WO2001089540A1

WO2001089540A1 - Use of egg white and egg white fractions for tissue regeneration

Info

Publication number: WO2001089540A1
Application number: PCT/NL2001/000395
Authority: WO
Inventors: Jeroen Johannes Maria Van Den Berg; Noortje Martina Augustina Van Beek; Gerrit Jan Witte; Gerrit Jan Beumer; Maria Cornelia Rudolfje Poland
Original assignee: N.V. Nutricia
Priority date: 2000-05-23
Filing date: 2001-05-23
Publication date: 2001-11-29
Also published as: AU2001260794A1

Abstract

The present invention relates to the use of egg white and/or fractions thereof for the manufacture of a preparation for tissue regeneration. In particular whole egg white is used. With the preparation of the invention several stages of tissue regeneration can be treated, such as initiation and/or acceleration of tissue healing, in particular stimulation of cell migration and/or stimulation of cell proliferation; prevention of wound infection; and prevention of tissue scar formation. The invention also relates to specific fractions of egg white. The preparation of the invention can be a topical preparation, a wound dressing or a specific preparation for the treatment and/or prevention of tissue damage of the gastrointestinal tract.

Description

Use of egg white and egg white fractions for tissue regeneration

The present invention relates to the use of egg white and/or fractions thereof for tissue regeneration, in particular wound healing. The invention also relates to preparations containing egg white and fractions thereof for the above mentioned use.

Recovery from various clinical and subclinical conditions involving tissue injury requires tissue regeneration. On a cellular level, the healing of injured tissue requires breakdown and removal of damaged tissue and replacement with fresh tissue. This process as a whole is a complex interplay of 'on' and 'off signals, e.g. for cellular migration and proliferation, enzymatic activity, and nutritional and immunological factors. It is of considerable importance to medicine to find ways of accelerating wound healing processes and improving impaired wound healing in conditions of chronic injury.

In order to create fresh replacement tissue, existing healthy cells have to grow and proliferate (divide). One aspect of wound healing/tissue regeneration, therefore, is to provide the healthy tissue with sufficient building blocks/nutrients to produce new cells. Egg white contains a number of proteins and other constituents that function to support the growth of the embryo by offering adequate nutrition. In fact, egg white has been suggested as a constituent of growth media for cells in culture.

Zou et al. (Zou, C, Kobayashi, K. and Kato, A. Effect of chicken egg white on the proliferation and neurite outgrowth of mammalian cells. JΛgric.Food Chem. 39 (1991) 2137- 2141) observed that chicken egg white may replace foetal bovine serum in cell culture media as egg white was also capable of stimulating the growth of cells in culture. They also observed morphological changes in some cell types under the influence of egg white, suggesting that egg white may promote cell differentiation. Effects of egg white on specific wound healing processes were not studied or speculated on.

Azuma et al. (Azuma, N., Mori, H., Kaminogawa, S. and Yamauchi, K. Presence of cell growth promoting peptides in chicken egg white extract. Agric.Biol.Chem. 53 (1989) 1291- 1295) found similar cell growth promoting activity in fractionated acidic egg white extracts, and attributed the observed DNA synthesis-promoting effect to some as yet unidentified peptides in the extract. No mention was made of observed or speculative wound healing activity.

Further, Miyagawa et al. (Miyagawa, S., Kamata, R, Matsumoto, K., Okamura, R.and Maeda, H. Inhibitory effects of ovomacroglobulin on bacterial keratitis in rabbits. Graefe's Arch.Clin.Exp.Ophtalmol. 229 (1991) 281-286) reported that ovomacroglobulin, a fraction of egg white, inhibited bacterial proteases and decreased the development of corneal damage caused by these proteases or by the bacteria producing them, S. marcescens and P. aeruginosa. They also found that ovomacroglobulin inhibited the growth of these bacteria and suggested that bacterial growth inhibition is involved in the observed protective effect. In subsequent studies, Miyagawa et al. (Miyagawa, S., Matsumoto, K., Kamata, R., Okamura, R. and Maeda, H. Spreading of Serratia marcescens in experimental keratitis and growth suppression by chicken egg white ovomacroglobulin. Jpn.J.Ophthalmol. 35 (1991) 402-410) confirmed that S. marcescens and P. aeruginosa need extracellular proteases for their growth, and that ovomacroglobulin exhibits an antibacterial effect by inhibiting these proteases.

EP0166130 describes the use of ovomacroglobulin from egg white as an additive for use in cell cultures, as it stimulates the growth of cells in culture. No reference is made to other properties (such as wound healing properties) of ovomacroglobulin or egg white as a whole.

The present inventors have now found that tissue regeneration activity can be found in whole egg white or specific egg white fractions. Egg white was found to be an excellent promoter of wound healing and tissue regeneration, displaying activity on several levels important to the healing/regeneration process. Importantly, although individual egg white components may be found to have a particular activity, we found that egg white as a whole or specific egg white fractions provide optimal activity better than individually isolated and purified egg white components.

The invention therefore provides the use of whole egg white and/or fractions thereof for the manufacture of a preparation for tissue regeneration. Tissue regeneration is here used as a broad term which also includes wound healing. The various processes important for tissue regeneration where egg white may play a role are described below:

• Cellular migration. As an initial phase in wound closure and reepithelialization, epithelial cells at the edge of the site of injury stretch and migrate into the damaged area. This process is initiated by specific cellular signals. Egg white can stimulate this early recovery phase.

• Cellular proliferation. New cells have to be made to replace damaged ones. Therefore, a later stage in wound healing/tissue regeneration is cell division. This process can be stimulated by egg white.

• Regulation of immune cell activation in the damaged tissue. These cells are important for removing cellular debris and infiltrating bacteria. They also produce signals for initiation of healing processes. However, overactivation may lead to uncontrolled inflammation contributing to tissue damage rather than repair. Egg white components can modulate the activity of immune cells such as macrophages and neutrophils.

• Prevention of wound infections: egg white components can kill or inhibit several pathogens.

• Angiogenesis. New tissue requires blood vessels to supply nutrients and remove waste products. Egg white may promote vascularization by stimulating the growth of vascular endothelium.

• Tissue scarring. Cystatin and other protease inhibitors in egg white may help prevent scar tissue formation which may occur during later stages of wound healing.

In view of the above processes which are all encompassed in the term "tissue regeneration", the present invention also provides for the use of whole egg white and/or a fraction thereof for

• initiation and/or acceleration of tissue healing, in particular stimulation of cell migration and/or stimulation of cell proliferation;

• prevention of wound infection;

• prevention of tissue scar formation.

Which (combination) of the above tissue regeneration processes may be stimulated and to what extent depends to a large degree on the pathophysiology as well as the cell types involved (e.g. dermal epithelial vs. oral epithelial vs. intestinal epithelial cells). In addition, the requirement for stimulation of a specific stage in the tissue regeneration process may vary from one physiological situation to the next. Thus, the formulation of an egg white product may be adapted to more specifically address the needs of the individual physiological targets.

Typical applications of the invention include, but are not limited to decubitus (bed sores); burn wounds; leg ulcers (including, but not limited to, venous ulcers and diabetic ulcers); abrasions and bruises; diaper rash; cuts and surgical wounds; oral and pharyngeal tissue damage and ulceration; acne; intestinal ulceration and tissue damage, such as with gastric ulcers, in Inflammatory Bowel Disease, chemotherapy and radiotherapy.

Before use, any microbiological contamination is preferably removed from the whole egg white. This can be achieved by light heat pasteurization (58 °C), but in some cases this leads to a decreased activity of the egg white. Another method to remove microbiological contamination is by means of microfiltration, preferably over a filter of 0.2 μm. As the egg white avian egg white, preferably chicken egg white is used.

According to a first aspect of the invention whole egg white is used. It is believed that the use of whole egg white provides multi-factorial support and stimulation of the complex processes of wound healing and tissue regeneration that cannot be reproduced by applying combinations of individual, isolated egg white components.

Without wanting to be bound by any theoretical explanation it is believed that egg white may stimulate the individual processes described above by providing the specific starting signals they need. For example, egg white may provide triggers for epithelial cells at the edge of a wound area to transform from a quiescent state into an active state where they stretch and migrate into the wound. Similarly, egg white may provide the required 'on' and 'off triggers for other healing/regeneration processes. Furthermore, egg white is an excellent source of nutrients required in the processes described above. In summary, egg white as a whole is an excellent, well-balanced mixture of nutrients, regulatory factors and antipathogenic components that may all work together to provide optimal support for wound healing/tissue regeneration. According to a second aspect, egg white fractions are used. The advantage of the use of egg white fractions is that components that have no or relatively small activity are removed. As the fractions contain less inactive material they are more economical, i.e. smaller amounts of the preparations can be used. Another advantage is that specific fractions can be applied in the different processes involved in tissue regeneration as described above. Also, the fractions obtained can be used to enrich the whole egg white. Further, allergenicity of the product can be reduced by removal of known allergenic compounds, e.g. ovalbumin.

Preferably a fraction is used which is substantially free of all components having a molecular weight larger than 500 kDa, more preferably larger than 200 kDa. Such a fraction can be obtained by means of ultrafiltration of whole egg white. The ultrafiltration process consists of mixing liquid egg white with a suitable amount of water or any buffered salt solution in order to obtain a solution suitable for ultrafiltration. This solution is added to the ultrafiltration apparatus and pumped through a filter with an appropriate effective cut-off, e.g. 200 kD. The filtrate, containing egg white components smaller than the cut-off value, is collected and contains the wound healing activity.

A further preferred fraction is that which is substantially free of ovalbumin. This fraction can be obtained by anionic exchange chromatography using a suitable resin. In this process liquid egg white is mixed with a suitable amount of water or any buffered salt solution in order to obtain a solution appropriate for anion exchange. Anion exchange beads are mixed with the egg white solution in a ratio chosen such, that there is sufficient binding capacity on the anion exchange resin to bind all the ovalbumin in the egg white solution. After mixing, the suspension is allowed to settle or is centrifuged, and the supernatant containing wound healing activity is collected. The anion exchange procedure may also be conducted using an anion exchange column by standard procedures known to a person skilled in the art.

It is also possible to use the egg white fraction free of components having a molecular weight larger than 200 kDa as a starting material for the removal of ovalbumin.

Also preferred is a fraction from which ovomacroglobulin has been removed. As will be shown hereafter, the ovomacroglobulin fraction has hardly any effect on wound healing. According to a third embodiment, whole egg white or one of the above described egg white fractions enriched with conalbumin and/or lysozyme is used. Conalbumin and lysozyme are egg white fractions that can be obtained by ion exchange chromatography or that are available commercially.

The present invention also relates to pharmaceutical preparations containing egg white and/or a fraction thereof and a pharmaceutically acceptable carrier. Suitable preparations are topical preparations such as a cream, paste, powder, gel, ointment or spray. These preparations further contain excipients and additives suitable for the specific type of preparation.

The invention also relates to wound dressings containing egg white and/or a fraction thereof.

The preparations can also be oral preparations, such as nutritional preparations. These preparations can be used for the treatment and/or prevention of tissue damage of the gastrointestinal tract and the oropharyngeal area. In this case it is important that the egg white reaches its destination intact, i.e. that it is not attacked by digestive enzymes in the stomach such as pepsin. This can be achieved by means known to a skilled person, such as manipulation of the pH in the stomach, mixing with a protein that is preferentially digested, encapsulation, microencapsulation, liposome formation, incorporation of a protease or pepsin inhibitor, etc. or combinations of these methods.

The invention is further illustrated by means of the attached figures, wherein

Figure 1 shows wound healing of intestinal epithelium stimulated by egg white;

Figure 2 shows concentration dependent stimulation of intestinal wound healing by egg white.

Figure 3 shows the healing effect of egg white in a skin model.

Figure 4 shows the effect of removal of ovomacroglobulin on wound healing properties of egg white in a skin epithelial cell model.

Figure 5 shows the effect of individual egg white components on wound healing in skin epithelial cell model.

Figure 6 shows anti-inflammatory properties of egg white and egg white constituents. Experimental Materials and methods

Egg white and egg white proteins

Hen egg white was obtained fresh from a commercial supplier as a pasteurized fluid in sealed container. Prior to testing in wound healing assays, egg white was centrifuged at 10,000 rpm for 10 min as needed to remove contaminating solids. The egg white was lyophilized and stored in sealed containers under Nitrogen. Conalbumin and lysozyme were obtained commercially.

Preparation of ovomacroglobulin-rich and ovomacroglobulin-free fractions from egg white

For the preparation of ovomacroglobulin and non-ovomoacroglobulin fractions from egg white, the procedure described in EP 0274532 and EP 0166130 was followed. Fresh hen egg white (100 g) was suspended in 100 ml Tris buffer (25mM Tris, 1% NaCl, pH=7.6). Polyethylene glycol (MW=6,000, Fluka) was added to a final concentration of 2%. The mixture was centrifuged at 10,000 rpm for 10 min. Polyethylene glycol was added to the supernatant in such an amount as to obtain a final concentration of 10%. This was centrifuged again for 10 min at 10,000 rpm. The ovomacroglobulin-containing pellet was stored at 4 °C without further purification.

The non-ovomacroglobulin supernatant was filtered over a 500 kD MWCO filter (Millipore Biomax-500) to further exclude any potential ovomacroglobulin, having a molecular weight of 700-800 kD. The >500 kD retentate was stored at 4 °C until testing. A portion of the <500 kD filtrate was further subjected to microfiltration over a 100 kD MWCO filter (Centricon 100). All fractions thus obtained were stored at 4 °C and subsequently tested in wound healing test systems within three days of purification.

Intestinal wound healing: migration and proliferation A confluent monolayer of rat intestinal epithelial cells (IEC-6) was grown in 12 well-plates in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4.5 g/1 glucose and 10% FCS (37 "C, 5% CO₂) (final concentrations). In this model intestinal epithelium, a wound was made by controlled scratching with a sterile pipette tip. After making the epithelial wound in this way, cellular debri was washed out of the wound area and fresh medium with 0.1 % FCS was added. After addition of test components, the test epithelium was further incubated in medium containing 0.1% FCS and wound healing was assessed as described below.

At different time points, the epithelial wound was examined microscopically, and the open wound area was calculated automatically using an image analysis program. The wound healing potential of test components was expressed as a healing stimulation index relative to the control situation (medium plus 0.1% FCS). During the first few hours after application of the wound, the stimulation index calculated in this way reflects the intrinsic capability of the epithelial cells for initial wound closure, involving processes such as cellular elongation and migration into the wound area. Microscopic analysis after longer recovery periods reveals wound healing potential of test components as a sum of elongation, migration, and proliferation events.

Stimulation of skin wound healing: migration and proliferation

Primary human skin fibroblasts were stored in liquid nitrogen. For the wound healing stimulation assay, 1,000 cells were added to each well in a 96-well plate using DMEM supplemented with 7.5% FCS. Total volume was 100 μl per well. After overnight incubation (37 °C, 5% CO₂), the fibroblasts had adhered to the sides of the well. The medium was removed by aspiration and was replaced with 100 μl of DMEM supplemented with 1% FCS and containing components to be tested for stimulatory properties. Each experimental condition was tested in triplicate. Incubation of the cells was continued for 48 hrs, after which BrdU label was added (10 μl label in 1 ml PBS, 10 μl per well) according to the commercial kit protocol (Boehringer Mannheim). Incubation was continued overnight, after which BrdU incorporated into cellular DNA was measured by ELISA according to the manufacturer's instructions. Stimulation of wound healing by test components is expressed as a healing index relative to the control situation.

In another assay, skin wound healing was assessed in a more acute time frame in order to selectively evaluate cell migration and elongation phenomena. Cells from a human epithelial cell line (SVK14) were grown to confluence in 12- well plates in DMEM. Prior to incubation with test components, the cells were incubated overnight with DMEM supplemented with 0.2% FCS. A wound was then made by controlled scratching with a sterile pipette tip. After making the epithelial wound in this way, cellular debri was washed out of the wound area and fresh medium with 0.2 % FCS was added. After addition of test compounds, cells were incubated for another 2.5 hrs. Stimulation of wound closure was assessed by microscopic examination of the wound and automatic calculation of the wound area using an image analysis program. Stimulation of wound healing by test compounds is expressed as a healing index relative to the control situation.

Antibacterial activity of egg white constituents

To examine the potential of egg white to prevent and/or treat bacterial wound infections, a number of bacterial pathogens were examined for their susceptibility to egg white constituents. Selected colonies from a nutrient agar plate were resuspended in 5 ml LB medium. This culture was incubated for 6 hrs at 37 °C in air with continuous shaking. O.D. (600 nm) was measured, and the culture was diluted to 1 x 10⁵ CFU/ml by referring to a standard curve. A test inoculum (1-5 x 10⁴ cells/ml) was added to a Bioscreen 100-well plate subsequent to addition of medium and compound to be tested. The mixture was incubated for 16-20 hrs at 37 °C in air in a Bioscreen incubator/reader (LabSystems, Helsinki, Finland); OD (600 nm) was read every ten minutes (preceded by shaking). From the collected OD measurements, growth curves were plotted. Growth inhibitory effects are estimated by calculating the area under the curve for a sample incubation compared to a control.

Modulation of immune cell actvity

THP-1 human monocytes were cultured in RPMI 1640 medium. Differentiation to macrophages was stimulated by incubation of the cells with vitamin D3 for 44-48 hours. Production of pro-inflammatory cytokines by the monocyte/macrophage cell culture was initiated by stimulation with bacterial endotoxin (LPS) for 5 hours in the presence or absence of test compounds. Levels of tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) were measured using commercial ELISA kits.

Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats after centrifugation of fresh blood drawn from healthy volunteers. They were cultured and challenged with LPS as described above for THP-1 cells, without pre-incubation with vitamin D₃. Examples

The intestinal epithelial wound healing model and results obtained with chicken egg white are illustrated in Fig.l. In the control incubation, partial epithelial wound closure was observed. With egg white at the indicated (high) protein concentration, complete closure of the wound gap was achieved in 6 hrs. Automated calculation of the wound area by an image analysis program such as NIH Image allows for quantitative evaluation of test compounds, expressing their wound healing potential as stimulation of cellular migration (wound closure) relative to the control situation.

Results of this kind of analysis are shown in Fig.2, where total hen egg white is shown to stimulate intestinal wound healing in a dose-dependent manner. A wound healing index was calculated from microscopic assessment of wound closure by cellular migration after 6 hours of incubation (see text). Results are given as mean ± s.d. of 3 independent experiments. At a total protein concentration in egg white of 10% = 100 mg/ml, the tested range of egg white protein concentrations represents dilutions of 50 - l,000x relative to original liquid egg white. Some individual egg white constituents were also tested and found to stimulate wound healing in this migration assay. Lysozyme resulted in slight stimulation of cellular migration, while conalbumin gave considerable stimulation equal to approx. 75 % of egg white protein at comparable concentrations.

Egg white also stimulated the long-term healing process in human dermal fibroblasts measured by BrdU incorporation into cellular DNA, indicative of cellular proliferation. Fig.3 shows increasing stimulation of proliferation with increasing egg white concentrations. Results presented are the average of two independent experiments and are expressed as a healing index relative to the control incubation (no egg white). At a total protein concentration in egg white of 10% = 100 mg/ml, the tested range of egg white protein concentrations represents dilutions of 5 - 500x relative to original liquid egg white.

Table 1 shows that egg white promotes wound closure in the in vitro setting using human skin fibroblasts as described in Materials and Methods. Results are given as a cellular migration index relative to the control situation (medium + 0.2 % FCS) = 100 %. Stimulation of wound closure was found at egg white protein dilutions of up to 500 times relative to liquid egg white. Table 1. Wound closure in a skin model as stimulated by egg white.

The wound healing properties of ovomacroglobulin were also tested. Fractionation of liquid egg white according to the first steps in a published protocol to obtain ovomacroglobulin yielded an ovomacroglobulin-containing fraction and a non-ovomacroglobulin fraction.

The results are shown in Fig. 4. In Fig. 4, fraction 1 is egg white before first centrifugation step in purification protocol; fraction 2 is supernatant after centrifugation (ovomacroglobulin- free); fraction 3 is pellet after centrifugation (ovomacroglobulin fraction), no further clean-up; fraction 4 is supernatant fraction after ultrafiltration with 500 kDa cut-off, i.e. components <500 kDa; fraction 5 is supernatant fraction, components >500 kDa. Fraction 1 (total egg white) was tested in lOx dilution, i.e. total protein concentration of approx. 1% = 10 mg/ml. For fractions 2-5, the volume after treatment was adjusted to the total volume before treatment in order to facilitate direct comparison between fractions. Fig.4 shows that most of the wound healing stimulatory activity was associated with the non-ovomacroglobulin fraction.

Fig. 5 shows the effect of individual egg white components on wound healing in a skin fibroblast cell model. Human primary dermal fibroblasts were used in the BrdU incorporation assay as described in Materials and Methods. Control: medium plus 1% FCS; Egg white: whole egg white, protein concentration 2 mg/ml; conalb.: conalbumin, 1 mg/ml; ovomuα: ovomucoid, 1 mg/ml; lysozyme: lysozyme, 1 mg/ml; ovalb.: ovalbumin, 1 mg/ml. Results shown are the average ± s.d. of two independent experiments, where each experimental condition was determined in triplicate.

Microbial infections often complicate and inhibit the wound healing process. Some egg white constituents may help prevent wound infections by exerting antimicrobial effects. Conalbumin inhibits the growth of several pathogens in vitro (Table 2). Lysozyme also exhibits antibacterial properties, mainly against Gram-positive species. We also found that the combination of conalbumin and lysozyme acts synergistically against some pathogens (data not shown).

Table 2. Inhibition of bacterial growth by conalbumin

Numbers given are growth inhibition indices, representing the percentage growth inhibition from area-under-the-curve calculations of bacterial growth curves (see Materials and Methods).

Activation of immune cells in the early stages of wound healing is important for removal of cellular debris and preventing bacterial infections. However, in cases such as chronic leg ulcers the continued inflammatory state of the wound tissue inhibits the subsequent healing and regeneration stages. Fig. 6 shows anti-inflammatory properties of egg white and egg white constituents. Experiments were conducted with peripheral blood mononuclear cells (PBMCs) obtained from a healthy volunteer, or with a THP-1 human monocyte/macrophage cell culture. Cells were stimulated with LPS, and production of pro-inflammatory cytokines measured using specific ELISA methods as described in Materials and Methods. The figure shows the reduction in production of tumor necrosis factor-α (TNF) in PBMCs by egg white protein and in THP-1 cells by conalbumin (con) and lysozyme (lys) derived from egg white.

From this figure it can be concluded that egg white and some individual egg white constituents reduce inflammatory reactions in vitro systems involving monocyte/macrophages and peripheral blood mononuclear cells. Thus, egg white may be able to reduce the inflammatory state of such chronic conditions and initiate proper healing of these wounds.

Claims

1. Use of egg white and/or fractions thereof for the manufacture of a preparation for tissue regeneration.

2. Use according to claim 1, wherein whole egg white is used.

3. Use according to claim 1 or 2, for initiation and/or acceleration of tissue healing, in particular stimulation of cell migration and/or stimulation of cell proliferation;.

4. Use according to claim 1 or 2, for prevention of wound infection.

5. Use according to claim 1 or 2, for prevention of tissue scar formation.

6. Use according to any of claims 1 or 3 to 5, wherein a fraction of egg white is used, substantially free of components having a molecular weight larger than 500 kDa, preferably larger than 200 kDa.

7. Use according to any of claims 1 or 3 to 6, wherein a fraction of egg white is used, substantially free of ovalbumine.

8. Use according to any of claims 1 to 7, wherein whole egg white or an egg white fraction enriched with conalbumin and/or lysozyme is used.

9. Use according to any of the preceding claims, wherein avian egg white, preferably hen egg white is used.

10. Pharmaceutical composition containing egg white and/or a fraction thereof and a pharmaceutically acceptable carrier.

11. Topical preparation containing egg white and/or a fraction thereof and a carrier suitable for a topical preparation.

12. Topical preparation according to claim 11, which is a cream, paste, powder, gel, ointment or spray.

13. Wound dressing containing egg white and/or a fraction thereof.

14. Use of egg white and/or a fraction thereof in the manufacture of a nutritional preparation for the treatment and/or prevention of tissue damage of the gastrointestinal tract.

15. Oral preparation containing a fraction of egg white, substantially free of components having a molecular weight larger than 500 kDa, preferably larger than 200 kDa.

16. Oral preparation containing a fraction of egg white, substantially free of ovalbumine.

17. Oral preparation containing whole egg white enriched with conalbumin and/or lysozyme or an egg white fraction enriched with conalbumin and/or lysozyme.