CA2381830A1 - Compositions comprising thylakoids useful in the modulation of the inflammation process - Google Patents

Abstract

This invention relates to the use of a thylakoid extract that is preferably stabilized and activable for treating inflammation. Different types of cell or tissue targets and inflammatory stimuli have been used to evalute the performance of the extract, which, in all cases successfully modulate inflammation.

Description

TITLE OF THE INVENTION
Compositions comprising thylakoids useful in the modulation of the inflammation process FIELD OF THE INVENTION
The invention relates to the use of compositions comprising thylakoids, in specific formulations that ensure the integrity and stability of said thylakoids, to regulate or inhibit inflammation, through their modulating activity on cytokines involved in the inflammation process.
BACKGROUND OF THE INVENTION
Inflammation is a process well known for its implication in acute and chronic diseases and disorders in the biomedical field. Although inflammation is a natural process associated with cell and tissue defense and regeneration, disorganized inflammation can contribute to (or is implicated in) many processes that are harmful to cells and tissues.
Inflammation is the body's reaction to infectious agents, antigen challenge or physical, chemical or traumatic injury (Stvrtinova et al., 1995). The main purpose of inflammation is to bring fluid, proteins, and cells from the blood into the damaged tissues. The main features of the inflammatory response are (i) vasodilation (widening of the blood vessels to increase blood flow); (ii) increased vascular permeability that allows diffusible components to enter the tissues; (iii) cellular infiltration by chemotaxic, or directed movement of inflammatory cells through the walls of blood vessels into the site of injury;
(iv) changes in biosynthetic, metabolic, and catabolic profiles of the affected tissues;
and (v) activation of cells of the immune system as well as enzymatic systems of the blood plasma.
In general, the inflammation response is quite efficient in managing and repairing damages induced by injury or infectious agent. The degree to which these phenomena occur is normally proportional to the severity of the injury or the extent of the challenge. However, inflammation can become harmful to tissues when it develops in a disorganized, disproportionate or undesired manner and can lead to diseases and disorders.

The acute inflammation response is short-lasting and involves all of the previously mentioned features of inflammation. Acute inflammation, when it proceeds in a disorganized fashion, can cause many harmful effects such as the digestion/destruction of normal tissues, excessive swelling that may lead to obstruction of blood flow, resulting in ischemia damage, hypersensitive reaction to non threatening entities (e.g. allergens), etc.
The chronic inflammation reaction may be seen a long-lasting inflammation, where the inflammatory agent is continually present. In this context, chronic inflammation is essentially observed under conditions of delayed hypersensitivity. However, chronic inflammation may be seen is cases where the inflammatory agent is not continuously present, as is the case of in asthma, arthritis or inflammatory bowel disease, and it may also be related to neurological or genetic disorders.
The process of inflammation is driven and modulated by a complex interplay between products of the plasma enzyme systems, lipid mediators (arachidonic acid metabolites such as prostaglandins and leukotrienes), vasoactive mediators released from inflammatory cells, and, in particular, cytokines. Cytokines play a critical role in the inflammatory response. They are produced at the onset of inflammation development and are responsible for the eventual outcome of the inflammation process as well as its resolution. When injury or challenge occurs, cytokines are released from inflammatory cells (mast cells, basophils, endothelial cells, macrophages and neutrophils). The release of many different cytokines is activated during this process including the pro-inflammatory interleukins IL-1, IL-6, IL-8, IL-12, and tumor necrosis factor (TNF-a).
In order to counteract an exaggerated inflammation, anti-inflammatory cytokines such as IL-4, IL-10, IL-13, and transforming growth factor (TGF-~3) are also produced.
Pro- to anti-inflammatory cytokines will determine the eventual outcome of inflammation by their relative proportions, their affinities, and their interactions. More accurately, an appropriate balance and interaction of pro-to anti-inflammatory cytokines will modulate the inflammation process in order to deal with the injury or challenge in the most efficient manner. In order to limit or prevent the damaging effects of inflammation, the immune system is normally well equipped with methods to regulate the balance of pro- and anti-inflammatory cytokines. However, many diseases or disorders will occur when the injured tissue is unable to create this appropriate cytokine balance and interaction (Feghali and Wright, 1997). The onset of the inflammation process is, therefore, not attributable to a single cytokine. For example, an elevation in pro-inflammatory cytokines will not necessarily cause exaggerated inflammation if it is accompanied by an elevation in anti-inflammatory cytokine levels.
Although many cytokines are involved in the inflammation process, some cytokines have a central role in the process and have recently been examined as possible targets for anti-inflammatory products. For example, the pro-inflammatory cytokine TNF-a has been clearly established as playing a pivotal role in many chronic inflammatory diseases and has been targeted for such therapies as monoclonal antibodies, soluble TNF-a receptors, TNF-converting enzyme, and other anti-TNF-a therapies (Lewis and Manning, 1999).
The anti-inflammatory cytokine IL-10 also plays a critical role in the inflammation process to down-regulate the acute inflammation response.
Because of this property, IL-10 has been actively studied as a therapeutic means of controlling inflammation related diseases through gene therapy (Lewis and Manning, 1999; Sacca et al., 1997) A thylakoid extract that has anti-oxidant properties, as described in co-pending application PCT/CA00/01541, has been tested for its capacity as a modulator of cytokines. This extract is provided in the form of specific formulations that ensure the integrity and stability thereof. To simplify terminology, the terms "thylakoids", "thylakoid extract", and "extract" are used hereinbelow and are meant to cover all the specific formulations comprising thylakoids.
TNF-a and IL-10 have been selected as preferred examples of cytokines that are systematically involved in inflammation, notwithstanding the nature of the causative agent or the nature of the tissue or system.
There is an increasing body of literature suggesting that these two cytokines are involved in the expression of inflammatory diseases and disorders examplified but not limited to those affecting the following tissues:

Skin: psoriasis (Reich et al., 2001 ), cutaneous inflammation (Berg et al., 1995), atopic dermatitis (Lee et al., 2000);
Brain: encephalitis (Deckert et al., 2001 );
Gastrointestinal tract: inflammatory bowel disease (Gasche et al., 2000), Crohn's disease (Narula et al., 1998), colitis (Moriguchi et al., 1999);
Eye: infected cornea (Yan et al., 2001 );
Lung: hypersensitivity pneumonitis (Gudmundsson et al., 2000), chronic lung inflammation (Jones et al., 1996);
Multiorgan: ischemia-reperfusion injury (Daemen et al., 1999);
Autoimmune disease: rheumatoid arthritis (Maini et al., 1997; van Roon et al., 1996); and Hyper-reactivity: asthma (Thomas, 2001 ) The state of the art and the availability of a performing and stabilized thylakoid extract prompted the present inventors to test the extract against IL-10 and/or TNF-a expression.
SUMMARY OF THE INVENTION
The present invention relates to the use of a thylakoid extract, in nutraceutical, cosmeceutical, and pharmaceutical applications, in the modulation of expression of cytokines involved in the inflammatory response, which can cause diseases or disorders stemming from disorganized, disproportionate or undesirable inflammatory response.
This invention more specifically relates to the use of a thylakoid extract as an efficient and long-lasting modulator of both pro- and anti-inflammatory cytokines.
More precisely, the invention relates to the use of a thylakoid extract in the regulation of pro-inflammatory cytokines such as TNF-a and anti-inflammatory cytokines such as IL-10, as well as the relative proportions (balance) between these two inflammatory cytokines.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. Effect of thylakoid extract on cytokine expression (% relative to control) in alveolar macrophages when induced with LPS
Fig. 2. Effect of thylakoid extract on IL-10 expression in rat ear following inflammation induction by arachidonic acid.
Fig. 3. Effect of thylakoid extract on TNF-a expression in mouse skin following inflammation induction by UV-irradiation.
Fig. 4. Effect of 18 h pretreatment of FRTS (0.006 and 0.05%) and/or budesonide (10'$M) on IL-10 release 24 h after adding LPS (100 ng/mL).
Fig 5. Modulation of alveolar macrophage IL-10 production by FRTS/1 (0.006%) and budesonide (10-$M).
Fig. 6. Modulation of alveolar macrophage IL-10 production by FRTS/1 (0.05%) and budesonide (10'8M).
Fig. 7. Effect of 18 h pretreatment of FRTS (0.006 and 0.05%) and/or budesonide (10'8M) on TNF release 24 h after adding LPS (100 nglmL).
DETAILED DESCRIPTION OF THE INVENTION
The thylakoid extract is an active extract obtained from photosynthetic organisms, which has the ability to modulate inflammatory cytokines and/or their balance in tissues submitted to stresses that induce an inflammatory response. In particular, the thylakoid extract has demonstrated its competence in the modulation of the pro-inflammatory cytokine TNF-a and the anti-inflammatory cytokine IL-10, thus providing protection against the damages caused by the inflammatory response or, on the opposite, increasing inflammation to improve the body reaction against a danger (such as a tumor or an intruder).
The thylakoid extract of the present invention is considered as a modulator of cytokine bafance.Such a balance involves, on the one hand, pro-inflammatory cytokines comprising TNF-a, and, on the other hand, anti-inflammatory cytokines comprising IL-10. A modification of this balance accompanies other biological, biochemical, and/or physiological evidence of inflammation damage following stress induced by inflammatory agents such as physical, traumatic or infectious agents.
The thylakoid extract is particularly considered to significantly prevent or alleviate the damage due to disorganized inflammation through the modulation of inflammatory cytokines in favor of anti-inflammatory cytokines.
The thylakoid extract can regulate the inflammatory response through the modulation of pro- and anti-inflammatory cytokines at concentrations of 0.0001 to 1 %. For topical uses, maximum efficacy is obtained with 0.001 to 0.1 % thylakoid extract for an application of 2 p,1 per cm2, which provides an effective amount of 2 Ng to 200 Ng of extract per cm2 of surface. Generally, topical formulations would comprise from 0.1 pg to 1 mg of extract per cm2 of skin or mucosae. This extract is used to prevent or alleviate symptoms of inflammation damage to a degree that is comparable or superior to that of recognized anti-inflammatory therapies/products.
EXAMPLES
A. In vitro model: Evaluation of thylakoid extract on cytokine production in alveolar macrophages The alveolar macrophage is one of the major inflammatory cells of the lung and plays an important role in a variety of diseases (Crystal, 1991).
The macrophage protects the lung by regulating inflammatory and immune responses through cytokine production. However, these cytokines may also cause the tissue injury associated with many inflammatory diseases of the lung (Crystal, 1991 ).
Lipopolysaccharides (LPS), a major component of the outer membrane of Gram-negative bacteria is a potent activator of monocytes/macrophages that induces production of several cytokines, including the pro-inflammatory cytokine tumor necrosis factor (TNF-a) and anti-inflammatory cytokine interleukin-10 (IL-10) (Barnes and Lim, 1998).
Inflammatory stimuli activate the transcription of nuclear factor KB (NFxB), resulting in the increased transcription of many inflammatory genes and the release of inflammatory mediators from macrophages such as TNF-a. The same stimuli cause the delayed synthesis of IL-10, which inhibits the expression of these inflammatory-system genes, thus terminating the inflammatory response (Barnes and Lim, 1998).
In asthma, a chronic inflammatory disease of the airways, the IL-10 signal is reduced, leading to increased, lasting and more pronounced inflammation.
Experiment 1: The use of thylakoid extracts on TNF-cx and IL-10 expression in alveolar macrophages induced by LPS.
Protocol:
Rat alveolar macrophages (cell line NR8383) were treated with thylakoid extracts (0%, O.U006%, 0.003%, and 0.006%) in the presence of 10 ng/ml LPS for 20 hours. TNF-a and IL-10 contents were measured using an immunoassay kit for rat TNF-a and IL-10 (BioSources, Camarillo, CA).
Results:
TNF-a demonstrated a slight increase (less than 15%), when compared to the control, in all thylakoid concentrations used in this study (Fig. 1 ).
IL-10 revealed a significant increase in expression when compared to the control.
IL-10 expression was increased by a minimum of 137% and as much as 162% for the 0.0006 and 0.006% thylakoid treatments, respectively (Fig. 1 ).
Discussion and conclusions:

.
The thylakoid extract has clearly demonstrated its modulation ability in cytokine expression and balance on alveolar macrophages. The thylakoid extract produced a limited increase (less than 15%) of the pro-inflammatory cytokine TNF-a while stimulating IL-10 expression greater than 2-fold, when compared to the control. Overall, these results confirmed a shift in cytokine balance towards the anti-inflammatory cytokine (IL-10) when compared to TNF-a, demonstrating its potential as an inflammation modulator.
The above observed effects on alveolar macrophages lead to the assumption that the thylakoid extract is useful to treat a disease or disorder involving macrophages, or in the making of a medication for such a purpose. By simple extrapolation, it is even possible to envisage that a dose capable of achieving about 1 Ng to 100 mg of the extract per liter of body fluid (plasma, blood, extracellular water or total body water content, depending on the distribution) could be effective for systemic purposes. A dose in the milligram range would be preferred.
B. In vivo models: Evaluation of topical application of thylakoid-comprising cream on physical and biochemical parameters of skin inflammation.
As previously mentioned, acute inflammation is associated with numerous diseases and disorders. Among these diseases and disorders are skin inflammation that produces physiological observable characteristics such as redness and oedema, which are accompanied by biochemical parameters such the regulation of inflammatory cytokines. Recognized means of evaluating acute inflammation and cytokine regulation are comparative studies involving treatments that induce inflammatory stress such as with arachidonic acid, which is a lipidic mediator of the inflammatory response (Griswold et al., 1998), or such as with ultraviolet (UV) rays (Brink et al., 2000).
Experiment 2: The ability of the thylakoid extract in reducing or preventing the physical and biochemical signs of inflammation was investigated on rat ears with arachidonic acid as the inflammation stress inducer.
Protocol:

Male star rats (Charles River laboratories) were kept in individual cages, at 20°C and 55% relative humidity with 12-h light/12-h dark cycle, in a facility that met the Canadian Council on Animal Care (C.C.A.C.) guidelines. The protocol was approved by the Animal Care Committee of the Universite du Quebec a Trois-Rivieres. The rats were subjected to an 18 hour starving period prior to treatments.
Three groups of rats were treated in the following manner:
(Treatment 1 ) No application of cream (n =3);
(Treatment-2) Neutral base cream (n =3);
(Treatment-3) Thylakoid extract (0. 1 %) in neutral base cream (n = 3).
All treatments were applied 16 hours, 8 hours, and 1 hour prior to the inflammatory stress at a dose of 2 NI/cm2.
Following treatment, rat ears were subjected to an inflammatory stress by a topical application of arachidonic acid (Sigma Chemicals Co.). 30 p,L of arachidonic acid (0.1 mg/~l_) in an acetone solution was applied. The arachidonic acid solution was reapplied 15 minutes after the initial application (Griswofd et al., 1998). The right ear of each rat was treated with arachidonic acid whereas the left ear served as the unstressed control.
After one hour, the rats were sacrificed and a punch of 6 mm in diameter of both the left and right ears was sampled. Weight of ear punches was recorded and ear punch thickness was measured by an electronic digital caliper 0.01 mm (Traceable).
IL-10 was measured using a solid phase sandwich enzyme linked-immuno-sorbent assay (ELISA) (BioSource International) according to the manufacturer's specifications.
Results:
Arachidonic acid application caused an inflammatory effect in rat ears that were revealed by the presence of redness and oedema.

Results of oedema parameters indicate that both weight and thickness of rat ear punches decreased with a preventive application of thylakoid extracts. When compared 'with average ear punch weight of the thylakoid extract treatment, there was a 43.1 % increase in weight in the non protected treatment (Treatment-1 ) and a 69.2% increase in weight in the neutral base cream treatment (Treatment-2). Furthermore, when compared with average ear punch thickness of the thylakoid extract treatment, there was an increase of 138.0% of thickness of the non protected ear (Treatment-1 ) and an 89.6% increase of ear thickness in the neutral base cream (Treatment-2).
Fig. 2 summarizes the observed results of IL-10 expression in treated rat ears. Treatment-1 and Treatment-2 demonstrated an increase in IL-expression of 25.6 and 23.2%, respectively, when compared to the ear that did not receive arachidonic acid (unstressed control). Conversely, rat ears receiving the thylakoid treatment revealed a decrease of 13.7% in IL-10 expression.
Discussion and conclusions:
The thylakoid extract has an effect on both the physical and biochemical parameters associated with inflammation. The thylakoid extract reduces swelling of rat ears and did not cause an increase in IL-10 expression.
Theses results suggest that the thylakoid extract has a protective effect towards inflammation in that it does not require the production of IL-10 as an agent of down-regulation of the inflammation process. The fact that a decrease of IL-10 is observed in in vivo studies indicates that a lesser production of pro-inflammatory cytokines occurred, which leads to a lesser production of anti-inflammatory cytokines.
Experiment 3: The ability of the thylakoid extract in reducing or preventing the physical and biochemical signs of inflammation was investigated in mouse dorsal skin with UVA and UVB as the inflammation stress inducer.
Protocol:
Hairless mice (5 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). Mice were housed at the Animal Facility of the Institute for Biological Sciences (IBS) and maintained under standard conditions (2311°C, 4216% relative humidity, 12:12-h light-dark cycle). Lights were automatically switched on daily at 7AM and switched off daily at 7 PM. Mice were fed Purina chow diet (24% protein, 4% fat, and 4.5% fiber) and water ad libitum.
Mice were randomly assigned into irradiation and treatment groups as follows:
Group I: UV-irradiated animals pre-treated with a preparation of topical ointment containing the thylakoid extract (n=5).
Group II: Animals received topical application of the cream containing the thylakoid extract during UV irradiation (post-treated) (n=5).
Group III: UV-irradiated animals (n=5) treated with the preparation of topical ointment without the thylakoid extract.
Group IV: Control A: UV-irradiated animals without any topical treatment (n=5).
Group V: Control B: f~on-irradiated animals without any topical treatment (n=5).
Mice were treated with 0.1 % thylakoid-comprising cream for a final application of 2 NUcm2 .
The mice were placed in a plastic cage without a lid. Two Westinghouse FS40 sunlamps (spectral irradiance: 280-400nm, 80% UVB and 20% UVA). Black ray ultraviolet meter was used for the measurement of intensity of light. The fluence at 60 cm from the dorsal surface of the mice was 0.48-0.50 mJ/cm2. The mice were given a single exposure of 200 mJ UV light/cm2 (acute dosage) for 10 min. This approach has been adopted to take into account minor differences in UV absorption characteristics (optical density differences) in 320 nm range that might optically affect UV light irradiation conditions.
After irradiation treatments, mice were kept under the conditions mentioned above for one week. Epidermal observation and photographs were taken to assess redness and dryness.

All the mice from each of the 5 groups were sacrificed. 1 cm2 skin was then removed from each mouse and kept in liquid nitrogen. Two skin pieces from each group were ground in liquid nitrogen and the powder was dissolved in RIPA buffer containing a cocktail of protease and phosphatase inhibitors. The samples were homogenized in a mechanical homogenizes at 4°C and centrifuged at 2500 rpm for 5 min at 4°C. The supernatants were used as cytosolic extracts to study the expression of various epidermal markers.
Samples containing 35 wg total proteins were subjected to protein separation by electrophoresis and the proteins were transferred on nitrocellulose membranes. Western blots were analyzed following immuno staining with antibodies against TNF-a (rabbit polyclonal antibody; Santa Cruz Biotechnology Inc.).
Results:
Results of skin irritation are summarized in Table 1. Irradiated mice with a pre- or post-treatment of the thylakoid extract showed no skin irritation or symptoms of inflammation whereas irradiated mice without the extract (with or without neutral base cream) exhibited redness and dry skin.
Table 1. Effect of 0.1 % thylakoid-comprising cream on the physical symptoms of UV-irradiated mice Treatment Symptoms Pre-treated with thylakoid extract, irradiated None Post-treated with thylakoid extract, irradiated None Neutral cream base, irradiated Redness and dry skin Untreated, irradiated (Control A) Redness and dry skin Untreated, non irradiated (Control B) None Following irradiation, TNF-a was less abundant in both pre- and post-treated thylakoid extract treatments when compared to the non treated or neutral base cream treated mice (Fig. 3). The expression of the pro-inflammatory cytokine TNF-a in the thylakoid extract treated groups are similar to those of the non-stressed control B.
Discussion and conclusions:
The thylakoid extract protected the animals from UV irradiation damages. Even after one week, the non-protected irradiated mice continued to demonstrate redness and skin irritation whereas none of the thylakoid extract treated mice demonstrated these symptoms.
The thylakoid extract clearly exerted a protective effect against inflammation induced by U~/-irradiation of exposed mouse skin. It lowers the level of the pro-inflammatory cytokine TNF-a in irradiated mouse skin.
In view of the foregoing results, it is envisageable that the thylakoids extract is useful for the treatment of inflammation caused by a diversity of pro-inflammatory stimuli.
The modulating activity of the extract (PureCell Complex) on the inflammatory process was indeed investigated. Lipopolysaccharides (LPS) stimulated alveolar macrophages were treated with various concentrations of PureCell Complex alone and in combination with commercial anti-inflammatory ingredients, such as budesonide, and pro- and anti-inflammatory cytokine levels, tumor necrosis factor (TNF) and interleukine-10 (IL-10) respectively, were measured in cell free supernatants at different times after the treatment.
Results (Figures 4 to 7) demonstrated and confirmed that PureCell Complex modulates the synthesis of some cytokines in a dose-dependent manner. Furthermore, PureCell Complex stimulated the release of IL-10 when given in pre- and post-treatment to LPS and a synergistic effect was observed in combination with budesonide. In contrast, TNF release was inhibited by pre-treatment with PureCell Complex and/or budesonide. These data provide further evidence that PureCell Complex possesses anti-inflammatory properties and potentiates the effect of anti inflammatory agents.

Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
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Claims (10)

1. A method for modulating inflammation in a subject, which comprises the step of administering an effective amount of a thylakoid extract.

2. The method of claim 1, wherein the modulation of the inflammation is a reduction of inflammation.

3. The method of claim 1 or 2, wherein the modulation of inflammation involves the modulation of the production of pro- or anti- inflammatory cytokines, or both.

4. The method of claim 3, wherein said pro-inflammatory cytokines comprise TNF-.alpha..

5. The method of claim 3, wherein said anti-inflammatory cytokines comprise interleukine-10.

6. The method as defined in any one of claims 1 to 5, wherein the administration is topical.

7. The method of claim 6, wherein said effective amount is about 0.1 µg to mg of extract per cm2 of surface of skin or mucosae.

8. The method of claim 6, wherein said effective amount is about 1 µg to µg of extract per cm2 of surface of skin or mucosae.

9. A composition comprising an anti-inflammatory amount of a thylacoid extract and of an anti-inflammatory agent.

10. A composition as defined in claim 9, wherein said anti-inflammatory agent is budesonide.