal delivery systems. Examples of suitable transmucosal delivery systems may be found in co-owned international patent applications PCT/IL02/00402 (published as WO 02/094300) and PCT/IL2003/00159 (published as WO 04/047813) . Thus, in addition to the active ingredients, the compositions of the present invention may also contain one or more excipients including, but not limited to zinc, zinc oxide, silicones, calcium silicate, aluminum hydroxide, polyethylene glycols, methacrylate polymers and co-polymers, antiseptic substances including (but not limited to) povidone derivatives, carbomers, magnesium stearate, fats of animal or vegetable origin, oils, waxes gums, starch and cellulose or cellulose derivatives. Further details regarding the manufacture of suitable formulations and the excipients used therein may be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co, Easton, Pa, USA (1980) .

The compositions of the present invention are based on the combined use of growth factors with plant extracts. In one embodiment, the growth factor may be a growth factor that has been purified from a body tissue, body fluid or other natural source. However, it is to be noted that such an approach suffers many practical limitations that present obstacles for producing commercially-viable quantities of the desired growth factor proteins . The growth factors of the present invention may therefore be more conveniently prepared by means of recombinant biotechnological means, whereby the gene for the growth factor protein of interest is isolated and inserted into an appropriate expression vector system (such as a plasmid or phage), which is then introduced into a host cell that will permit large-scale production of said protein by means of, for example, overexpression.

As a first stage, the location of the growth factor genes of interest may be determined by reference to a nucleic acid database such as Genbank (accessible on the internet at http: //www.ncbi . nlm.nih. qov/Genbank/GenbankSearch .html) . The selected sequence may, where appropriate, be isolated directly from the relevant host genome .by the use of appropriate restriction endonucleases, or more effectively by means of PCR amplification. Suitable techniques are described in, for example, US patent Nos . 4,683,195, 4,683,202, 4,800,159, 4,965,188, as well as in Innis et al. eds., PCR Protocols: A guide to method and applications.

Following amplification and/or restriction endonuclease digestion, the desired gene or gene fragment is ligated either initially into a cloning vector, or directly into an expression vector that is appropriate for the chosen host cell type. In many cases, Escherichia coli is the most useful expression host. However, many other cell types may be also be usefully employed including other bacteria, yeast cells, insect cells and mammalian cell systems.

High-level expression of the desired protein within the host cell may be achieved in several different ways (depending on the chosen expression vector) including expression as a fusion protein (e.g. with factor Xa or thrombin) , expression as a His-tagged protein, dual vector systems, expression systems leading to incorporation of the recombinant protein inside inclusion bodies etc. The recombinant protein will then need to be isolated from the cell membrane, interior, inclusion body or (in the case of secreted proteins) the culture medium, by one of the many methods known in the art.

All of the above recombinant DNA and protein purification techniques are well known to all skilled artisans in the field, the details of said techniques being described in many standard works including "Molecular cloning: a laboratory manual" by Sambrook, J., Fritsch, E. F. & Maniatis, T., Cold Spring Harbor, NY, 2^nd ed., 1989, which is incorporated herein by reference in its entirety.

In many cases, suitable recombinant growth factor preparations may be obtained commercially from a variety of manufacturing companies, as is well known to the skilled artisan.

It is to be noted that the term "herbal extract" or "plant extract" is used herein to include all of the many types of preparations containing some or all of the active ingredients found in the relevant plants. Thus the extracts may be produced by cold extraction techniques using a variety of different extraction solvents including, but not limited to, water, fatty solvents (such as olive oil), and alcoholic solvents (e.g. 70 % ethanol) . Cold extraction techniques are usefully applied to softer parts of the plant such as leaves and flowers, or in cases wherein the desired active components of the plant are heat labile. Alternatively, the aforementioned solvents may be used to produce extracts of the desired plants by a hot extraction technique, wherein said solvents are heated to a high temperature, the precise value of said temperature being dependent on the properties of the chosen solvent, and maintained at that temperature throughout the extraction process. Hot extraction techniques are more commonly applied to the harder, tougher parts of the plant, such as bark, woody branches and larger roots. In some cases, sequential extractions need to be performed in more than one solvent, and at different temperatures. Standard procedures for producing plant extracts (including hot extraction, cold extraction and other techniques) are described in many publications which are well known to the skilled artisan, one example of which is "Making plant medicine" [author: R. Cech, pub. by Horizon Herbs, 2000] .

In the context of the present invention, the terms "herbal extract" and "plant extract" and the like also include within their range of meaning solid plant material which has been chopped, ground or pulverized or otherwise subjected to a method aimed at reducing the particle size of the plant material.

It is also to be noted that, in the context of the present invention, the terms "herbal extract" and "plant extract" and the like also include within their range of meaning fractions obtained from such extracts. Many standard chemical and biochemical laboratory techniques (e.g. liquid chromatography methods such as HPLC in all of its various modes) may be used to prepare such fractions by separating the components of the plant extracts on the basis of electrical charge, molecular size, ligand affinity, and so on.

As described above, compositions may be prepared by combining TGFb (or other growth factor) with a single extract of Sambucus nigra or with Sambucus nigra plus extracts of one or more different plant species. In the latter case, the composition may be prepared using different ratios of each extract. For example, the aforementioned three-herb composition may comprise extracts of Centella asiatica, Echinacea purpurea and Sambucus nigra in the following range of weight ratios:

0.5-3 : 0.1-3 : 2-15

More preferably, these extracts are present in the weight ratio of 1.5 : 1.5 : 7

In order to treat a patient with a composition containing TGFb or other growth factor and either a single herbal extract or a mixture of herbal extracts as described hereinabove, it is necessary to administer said composition in a therapeutically-effective amount, that is, in an amount that will provide a concentration of TGFb and the herbal extract (s) at the treatment site that is capable of exerting the desired therapeutic effect. To be effective, TGFb can be administered either as a soluble protein together with the herbal material or via gene therapy where a vector encoding TGFb is administered to the patient in order to induce TGFb expression at the site of treatment. In addition, TGFb may be administered systemically for example via intravenous injection and the herbal material applied topically in order for the synergistic effect to be obtained at the treatment site. It has been found, in general terms, that the compositions of the present invention need to be administered in amounts such that, typically, picogram to milligram amounts of TGFb are administered with topical doses containing between 0.01 mg and 100 mg (dry weight) of the Sambucus nigra extract or other herbal extracts, the precise values depending on the particular combination used, and on the mode of topical and/or systemic delivery. In the case of compositions intended primarily for topical use, it is necessary to administer said compositions for a period of time that is sufficient to allow optimal contact of the therapeutically effective amounts of the composition with the lesions to be treated. When the compositions and medicaments are to be given by incorporation into a controlled-release intra-oral device (e.g. as described in WO 04/047813) , said device needs to remain in contact with the lesion to be treated for a period of between 1 and 5 hours. This treatment may be repeated up to 5 times each day, as required, and as determined by a competent clinician. In certain cases, the compositions of the present invention may be incorporated into controlled- release devices and formulations that permit slow release of the active components over a longer period of time than that indicated above. Thus, such slow-release formulations and devices may be used to cause the release of said active components over the cause of time periods including (but not limited to) 3 days or 7 days.

One possible treatment regime may involve administration of a growth factor such as TGFb systemically, while the herbal component of the composition of the present invention is administered in the form of a mouthwash. In this case, the mouthwash should be taken in quantities of between 5 ml and 15 ml and allowed to remain in contact with the lesions to be treated for periods of between 30 seconds and one minute. This treatment regime may be repeated up to 5 times per day. Lozenges, pastilles, candies and other solid, soluble formulations are to be placed in the mouth, if possible in close proximity to the lesions to be treated, and allowed to dissolve at the natural rate determined by the additives present in said formulations.

The following examples are provided for illustrative purposes and in order to more particularly explain and describe the present invention. The present invention, however, is not limited to the particular embodiments disclosed in these examples.

Example 1 Preparation of TGFb/Samfoucus composition

The TGFb plus Sambucus nigra mixture mentioned herein above and used in the experimental investigations reported herein below was prepared from recombinant human TGFb protein mixed with an extract of Sambucus nigra. This composition was prepared by mixing 200 microliters of a solution of TGFb with 1 ml of a 1:5 hydroalcoholic extract of Sambucus nigra or with ImI of a mixture of a 1:5 hydroalcoholic extract of Sambucus nigra, a 1:4 hydroalcoholic extract of Centella asiatica , and a 1:1 hydroalcoholic extract of Echinacea purpurea. The recombinant TGFb was purchased from R and D Systems (Minneapolis, MN) . The hydroalcoholic extracts of Sambucus nigra, Centella asiatica and Echinacea purpurea were purchased from Pentapharm (Basil, Switzerland) or from Finzelberg (Andernach, Germany) . It is to be noted that the abovementioned extract values of the form l:x indicate that 1 g of the_. plant material was dissolved in x liters of solvent. The term "hydroalcoholic extract" indicates that the plant material was extracted using ethanol at concentrations of between 25% and 75% in water .

Example 2 Enhancement of TGffb-induced IL-Il release by an extract of

Samhucus nigra alone, and by a composition containing Sambucus nigra, Centella asiatica and Echinacea, purpurea

In this study, TGFb-induced release of IL-Il was determined using a quantitative ELISA assay to measure IL-Il.

Cells and culture reagents :

Fibroblasts were cultured and sub-cultured after trypsinization (0.5% trypsin-EDTA in balanced salt solution without Ca ²⁺ /Mg ²⁺, 5 min) (Biological Industries, Israel) .

Fibroblasts were used between passages 3 and 8. All cells were maintained in a 5% C0₂/air humidified incubator at

37°C.

For this study, the human gingival fibroblast (HGF-I) cell- line, was used. Cells were cultured for 24 hours in cell culture medium as above.

Tumor growth factor beta (TGFb) was obtained from R& D Systems (Minneapolis, MN, USA) . The levels of secreted IL- 11 cytokine were quantified using the commercial ELISA kit (R& D Systems, Minneapolis, MN, USA, Cat. #DY218) employed according to the manufacturer's instructions.

Cytokine assay:

HGF-I cells were seeded in 96-microwell tissue plates at a density of 10⁴ cell/well in 200μl of DMEM growth medium, as above. Cells were cultured overnight at 37°C in a humidified atmosphere containing 5% CO₂. Cells were then stimulated with TGFb (2ng/ml) in starvation medium, as above, ± the herbal ingredients. Cells were incubated for an additional 24 hours at 37⁰C and 5% CO₂.

A sample of supernatant (100 μl) was withdrawn for the determination of cytokine content. Negative controls were included without the addition of TGFb or herbal extracts. The potential effect of increasing concentrations of TGFb ± herbal extracts on the production of IL-Il was measured by ELISA assay.

ELISA was performed, according to manufacturer's instructions. Optical densities were measured at λ= 450nm and 540nm using a Multiskan RC plate reader (Thermo Labsystems, Finland) and Genesis lite software (Thermo Life Sciences, Waltham, MA, USA) . The quantities were calculated using a standard curve of 7 serial concentrations of IL-Il. Duplicate sample points were measured.

The stimulatory effects of the addition of an extract of Sambucus nigra and (independently) of a composition containing a mixture of extracts of Sambucus nigra, Centella asiatica and Echinacea purpurea on IL-Il release by the cultured fibroblasts are summarized in Figure 1.

It may be seen from the results shown in Figure 1 that the effect of combining TGFb with Sambucus nigra alone or with Sambucus nigra plus Centella asiatica and Echinacea purpurea exerts an effect on the fibroblasts for the production of IL-Il that is much more than additive since the TGFb and Sambucus components on their own are present at sub-therapeutic or near-sub-therapeutic levels (in the case of Sambucus nigra lmg/ml) . In other words, there is an unexpected synergistic interaction between Sambucus nigra and the recombinant TGFb on the induction of fibroblast IL-Il. Synergy is preserved when Sambucus is present as a combination with other herbal extracts (as seen for the 4B2 preparation) .

Example 3

Enhancement of TGFb-induced fibroblast gene expression by an extract of Sambucus nigra

In order to investigate the molecular basis for the unexpected synergism observed with the growth factor-herbal composition combinations of the present invention, the effect of a Sambucus nigra extract on TGFb-induced gene expression in fibroblasts was investigated.

Fibroblasts were seeded in 24-well tissue culture plates in ImI of supplemented DMEM/10% FCS until confluence. Herbal extracts in ImI of DMEM, containing 0.1% BSA were added, and the cultures were incubated for 24 hr . Following extensive washing the cell layers were lysed and RNA was prepared using an RNA extraction kit (Superarray, Frederick, MD, USA) . cRNA was prepared and labeled (Superarray labeling kit) and hybridized to microarrays containing 113 extracellular matrix related oligonucleotides (Superarray) according to the manufacturer's protocols. We found that both TGFb and Sambucus nigra upregulated expression of several extracellular matrix-related genes. Synergistic upregulation of several genes was observed (Table 1) . The function of these genes is outlined in Table 2.

Table 1. Gene expression induced by exposure to TGFb, Sambucus nigra, or the combination of TGFb and Sambucus nigra. Fold induction is defined as the signal produced in cells exposed to the stimulant compared to the signal in untreated cells. For the combined treatment, expected values are shown in parentheses before the observed value.

Gene Description Function

Collagen, Adhesion The fibril-associated collagens type XII, molecule with interrupted triple helices

COL12A1 alpha 1 (FACITs) (types IX, XII, and

XIV) appear to connect fibrils to other matrix elements. Found in periodontal ligaments.

Collagen, Adhesion Collagen VI is a widely type VI, Molecule expressed and abundant

COL6A3 alpha 3 extracellular matrix protein that forms an extensive beaded microfibrillar network.

Chondroitin Adhesion Versican may provide a sulfate Molecule mechanism to stabilize proteoglycan hyaluronan associated with cell

2 (versican)

LorbZ membrane CD44: thus, forming a versican/HA pericellular matrix around the cell affecting adhesive and migratory capabilities .

Catenin Adhesion α-catenin has been best

(cadherin- Molecule understood as an important associated cytoplasmic component of the

CTNNAl protein) , classical cadherin complex alpha 1, responsible for cell-cell

102kDa adhesion.

Table 2. Description and function of genes induced by exposure to TGFb, Sambucus nigra, or the combination of TGFb and Sambucus nigra.

Example 4

Enhancement of PDGF-induced IL-Il release by an extract of

Sambucus nigra

The fibroblast response to platelet-derived growth factor (PDGF) with and without an extract of Sambucus nigra was investigated. As can be seen from Fig. 2, the addition of PDGF and Sambucus induced an amount of IL-Il that would not be expected based solely on an additive effect of the two stimulants .

We conclude from these results that Sambucus nigra extracts are synergistic with growth factors that influence processes involved in wound healing and tissue repair.

Example 5

Synergistic enhancement of PDGF-induced cell proliferation caused by an extract of Sambucus nigra

One of the earliest characterized effects of growth factors is their ability to increase the proliferation of cells that expressed receptors to those factors. The present inventors investigated whether S. nigra would also synergize with growth factors for this effect.

Cell proliferation was measured by the CellTiter-Glo Luminescent Cell Viability Assay (Promega, WI, USA) . The number of viable cells in culture was determined by quantification of ATP, which signals the presence of metabolically active cells present in the sample. The assay- system utilizes a thermostable luciferase, which generates a stable "glow-type" luminescent signal while simultaneously inhibiting endogenous enzymes released during cell lysis (e.g., ATPases) . Release of ATPases would interfere with the accurate measurement of ATP. The cell lines used in this assay were Hs68 (human foreskin fibroblasts) and HGF-I (human gingival fibroblasts) . These cells were grown in DMEM (containing 10% FCS, 4mM glutamine, 100 U/ml penicillin and lOOμg/ml streptomycin) 1- HGF-I: 24h after seeding 8xlO³ cells/well in a 96 well plate, cell culture medium was replaced by starvation medium (DMEM containing 2mM glutamine, ImM sodium pyruvate, 100 u/ml penicillin, 0. lmg/ml streptomycin, and 0.1% BSA) + PDGF (2ng/ml) ± S. nigra extract (0.125, 0.25 or 0.5mg/ml), for 24h.

2. Hs68: 24h after seeding 8xlO³ cells/well in a 96 well plate, cell culture medium was replaced by starvation medium (as described above) for a 2 hour synchronization step, after which basic FGF (b-FGF; 50ng/ml) ± S. nigra extract (0.16, 0.31, 0.63 or 1.25mg/ml) were added, for 24h.

After 24h incubation in the presence of the growth factor + S. nigra extract, plates were equilibrated to room temperature for 30 minutes. A volume of CellTiter-Glo reagent, equal to the volume of starvation medium left in each well (generally 50-100μl) , was added to each well, and the plates were mixed on an orbital shaker for 2 minutes to induce lysis. The plates were further incubated at room temperature for 10 minutes, after which the luminescence was recorded using a Genios fluorometer (Tecan, Salzburg, Austria) and analyzed using Magellan software (Tecan, Salzburg, Austria) .

The results obtained indicate a strong synergistic effect on fibroblast cell proliferation between 5. nigra and either PDGF or b-FGF (Figs. 3 and 4, respectively) . The lightly shaded bars (on the left side of each pair of bars) indicate the percentage increase in viable cell number obtained when only S. nigra is added to the fibroblasts; the deeply-shaded bars indicate the percentage increase in viable cell number after treatment with growth factor + S. nigra over the level obtained for the growth factor by itself. As can be seen, the proliferation increase in both cases is dependent on the concentration of S. nigra.

Example 6

Synergistic enhancement of growth factor-induced wound contracture caused by an extract of Sambucus nigra

In order to further test the synergistic enhancement of growth factor activity by S. nigra, the fibroblast- populated collagen lattice (FPCL) assay was used as a model of wound contracture. This method involves the introduction of cultured fibroblasts into a three-dimensional collagen matrix, leading to the eventual dynamic reduction in size of that matrix, caused by the reorganization and translocation of randomly oriented collagen fibrils. The FPCL model demonstrates organization of collagen by fibroblasts through compaction of collagen fibrils. The more rapidly the lattice contracts, the faster the translocation of collagen fibrils by resident fibroblasts.

The assay was prepared and performed as follows: three- dimensional collagen lattices were prepared by mixing fibroblasts with a liquid collagen solution at 4°C and polymerizing to form a firm gel at 37°C. This was accomplished by integrating human foreskin fibroblasts (Hs68) at a density of 5xlO⁴ cells per lattice within collagen gels (5mg/ml Cultrex 3-D Culture Matrix™, R&D) . Each collagen gel contained 5% PBS, 1.2% NaOH, 18.8% sterile H₂O, 25% collagen solution (1.2mg/ml final concentration) and 50% DMEM (containing 10% FCS, 4mM glutamine, 100 U/ml penicillin and lOOμg/ml streptomycin) containing the cell suspension. Four ml of gel solution per well were then poured into 6-well tissue culture plates (Greiner bio-one) and placed in a 37 °C incubator for Ih to polymerize. The collagen gels were then detached from the plates and allowed to undergo free contraction in the presence or absence of compounds added to serum-containing culture medium. In the present experiments, collagen lattices were incubated with S. nigra extract (0.1 to 2.0mg/ml) either separately or in combination with one of several growth factors, in order to determine synergy between S. nigra extract and a given growth factor.

Controls were run, including collagen lattices that did not contain cells and lattices that contained only cells without other additions, and all samples were run in duplicate. Gel contraction was recorded twice daily using a CCD camera coupled to an imaging instrument (F-ChemiBIS 0.5, DNR Bio-imaging Systems) by measuring the gel diameter in experimental and control gels.

Fig. 5 presents the results obtained for Hs68 fibroblasts when exposed to b-FGF, separately and in combination with S. nigra extract. As can be seen in this figure, the contraction rates obtained for the single tested materials

(second and third curves from top to bottom) are similar to the gel contraction rate induced by the cells themselves

(cells only; upper curve) , whereas the combination of the two materials gives a different contraction pattern altogether (lowest curve) . b-FGF (at lOng/ml) had a rapid synergistic effect with S. nigra extract (at 0.1mg/ml), first observed at the 0.5 day time point, carrying through day 5; by day 7 the extent of contraction was identical for all samples. BMP-7 also demonstrated synergy in this model (data not shown) .

Example 7

Synergistic enhancement of growth factor-induced regulation of gene expression caused by an extract of Sambucus nigra

The role of several growth factors in the regulation of gene expression with and without the addition of S. nigra was investigated. Gene expression was measured by labeling and hybridization of cRNA from treated cells to gene microarrays representing about 113 genes in a particular pathway per array. Included in the study were arrays of genes involved in the construction of the extracellular matrix (ECM) , genes involved in osteogenesis, and genes involved in angiogenesis .

Sample Preparation:

Hs68 (human foreskin fibroblast) , HGF-I (human gingival fibroblast) and FHs738Lu (fetal lung fibroblast) cell lines, all obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) , were used in this assay. Cells lines were grown in cell culture medium (DMEM supplemented with 10% heat-inactivated FBS, 4mM glutamine, and ImM sodium pyruvate) . Hsβ8 and FHs738Lu were seeded at a density of 2.5 x 10⁵ cells/well; HGF-I was seeded at a density of 2.0 x 10⁵ cells/well. Two ml of cell culture were deposited per well in 6 well plates and cells allowed to adhere and grow at 37°C, 5% CO₂, for 17 - 19 hours. Cell culture medium was then removed and replaced with starvation medium (DMEM containing 2mM glutamine, ImM sodium pyruvate, 100 u/ml penicillin, 0. lmg/ml streptomycin, and 0.1% BSA) ± S. nigra extract + growth factor, and cells were thus incubated for 6h.

The growth factors that were assayed include BMP-2 (tested at 25, 50 and lOOng/ml) , BMP-4 (tested at 50 and lOOng/ml) , BMP-7 (tested at 75ng/ml) and basic FGF (b-FGF; tested at 10 and 25ng/ml) . S. nigra extract was tested at 0.1 and 0.2mg/ml. Two ml of starvation medium were removed after the 6h incubation and RNA was extracted from the cells in the adherent cell layer, as per the protocol for RNA extraction for the Qiagen RNAeasy mini kit (Qiagen, Germantown MD, USA, Cat. # 74104) .

cRNA creation and hybridization to arrays: cRNA was created from the RNA extracted from the treated cells (see above), according to the instruction manual of the Ambion MessageAmp II aRNA amplification kit

(Ambion/Applied Biosystems, Austin, TX, USA, Cat. #1751) . Four μg of the resultant Bio-16-UTP-labeled cRNA was then used to hybridize to the individual arrays overnight. The hybridization and washing is standardized and was performed according to the SABiosciences manual

(Superarray/SABiosciences, Frederick, MD, USA, Cat. #EHS- 0##) . The images were captured by a CCD camera and then analyzed with the GEArray Expression Analysis Suite 2.0 software (http: //geasuite . superarray. com/index. jsp) . The arrays tested were EHS-013 (ECM-related genes), EHS-026

(osteogenesis-related genes) and EHS-024 (angiogenesis- related genes) . Analysis :

In each assay at least two wells of untreated cells served as a control, the average value providing a baseline level of gene expression for the particular cell line and culture conditions . The value obtained for each well containing treated cells was then divided by the average value obtained for the control, to allow determination of the level of up-regulation or down-regulation of gene expression in treated cells, compared to untreated cells, for each condition tested. To determine if synergy was achieved, the value obtained for a sample treated with a growth factor alone and the value obtained for a sample treated with S. nigra extract alone were compared to the value obtained for the sample treated with the growth factor + S. nigra extract. Synergy is observed when:

1. the value obtained for the combination is >20% greater than the value obtained by adding the values obtained for each of the two separate materials;

2. the growth factor alone and S. nigra extract alone each reduces gene expression and the combination results in up-regulation in gene expression;

3. the growth factor alone and S. nigra extract alone each induces gene expression and the combination results in down-regulation in gene expression

The role of several growth factors in the regulation of gene expression with and without the addition of S. nigra was investigated. Gene expression was measured by hybridization to gene microarrays representing about 113 genes in a particular pathway per array. We studied arrays of genes involved in the construction of the extracellular matrix (ECM) , genes involved in osteogenesis, and genes involved in angiogenesis .

Figs. 6-18 summarize the genes that are synergistically regulated by S. nigra with one of the following growth factors: BMP-2, BMP-4, BMP-7 or b-FGF. Figs. 6-14 present data obtained with the ECM gene array, Fig. 15 presents data obtained with the osteogenesis gene array and Figs. 16-18 present data obtained with the angiogenesis array.

In the ECM gene array, each of BMP-7 and S. nigra extract, administered separately to the fibroblasts, caused down- regulation of the fibronectin gene whereas, when administered together, they acted synergistically to mildly up-regulate fibronectin gene expression (Fig. 6) . Similarly, in probing an osteogenesis gene array, each of BMP-7 and S. nigra extract, administered separately to the fibroblasts, down-regulated expression of the Twist2 gene whereas, when administered together, the gene expression was normal (Fig. 9) . Note particularly that each of BMP-7 and S. nigra extract, administered separately to the fibroblasts, had little effect on the regulation of the NFKBI gene (plO5 domain, precursor of p50) ; however, when administered together BMP-7 and S. nigra extract significantly increased NFKBI gene expression (Fig. 15) .

BMP-2, in combination with S. nigra extract, synergistically up-regulated the expression of an entire collection of genes involved in ECM production (Figs. 7- 10) . Synergy in the expression of the largest number of ECM-related genes is observed when fibroblasts are incubated in the presence of 50ng/ml BMP-2 + 0. lmg/ml S. nigra extract .

BMP-4, in combination with S. nigra extract, synergistically up-regulated the expression of an entire collection of genes involved in ECM production (Figs. 11- 14) . Synergy in the expression of the largest number of ECM-related genes is observed when fibroblasts are incubated in the presence of 50ng/ml BMP-4 + 0.2mg/ml S. nigra extract.

b-FGF, in combination with S. nigra extract, synergistically down-regulated, the expression of an entire collection of genes involved in angiogenesis (Figs. 16-18) . A smaller number of genes were synergistically up- regulated. Synergy in the expression of the largest number of ECM-related genes is observed when fibroblasts are incubated in the presence of 25ng/ml b-FGF + 0. lmg/ml S. nigra extract .

Example 8

Synergistic enhancement of keratinocyte growth factor- induced epithelial cell migration caused by an extract of

Sambucus nigra

Keratinocyte Growth Factors KGF-I (FGF-7) and KGF-2 (FGF- 10) are members of the 23-member fibroblast growth factor family. These factors are up-regulated in the epithelialization-phase of wound healing and are responsible for the migration, invasion and proliferation of epithelial cells. In the present study, the effect of KGF -1 alone and in combination with S. nigra on the migration of Normal Human Epidermal Keratinocytes (NHEK) was investigated.

Cells and culture reagents :

NHEK cells were obtained from Promocell (Promocell GmbH, Heidelberg Germany) and were grown in Keratinocyte Growth Medium with the relevant supplement mix obtained from Promocell as well. The supplement mix contained: 0.4% Bovine pituitary extract, 125ng/ml recombinant human epidermal growth factor, 5μg/ml human insulin, 0.33μg/ml hydrocortisone, lOμg/ml human transferrin, 0.39μg/ml epinephrine and 0.15mM CaCl₂. The cells were maintained in a 5% CO₂/air-humified incubator at 37°C. NHEK cells were subcultured by using the solutions: Hanks, Trypsin and TNS (Trypsin Neutralizing Solution) . The recommended seeding density in a 96 well tissue culture plate is 5xlO⁴ cells/well. Culture age is expressed as population doubling level (PDL), rather than passage number. The PDL recommended by the manufacturer is ≤ 15, where each PDL is different according to the initial seeding density. All experiments were performed at PDL between 12 and 15.

The test was performed by adding KGF-I (Cat. #251-KG, R& D Systems, Minneapolis, MN, USA) at a concentration of lOng/ml and 5. nigra extract at concentrations of 0.02 - 1.25mg/ml after cells reached confluence (24h) . The signal was recorded regularly until 48h.

Other materials :

Recombinant human KGF-I was obtained from R& D Systems (Minneapolis, MN, USA) . CeIl migration assay:

The migration assay was performed using an Oris Cell Migration Assay Kit (Platypus Technologies, Inc., Madison, Wisconsin, USA) . This assay is formatted for a 96-well plate and is used to monitor cell migration. The assay utilizes seeding stoppers that restrict cell seeding in the inner region of the well. Removal of the stoppers reveals the unseeded region to where the seeded cells around the periphery may then migrate. At the end of the experiment all cells are labeled with a florescent marker and a specially designed mask is applied to the plate bottom which restricts the visualization of the well contents to the previously unseeded region. In this way, the part of each well that is assayed for a fluorescent signal is only that part where there were no cells were present at time zero (to) , and to where any cells detected must have migrated.

Fig. 19 illustrates the effect on keratinocyte migration of KGF alone (10ng/ml) or S. nigra alone (1.25 mg/ml) , or the two together at those concentrations. It may be clearly- seen from this graph that the addition of the S. nigra extract causes synergistic enhancement of the pro-migratory activity due to KGF.

While specific embodiments of the invention have been described for the purpose of illustration, it will be understood that the invention may be carried out in practice by skilled persons with many modifications, variations and adaptations, without departing from its spirit or exceeding the scope of the claims.

Claims

1. A composition comprising one or more growth factors and an extract of the plant species Sambucus nigra.

2. The composition according to claim 1, further comprising extracts of one or both of Centella asiatica and Echinacea species .

3. The composition according to claim 2, comprising one or more growth factors and extracts of Sambucus nigra, Centella asiatica and Echinacea species.

4. The composition according to claim 1, wherein the one or more growth factors are selected from the group consisting of PDGF, FGF, BMP-2, BMP-4, BMP-7, TGF-beta and KGF.

5. A method for treating diseases or conditions characterized , by reduced levels of collagen and/or other extracellular matrix components in the affected tissues, wherein said method comprises administering a synergistic combination of one or more growth factors together with an extract of Sambucus nigra with or without additional herbal extracts to a mammalian subject in need of such treatment.

6. A method for treating diseases characterized by- degeneration of collagen and/or other extracellular matrix components, wherein said method comprises administering a synergistic combination of one or more growth factors together with an extract of Sambucus nigra with or without additional herbal extracts to a mammalian subject in need of such treatment .

7. The method according to claim 5 or claim 6, wherein the mammalian subject is a human subject.

8. The method according to claim 5 or claim 6, wherein the synergistic combination contains an extract of 5. nigra as the sole herbal extract.

9. The method according to claim 5 or claim 6, wherein the synergistic combination comprises extracts of S. nigra together with extracts of Centella asiatica and/or Echinacea species .

10. The method according to claim 5 or claim 6, wherein the growth factor is selected from the group consisting of PDGF, FGF, BMP-2, BMP-4, BMP-7, TGF-beta and KGF.

11. The method according to claim 5 or claim 6, wherein the disease or condition to be treated is selected from the group consisting of: wound healing, degenerative, inflammatory, or traumatic bone and joint diseases, keratoconjunctivitis, age related skin changes and oral mucositis.

12. The method according to claim 5 or claim 6, wherein the one or more growth factors and the herbal extract (s) are administered in a single composition.

13. The method according to claim 5, or claim 6, wherein the one or more growth factors and the herbal extract (s) are administered separately.

14. The method according to claim 13, wherein the growth factor (s) and the herbal extract (s) are administered by the same route.

15. The method according to claim 13, wherein the growth factor (s) and the herbal extract (s) are administered by different routes.

16. Use of a composition comprising one or more growth factors and an extract of the plant species Sambucus nigra in the preparation of a medicament.

17. The use according to claim 16, wherein the composition further comprises one or more extracts of Centella asiatica and Echinacea species.

18. The use according to claim 16, wherein the growth factor is selected from the group consisting of PDGF, FGF, BMP-2, BMP-4, BMP-7, TGF-beta and KGF.

19. The use according to claim 16, wherein the medicament is suitable for use in treating diseases or conditions characterized by reduced levels of collagen and/or other extracellular matrix components in the affected tissues.

20. The use according to claim 16, wherein the medicament is suitable for use in treating diseases characterized by degeneration of collagen and/or other extracellular matrix components .