US20020001625A1

US20020001625A1 - Growth-promoting agent

Info

Publication number: US20020001625A1
Application number: US09/912,040
Authority: US
Inventors: Francis Ballard; Geoffrey Francis; Geoffrey Regester; Leanna Read; David Belford
Original assignee: Gropep Ltd
Current assignee: Novozymes Biopharma DK AS
Priority date: 1990-07-13
Filing date: 2001-07-24
Publication date: 2002-01-03
Anticipated expiration: 2011-07-09
Also published as: US6319522B1; US6447808B2

Abstract

The invention relates to pharmaceutical or veterinary compositions for the treatment of surface wounds; pharmaceutical or veterinary compositions for the treatment of gastrointestinal injuries, diseases or ulcers; methods of treating surface wounds in animals, including humans; and methods for the treatment of gastrointestinal injuries, diseases or ulcers which compositions and methods include compositions of milk product extracts including growth factors with basic to approximately neutral isoelectric points.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation-in-Part of U.S. Ser. No. 07/956,759, filed Dec. 7, 1992. Application Ser. No. 07/956,759 was a national stage application based on PCT/AU91/00303, filed Jul. 9, 1991. The parent application claims priority from Australian Provisional Patent Application PK 1170, filed Jul. 13, 1990. The present application also claims priority from Australian Provisional Patent Application PN2712, which was filed May 2, 1995. Priority is claimed from the above recited applications, to the extent entitled.[0001]

FIELD OF THE INVENTION

This invention relates to the growth of animal cells in a cell culture composition. More specifically it relates to the provision of a cell culture composition including a milk product extract composition. This invention also relates to the growth of animal cells where that growth is associated with the repair of surface wounds or the repair of gastrointestinal injuries, diseases or ulcers, by the application of a composition including a milk product extract composition.

BACKGROUND OF THE INVENTION

Animal cells are grown in culture to provide a number of pharmaceutical, diagnostic and veterinary products including human vaccines, lymphokines, hormones, monoclonal antibodies, other pharmaceutically active protein products, veterinary hormones and for research and development and diagnostic purposes.

The growth of animal cells requires a defined isotonic medium that contains salts, nutrients, lipid precursors, nucleic acid precursors, vitamins and amino acids that are formulated to mimic the medium that would normally bathe those cells in vivo. Examples in common use include Eagle's Minimal Essential Medium, Dulbecco's-modified Eagle's Minimal Essential Medium (DMEM), Medium 199, RPMI 1640 medium and Ham's F12 Medium. However, virtually no animal cells will grow in such a medium, but require the co-addition of serum. Fetal bovine serum is frequently used as it is more effective than serum obtained from post-natal animals and it contains only minimal concentrations of immunoglobulins which otherwise could have undesirable effects.

The supply of fetal bovine serum is limited by the number of pregnant cows slaughtered. It also has undesirable lot-to-lot variations and may include toxins. Particular concern surrounds its use for the eventual production of recombinant proteins and other pharmaceuticals for human use because the serum may also contain viruses that are harmful to humans and may be carried through a purification protocol that yields the desirable product. Principally for these reasons, extensive efforts have been directed towards the replacement of serum by pure ingredients. Examples of such ingredients are growth factors, hormones and cell attachment factors. Unfortunately, the requirements of each cell type being grown are different and are difficult to establish. Frequently it has not proved possible to achieve equivalent growth properties or equivalent yields of cell products with “serum-free”, media as can be obtained with medium containing fetal bovine serum.

The limited availability of fetal bovine serum, its lot-to-lot variability, its resultant considerable cost as well as the deficiencies of “serum-free” media described above have prompted the investigation of other biological fluids as potential replacements in cell culture media. Some progress has been reported in the prior art with bovine milk and bovine colostrum as evidenced by the following selected reports: M. Klagsbrun: “Human milk stimulates DNA synthesis and cell proliferation in cultured fibroblasts” (Proc. Natl. Acad. Sci. USA 75, 5057, 1978); M. Klagsbrun & J. Neumann: “The serum-free growth of Balb/c 3T3 cells in medium supplemented with bovine colostrum” (J. Supramol. Struct. 11, 349, 1979).

The prior art also includes U.S. Pat. No. 4,440,860 to M. Klagsbrun which describes “compositions and methods for promoting cell growth featuring, in one aspect, cell culture media containing milk or colostrum and fibronectin; fibronectin is preferably pre-coated onto the culture substrate” and Japanese Patent JP 59166879 to Morinaga “A culture medium for cell incubation-containing milk or milk components”. Ultrafiltrates of milk whey have also been used to support the growth of cultured cells, as in O. Damerdji et al. “Utilization of whey fractions as a substitute for fetal calf serum in culture media” (Biotech. Tech. 2, 235, 1988).

Despite this progress a successful alternative to fetal bovine serum is yet to be located.

It is accordingly an object of the present invention to overcome, or at least alleviate one or more of the difficulties or deficiencies related to the prior art.

SUMMARY OF THE INVENTION

Accordingly in a first aspect of the present invention there is provided a milk product extract composition including a plurality of cell growth stimulating factors, extracted from milk product, in concentrated form; said factors having basic to approximately neutral isoelectric points.

By the term “milk product extract” we mean an extract from human or animal milk product in which the salt and/or main protein constituents thereof are reduced or eliminated. Examples of milk product extracts include cheese whey extracts, skim milk extract and acid (casein) whey extract, and extracts of colostrum. Milk products of the invention include cheese whey, skim milk, acid (casein) whey, and colostrum.

The range of isoelectric points described by the term “basic to approximately neutral isoelectric point” includes isoelectric points between about 6.0 and about 10.5 or between approximately 6.0 and approximately 10.5, preferably between 6.0 and about 10.5, preferably between 6.0 and approximately 10.5.

The present invention will be more fully described with reference to the preferred cheese whey extracts. However, this is illustrative only and should not be taken as a restriction on the generality of the invention.

Preferably the milk product extract composition is a cheese whey extract composition.

The cheese whey extract composition may be formed from cheese whey wherein the salt and/or main protein constituents thereof are reduced or eliminated.

The milk product extract composition may include reduced amounts of alpha lactalbumin, beta lactoglobulin and casein compared with said milk product. Preferably, the milk product extract includes less than approximately 1% w/w of the salt present in the original milk product. The milk product extract may include less than approximately 0.5% of the casein, alpha lactalbumin, beta lactoglobulin, immunoglobulin and/or albumin present in the original milk product.

The milk product extract composition according to this aspect of the present invention may be utilized in the promotion of cell growth and proliferation in vitro as discussed below. The milk product extract composition may be utilized in stimulation of surface wound repair in vivo, or for the treatment of gastrointestinal injuries, diseases or ulcers, in mammals as discussed below.

Surprisingly, the milk product extract composition may support the growth of animal cells at lower protein concentrations than achieved with fetal bovine serum, yet with an efficacy comparable to fetal bovine serum for several cell types.

Alternatively, the milk product extract may be used as a supplement to media containing low concentrations of fetal bovine serum in order to achieve better growth rates of cultured cells and to conserve the use of fetal bovine serum.

Cheese whey is a by-product of the cheese industry that has had essentially all the fat and casein removed during cheese manufacture. At the present state of the art cheese whey is essentially valueless, and indeed it may represent a net cost to the industry since it is a potential pollutant.

Cheese whey for example is a low protein, high salt product available in tonne amounts from cheese manufacture. The main protein constituents present in cheese whey are alpha lactalbumin (αLA) and beta lactoglobulin (βLG), which usually account for more than 90% of the proteins present. Significant amounts of serum albumin, immunoglobulins and residual casein may be present. All of these proteins have acidic isoelectric points. In contrast, the main protein factors that stimulate the growth of animal cells have basic isoelectric points. Examples include the growth factors basic FGF, IGF-I, des(1-3)IGF-I and PDGF. It is postulated that the extraction of the basic factors present in milk products such as cheese whey in the virtual absence of the otherwise abundant acidic proteins may account for the surprising efficacy of the milk product extract composition.

Accordingly in a further aspect of the present invention, there is provided a method for preparing a milk product extract composition including a plurality of cell growth stimulating factors, extracted from milk product in concentrated form; said factors having basic to approximately neutral isoelectric points, which method includes

providing

a source of milk product;

a cationic exchange resin; and

a buffer solution;

contacting the milk product with the cation exchange resin such that the more basic components of the milk product are absorbed thereon;

eluting the cationic exchange resin with the buffer solution; and filtering the eluate to remove salt therefrom.

The desorption of the basic proteins from the ion exchange resin leads to a preparation enriched in cell growth stimulating factors. The eluate may be concentrated and filtered utilizing any suitable technique. The eluate may be concentrated for example by conventional ultrafiltration methods or other procedures to yield a mixture of proteins which supports the growth of animal cells when added to protein-free media such as DMEM.

The source of milk product may be a milk product filtrate substantially free of insoluble material. Accordingly the preparation method may include the preliminary step of

filtering the milk product to remove insoluble materials therefrom.

The milk product may be filtered through a suitable sieve. The milk product may be filtered through a hollow fiber cartridge of defined porosity.

The cationic exchange resin may be of any suitable type. A Sepharose-based cation exchange gel may be used. The contacting step may be conducted at neutral to basic pH. The contacting step may be conducted at a pH of approximately 6.5 to 8.0.

The cationic exchange resin may be equilibrated with a suitable buffer at a pH of approximately 6.5 to 8.0. An aqueous sodium citrate buffer may be used. The elution steps may be conducted utilizing a suitable eluate. A salt solution may be used. A buffered saline solution may be used.

Thus in a preferred form of this aspect of the present invention the method of preparing a milk product extract composition may include treating milk product sequentially by:

subjecting the milk product to a filtration step, to remove insoluble materials therefrom;

adjusting the pH of the filtrate to between approximately 6.5 and 8.0;

contacting the filtrate with a cationic exchange resin;

eluting from the cation exchange resin at high ionic strength and high pH with a suitable buffer solution; and

subjecting the eluate to a concentration step and diafiltration step to remove salt therefrom.

Alternatively, the elution from the cation exchange resin is achieved at high ionic strength but without adjusting pH, such that the cell growth stimulating factors are recovered.

In this embodiment the cell growth stimulating factors are eluted with less extraneous protein.

In a further aspect of the isolation of a suitable extract from cheese whey, the eluant may be treated at high temperature and centrifuged. This modification removes additional protein. Accordingly, the method may further include subjecting the eluant to a heat treatment to reduce the content of extraneous protein.

The milk product extract composition may be sterilized and optionally freeze-dried for storage. The freeze-dried material may be dissolved in sterile saline for addition to cells in culture.

In a further aspect of the present invention there is provided a cell culture composition including an effective amount of a milk product extract composition including

a plurality of cell growth stimulating factors, extracted from milk product, in concentrated form; said factors having basic to approximately neutral isoelectric points; and

a culture medium.

The culture medium may be a substantially protein-free isotonic culture medium. The substantially protein-free isotonic culture medium may be Dulbecco's-Modified Eagle's Minimal Essential Medium (DMEM).

It has been found that an approximately equivalent growth rate of human skin fibroblasts to that achieved with 5% Fetal Bovine Serum may be achieved with approximately 20 μg of cell growth stimulating factors extracted from cheese whey according to the preferred aspect of the present invention per 100 μl of medium.

Alternatively a small but effective amount of fetal bovine serum may be utilized as the culture medium. It has been found that the addition of approximately 25 μg of cell growth stimulating factors per 100 μg of medium containing approximately 2% fetal bovine serum will increase the growth rate of Balb C/3T3 cells to that rate otherwise achieved with 10% fetal bovine serum.

Other additions may be made to the medium, depending on the cell type, including growth factors, attachment factors or low amounts of serum.

In a preferred form, the present invention provides a cell culture composition, as described above, wherein the milk product extract is present in media at a protein concentration of approximately 10 to 20,000 micrograms per ml, preferably 100 to 2,000 micrograms per ml.

Accordingly in a still further aspect of the present invention there is provided a method for culturing cells which method includes

providing

source of animal cells; and

a cell culture composition including an effective amount of a milk product extract composition including

a substantially protein-free isotonic culture medium; and

culturing the cells in the cell culture composition for a time sufficient, and at a temperature sufficient to achieve a predetermined cell concentration.

The cell culture method may be conducted at ambient temperature or above. A temperature in the range of approximately 35 to 40° C. may be used.

The cell culture process may be conducted in an incubator, for example a humidified incubator.

The cell culture method may be conducted on any suitable surface or in suspension. Tissue culture plates may be used.

The cell culture method may continue for a period of approximately 1 to 5 days depending on the cell concentration desired.

Although the method in particular applies to the growth of animal cells in vitro it can also be applied to animals, including humans, that have surface wounds.

It has been found that a composition including a milk product extract according to the present invention can improve surface wound repair in vitro and in vivo.

As used herein, the term “surface wounds” includes the following types of damage:

(a) ulcers of all kinds including pressure ulcers such as pressure sores, bed sores or decubitis ulcers, as well as ulcers associated with vascular disease such as venous ulcers, varicose ulcers, and ulcers associated with diabetes, autoimmune disease, sickle cell diseases or hemophilia;

(b) conditions that result from surgery such as debridement, skin grafting, partial thickness wounds that result after removal of skin for grafting, and full thickness incisions;

(c) therapeutically induced wounds including those induced during radiotherapy or in response to drugs;

(d) wounds associated with disorders of the central nervous system that may alter sensation or affect mobility;

(e) wounds that result from any exfoliative disease of the skin;

(f) wounds associated with either local or systemic infection such as yaws or HIV;

(g) congenital wounds such as spina bifida;

(h) pathological wounds that include wounds associated with skin cancers;

(i) traumatic and accidental wounds including penetrations, lacerations, abrasions, gouges and other trauma, and

(j) burns.

Accordingly, in a further aspect, the present invention provides a pharmaceutical or veterinary composition for the treatment of surface wounds, which composition includes:

an effective amount of a milk product extract composition including a plurality of cell growth promoting factors, extracted from milk product in concentrated form; said factors having basic to approximately neutral isoelectric points; and

a pharmaceutically or veterinarily acceptable diluent, carrier or excipient therefor.

In a particularly preferred form, the present invention provides a pharmaceutical or veterinary composition for the treatment of surface wounds which composition includes:

(a) a milk product extract that contains a mixture of cell growth factors with basic to approximately neutral isoelectric points, preferably isoelectric points between about 6.0 and about 10.5, preferably isoelectric points between 6.0 and about 10.5. The mixture of cell growth factors can be obtained from a milk product of an ungulate mammal by first subjecting that product to a cation exchange matrix under conditions whereby casein, alpha lactalbumin, and beta lactoglobulin present in the milk product are not absorbed to the matrix, after which the absorbed growth factor mixture is eluted and then concentrated; and

(b) a pharmaceutically or veterinarily acceptable diluent, carrier or excipient therefor.

The pharmaceutical or veterinary composition may further include an effective amount of at least one active ingredient.

The at least one active ingredient may be selected from antibiotics, antiseptics, other growth promotants, anaesthetics, and the like, and mixtures thereof.

The pharmaceutical or veterinary composition may be adapted for administration in any suitable manner. The composition may be adapted for internal or topical administration. The composition may be in an oral, injectable or topical form. Topical administration is preferred. The composition may take the form of a wash, lotion, cream, ointment or gel.

There are no limitations to the type of surface wound that may be treated, and these include, but are not limited to ulcers, conditions that result from surgery, therapeutically induced wounds, wounds associated with disorders of the central nervous system, any exfoliative disease of the skin, wounds associated with local or systemic infection, congenital wounds, pathological wounds, traumatic and accidental wounds, and burns.

Accordingly, in a further aspect of the present invention there is provided a method of treating surface wounds in animals, including humans, which method includes administering to a subject to be treated an effective amount of a pharmaceutical or veterinary composition, which composition includes

In a particularly preferred form, the present invention provides a method of treating surface wounds in animals, including humans, which method includes administering to a subject to be treated an effective amount of a pharmaceutical or veterinary composition, which composition includes:

In the above method, the milk product extract may be applied directly to wounds in a biologically acceptable carrier to ensure sustained release at sufficient concentration in the wound environment. Such carriers include any synthetic or biological polymer, glycosaminoglycan, or extracellular matrix molecule (eg. fibrin, collagen, gelatin, synthetic polymers, agarose, alginates, methylcellulose, hyaluronic acid, hydrocolloids, alginates). The carrier may be in the form of a gel. Alternatively the milk product extract could be administered in the form of a spray (in a biologically acceptable diluent, for example a buffer solution), powder, ointment, salve or irrigant or incorporated or impregnated into a dressing (absorbable and non-absorbable), transdermal patches or releasably associated with gauze, bandages, sutures, plasters, staples, prosthetic devices, screws or plates (biodegradable or non-biodegradable). Alternatively, the milk product extract may be incorporated into a toothpaste, gum or resin for chewing or provided as a mouth wash.

In another aspect the present invention relates to a wound support including an effective amount of a milk product extract composition including a plurality of cell growth promoting factors, extracted from milk product in concentrated form; said factors having basic to approximately neutral isoelectric points.

As used herein the term “wound support” includes any means which is used to support or secure a wound and includes a surgical securing means. The term includes plasters, dressings, sutures, staples and the like. The wound to be supported may be a wound created by surgery, or the result of accident or other injury. The milk product extract may be present on the surface of the wound support or may be impregnated in the wound support and is able to be released therefrom.

The milk product extract may have other molecules associated therewith to aid releasability, stability, solubility, activity and/or association with the wound support, including adjuvants, carriers, solubilizing agents, and growth factors. Furthermore, the milk product extract may be used in combination with other compounds or molecules which act in synergistic, agonistic and/or additive concert. There are no limitations to the nature of these ingredients except they should be pharmacologically and physiologically acceptable for administration and should not degrade the activity, or render harmfully toxic the active ingredients.

Preferably the milk product extract when used in the above described methods is administered in an amount from 0.01 to 10 mg/ml of fluid in the local environment at the wound side. The milk product extract may be delivered during the peri or post operative period. Alternatively the milk product extract may be impregnated or present on the surface of an adhesive dressing, occlusive plaster or surgical securing means such as sutures or staples.

It will be understood by those skilled in the art that the milk product extract may be administered for a time and under conditions sufficient to allow for wound repair.

The composition of the present invention may also be used to treat animals, including humans, that have gastrointestinal injuries, diseases or ulcers. It has also been found to improve the growth rate of gastrointestinal cells in vitro.

As used herein the term “gastrointestinal injuries, diseases or ulcers” includes the following types of damage to or diseases of the gastrointestinal tract:

(a) dental and oral wounds, including those associated with periodontal disease;

(b) peptic ulceration of the duodenum, stomach or esophagus;

(c) inflammatory bowel diseases such as ulcerative colitis or Crohn's disease;

(d) ulcers associated with stress conditions, for example burns, trauma, sepsis, shock, intracranial surgery or head surgery;

(e) damage to the lining of the alimentary tract resulting from radiotherapy and/or chemotherapy with agents such as mechlorethamine, melphalan, busulphan, cytarabine, floxuridine, 5-fluorouracil, mercaptopurine, methotrexate, thioguanine, bleomycin, actinomycin-D, daunorubicin, etoposide, mitomycin, vinblastine, vincristine, hydroxyurea or procarbazine;

(f) inadequate gut function or damage to the gut associated with prematurity such as narcotizing enterocolitis or poor gut motility;

(g) diarrheal conditions such as associated with bacterial, viral, fungal or protozoan infection, including AIDS;

(h) food intolerances such as coeliac disease;

(i) cancers of the gastrointestinal tract, including buccal cavity, esophagus, stomach or bowel;

(j) surgically induced damage such as following partial gut resection, short gut syndrome, jejunostomy, ileostomy, colostomy;

(k) damage due to esophageal reflux;

(l) conditions associated with loss of gut barrier function such as external burns, trauma, sepsis or shock;

(m) congenital conditions resulting in inadequate gastrointestinal function or damage such as volvulus and cystic fibrosis; and

(n) autoimmune diseases that affect the gut, such as Sjogren's Syndrome.

Accordingly, in a further aspect, the present invention provides a pharmaceutical or veterinary composition for the treatment of gastrointestinal injuries, diseases or ulcers, which composition includes:

a pharmaceutically or veterinarily-acceptable diluent, carrier or excipient therefor.

In a particularly preferred form, the present invention provides a pharmaceutical or veterinary composition for the treatment of gastrointestinal injuries, diseases or ulcers which composition includes:

There are no limitations to the type of gastrointestinal injuries, diseases or ulcers that may be treated, and these include, but are not limited to dental and oral wounds, peptic ulcers, inflammatory bowel diseases, ulcers associated with stress conditions, damage caused by radiotherapy and/or chemotherapy, inadequate gut function or damage associated with prematurity, diarrheal conditions, damage caused by food intolerance, cancer of the gastrointestinal tract, surgically induced damage, damage caused by esophageal reflux, conditions associated with loss of gut barrier function, congenital conditions resulting in inadequate gastrointestinal function or damage, and autoimmune diseases that affect the gut.

Accordingly, in a still further aspect of the present invention, there is provided a method for the treatment of gastrointestinal injuries, diseases or ulcers, which method includes administering to a subject to be treated an effective amount of a pharmaceutical or veterinary composition, which composition includes

an effective amount of a milk product extract composition including cell growth promoting factors, extracted from milk product in concentrated form and having a basic to approximately neutral isoelectric point; and

In a particularly preferred form, the present invention provides a method for the treatment of gastrointestinal injuries, diseases or ulcers, which method includes administering to subject to be treated an effective amount of a pharmaceutical or veterinary composition, which composition includes:

In the above method the milk product extract may be administered directly into the alimentary canal by oral delivery or other means of direct enteral administration, in order to maximize the effective dose reaching the affected tissue.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; as a mouthwash or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

It should be understood that in addition to the ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents.

The milk product extract may be administered at any appropriate time including prior to, during or after the gastrointestinal injuries, diseases or ulcers have become evident.

The milk product extract may be useful in combination with known therapeutic agents. If formulated as a fixed dose, such combination products may employ the milk product extract in an appropriate dosage range and the other pharmaceutically active agent within its approved dosage range. Compositions of the invention may be used sequentially with known therapeutic agents when a combination formulation is inappropriate.

When the milk product extract is administered to a human subject the daily dosage can be determined by the attending physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms. In general a suitable dose of the milk product extract of the invention will be in the range of 20 mg to 20 g per kilogram body weight of the recipient per day, preferably in the range of 200 mg to 2 g per kilogram body weight per day. However, the dose will also depend on the formulation and purity of the milk product extract used. The above mentioned doses are calculated on the basis of the cheese whey product extract described in Example 2 and could be modified accordingly by a person skilled in the art if a product of different activity or purity was used.

The present invention will now be more fully described with respect to the following examples. It should be understood, however, that the description following is illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above.

DESCRIPTION OF THE DRAWINGS

FIG. 1 Growth of (a) L6 myoblasts, (b) Balb C/3T3 fibroblasts and (c) SF1972 human skin fibroblasts in the presence of () fetal bovine serum (FBS), (∘) GFE prepared according to Example 1 or (▪) GFE-2 prepared according to Example 2. The details are described in Example 4. [0136]
FIG. 2 Growth promoting activities in human umbilical vein endothelial cells in the presence of cheese whey extract (GFE-2, Example 2). Data are expressed as the percentage [0137] ³H-thymidine incorporation above control cultures grown in serum-free medium only. The details are given in Example 7.
FIG. 3 Contraction of a fibroblast-populated collagen gel in response to cheese whey extract (GFE-2, Example 2). Fibroblast-induced contraction of the gel was assessed by counting the radioactivity remaining in the gel after 24 h culture (mean±SEM of triplicate determinations). The details are given in Example 8. [0138]
FIG. 4 Excisional wound closure in organ-cultured fetal rat skin in response to () FBS, (▪) DMEM and (▴) cheese whey extract (GFE-2, Example 2). Wound repair was assessed by recording the ability of the cheese whey extract at the indicated concentrations to promote repair of the excisional deficit over a 72 h period. Other details are given in Example 9. [0139]
FIG. 5 Strength of incisional wounds in normal rats after single dose treatment with either () a vehicle preparation (rat tail collagen at 1 mg/ml) or (▪) vehicle plus cheese whey extract (GFE-2, Example 2). Data are plotted as wound breaking strength (g) against time. Other details are given in Example 10. [0140]
FIG. 6 Contraction and epithelialization of excisional wounds on steroid-treated rats after treatment with either (▪) cheese whey extract (GFE-2, Example 2) or (♦) collagen vehicle alone, compared to wounds that were (□) untreated and left exposed. Wounds were treated with either vehicle preparation (1 mg/ml collagen) or vehicle plus cheese whey extract (2.5 mg/ml) daily for 10 days after wounding. A further group of control wounds received no treatment and were left exposed. Wound repair was measured by tracing both the full thickness wound margin and epithelial margin onto transparent sheets at various time points after wounding. FIG. 6([0141] a) shows full thickness wound area (MM²) vs time as a measure of wound contraction. FIG. 6(b) incorporates the epithelial component of excisional wound repair. Other details are given in Example 11.
FIG. 7 Growth promoting activities of cheese whey extract (GFE-2, Example 2) on IEC-6 (rat intestinal epithelial) cells. GFE-2 was added either (∘) alone or in the presence of (□) 1% FBS or (Δ) 5% FBS. Cell growth is expressed as a percentage of that obtained in the presence of 10% FBS. Other details are given in Example 12. [0142]
FIG. 8 Oral administration of cheese whey extract (GFE-2, Example 2) for 5 days to methotrexate-injected rats reduces small intestinal damage as assessed by (a) loss of mucosal crypt area (b) loss of mucosal villi area. Means±SEM, N=8. Other details are given in Example 13. [0143]
FIG. 9 Oral administration of cheese whey extract (GFE-2, Example 2) for 5 days to methotrexate-injected rats increases sucrose activity in the ileum. Means±SEM, N=8. Other details are given in Example 14. [0144]
FIG. 10 Oral administration of cheese whey extract (GFE-2, Example 2) for up to 12 days to methotrexate-injected rats reduces bacterial translocation across the gut. (a) Percentage of rats showing bacterial translocation, (b) Number of bacterial colonies per gram of intestinal lymph node. Means±SEM, N=8. Other details are given in Example 15. [0145]
FIG. 11 Daily treatment of the cheek pouch in hamsters with a milk product extract (GFE-2) reduces the severity of oral mucositis ulcers caused by 5-fluorouracil. [0146]
FIG. 12 Daily treatment of the cheek pouch in hamster with a milk product extract (GFE-2) reduces the body weight loss induced by 5-fluorouracil.[0147]

DETAILED DESCRIPTION

As examples of wound repair in vitro, the cheese whey extract promotes the growth of human skin fibroblasts and human endothelial cells, cell types that are major components of the skin. [0148]
In a further example of wound repair in vitro, the cheese whey extract stimulates the contraction of a fibroblast-populated collagen gel, a process that is analogous to the desired contraction of a wound in vivo. [0149]
In yet a further example of wound repair in vitro, the cheese whey extract induces the closure of a full thickness excisional wound in cultured fetal rat skin. [0150]
As an example of wound repair in vivo, it was found that the cheese whey extract enhanced the strength of an incisional wound in normal rats at 5 days or 7 days after a single treatment. [0151]
In a second example of wound repair in vivo, it was found that full thickness, excisional wounds on steroid-compromised rats repaired more rapidly when treated daily with the cheese whey extract than with the vehicle solution. In this example both wound contraction and wound epithelialization were improved by the treatment. [0152]
It has also been found that the cheese whey extract when administered to rats at the same time as the chemotherapy agent methotrexate can reduce the loss of mucosal crypts and villi in the jejunum and ileum regions of the gut. The treatment protocol can also lead to an increase in the activity of the digestive enzyme, sucrase, above that in animals that only receive methotrexate. [0153]
In a second investigation on rats having their gut damaged with methotrexate, the incidence of bacterial translocation across the gut is reduced at 9 and 12 days after treatment with the cheese whey extract. Moreover, the number of bacterial colonies detected in intestinal lymph nodes is reduced at 5 and 9 days after treatment with the cheese whey extract. [0154]
In a third investigation on male Golden Syrian hamsters, continuous topical application of GFE-2 to the hamster cheek pouch reduces the severity of 5-fluorouracil (5-FU)-induced chemotherapy-induced mucositis. [0155]
Some aspects of the compositions and methods desribed herein are described in U.S. patent application Ser. No. 07/956,759, filed Dec. 7, 1992, the complete disclosure of which is incorporated herein by reference. [0156]

EXAMPLE 1

Preparation of a Fraction From Cheese Whey (GFE) That is Enriched in Growth Promoting Activity

Pasteurized whey obtained as an end product of cheese manufacture was filtered through a 10 micron screen and a 0.2 micron Sartorius Microsart Sartocon II module to remove solids. The ultrafiltrate was adjusted to pH 6.5 and applied to a column of S-Sepharose Fast Flow S cation exchange resin (Pharmacia) that had been equilibrated with 50 mM sodium citrate buffer at pH 6.5. After washing the column with the same buffer the absorbed material was eluted by a solution of 1M NaCl containing 0.25 M NH[0157] ₄OH. This eluate was diafiltered against water until the conductivity reached 0 μg and then concentrated by ultrafiltration; both processes using a 3 kDa-excluding membrane. The resultant preparation was freeze-dried to produce the “GFE” product.
A preparation from 30 litres of cheese whey containing 18 g protein yielded a GFE extract containing 2.66 g protein. [0158]

EXAMPLE 2

Preparation of a Fraction From Cheese Whey That is Enriched in Growth-promoting Activity and Depleted in Extraneous Protein Including Lactoferrin (GFE-2)

Pasteurized whey was filtered and applied to a column of S-Sepharose and the column washed as in Example 1. Elution was accomplished with a solution containing 0.4M NaCl added to 10 mM sodium citrate pH6.5. This GFE-2 was diafiltered against water, concentrated and freeze-dried as described in Example 1. [0159]
A preparation from 30 litres of cheese whey which contained 18 g protein yielded a GFE-2 extract containing 0.56 g protein. [0160]

EXAMPLE 3

Preparation of a Modified GFE-2 Fraction That is Also Depleted in Extraneous Protein Including Lactoperoxidase (GFE-3)

The freeze-dried GFE-2 (Example 2) was dissolved at a concentration of 25 mg/ml and heated at 80° C. for 2.5 min. The heated sample was cooled rapidly and centrifuged. The clear supernatant was passed through a 0.22 μm filter before use. This solution contained 50% of the protein present in GFE-2 and approximately 10% lactoperoxidase. [0161]

EXAMPLE 4

Stimulation of the Growth of Cultured Cells by Cheese Whey Extracts (Examples 1, 2) Compared With Fetal Bovine Serum

Prior to the addition to culture media, the freeze-dried powders (GFE, GFE-2) were first suspended in Dulbecco's Phosphate-buffered saline and sterilized by passage through a 0.2 μm filter. [0162]
This example utilizes the cell lines L6 (rat myoblast), Balb C/3T3 (mouse fibroblast) and SF1972 (human diploid skin fibroblast). [0163]
Each cell line was subcultured on to 96-place tissue culture plates in Dulbecco-Modified Eagle's Minimal Essential Medium (DMEM) containing 5% fetal bovine serum and left in a 5% CO[0164] ₂, 37° C., humidified incubator overnight to ensure attachment of the cells. Sterile techniques were used throughout. The plates were thoroughly washed in DMEM to remove any residual serum and the whey extract (GFE or GFE-2) or fetal bovine serum (FBS) added at the indicated concentrations. The total volume in each well was 0.1 ml at 37° C., 5% CO₂and 100% humidity.
After a further 2 days the plates were washed, fixed and the cell numbers quantified using an automated methylene blue method (M. H. Oliver et al., J. Cell Sci. 92, 513, 1989). Growth is expressed as the percentage increase in absorbance units relative to the increase in absorbance produced by growing the cells in DMEM containing 5% fetal bovine serum (FIG. 1). [0165]
This example shows that in all three cell lines GFE and GFE-2 stimulate growth as well as fetal bovine serum. Moreover, in Balb C/3T3 and SF1972 cells GFE-2 is active at approximately one tenth the protein content as fetal bovine serum. [0166]

EXAMPLE 5

Stimulation of the Growth of Cultured Cells by Extracts of Cheese Whey Depleted in Extraneous Protein Including Lactoperoxidase (GFE-3, Example 3) Compared With GFE-2 (Example 2)

The experimental details were exactly as described in Example 4 except that the data are expressed as the protein content (μg/100 μl well) that achieved the same growth response as was achieved with 5% fetal bovine serum (see Table 1). [0167]

TABLE 1

Growth of Cells in the presence of GPE-2 or GFE-3

Concentration (μg/100 μl)

achieving growth equivalent to

Cell Type Extract 5% fetal bovine serum

L6 GFE-2 100

GFE-3 63

Balb C/3T3 GFE-2 15

GFE-3 6

SF1972 GFE-2 8

GFE-3 4

EXAMPLE 6

Growth Effects of Cultured Cells Produced by Supplementing Medium Containing 2% Fetal Bovine Serum With GFE-2 Extracts (Example 2)

The experimental details were exactly as described in Example 4 except that the human lung fibroblast line (HEL) replaced the human skin fibroblast line (SF1972). Data are expressed as absorbances achieved after growth of the cells for 2 days (see Table 2). [0168]

TABLE 2

Growth of Cells with GFE-2 added in the presence of 2%

fetal bovine serum

Fetal Increases in absorbance

Bovine GFE-2 Balb C/3T3 HEL

Serum (%) (μg/100 μl) L6 cells cells cells

2 0 0.618 0.126 0.16

5 0 0.998 0.270 0.21

10 0 1.309 0.502 0.34

2 5 1.010 0.294 0.32

2 25 1.108 0.585 0.38

2 50 1.157 0.698 0.38

2 100 1.370 0.799 0.37

EXAMPLE 7

Cheese Whey Extract Contains Factors That Promote DNA Synthesis by Cultured Human Endothelial Cells

GFE-2 was prepared as in Example 2. The GFE-2 was dissolved in M199 medium, passed through a 0.22 micron filter and serially diluted in the same medium. [0169]
This example utilizes human umbilical vein endothelial cells as a primary cell of wound repair. Primary cultures of human umbilical vein endothelial cells were isolated according to the method of Gimbrone et al. (J. Cell. Biol. 60, 673, 1974) and passaged in medium 199 containing 20% FBS, endothelial cell growth supplement, and heparin, and left in a 5% CO[0170] ₂, 37° C. humidified incubator overnight to ensure attachment of the cells. Sterile techniques were used throughout. The plates were then thoroughly washed in M199 to remove any residual serum and the whey extract added at the indicated concentrations. The total volume in each well was 1 ml. After a 24 h incubation ³H-thymidine was added to each well and allowed to incorporate into cellular DNA for a further 6 h. The cells were then washed thoroughly to remove unincorporated ³H-thymidine and the cell layer dissolved in 0.5M NaOH containing 0.1% Triton X ₁₃100. ³H-Thymidine incorporated into cellular DNA was measured by scintillation counting. DNA synthesis is expressed as the percentage increase in cpm above that produced by growing the cells in unsupplemented M199 (FIG. 2).
This example shows that the cheese whey extract contains factors that stimulate DNA synthesis by endothelial cells. The growth response exerted by cheese whey extract can be distinguished from transforming growth factor beta which inhibits the growth of endothelial cells (Muller et al. “Inhibitory action of transforming growth factor beta on endothelial cells”. Proc. Natl. Acad. Sci. USA 84, 5600,1987). [0171]

EXAMPLE 8

Cheese Extract Induces Contraction of a Fibroblast-populated Collagen Lattice

GFE-2 was prepared as in Example 2, and sterile filtered by passage through a 0.22 μm filter. [0172]
An in vitro model of wound contraction was prepared as follows. Human diploid skin fibroblasts were suspended in Dulbecco's-Modified Eagle's Minimal Essential Medium (DMEM) at a concentration of 200,000 cells per ml, and mixed with an equal volume of soluble rat tail collagen (2 mg per ml) containing [0173] ³H-inulin (10-20,000 cpm per ml). The mixture was poured into 24-place wells (1 ml per well) and allowed to gel by incubation at 37° C. for 30 minutes. The gels were then separated from the tissue culture plastic by reaming the margin of the gel with a 25 G needle. The fibroblast populated collagen matrix was then overlaid by DMEM containing cheese whey extract at the indicated concentrations and incubated for a further 24 hours at 37° C. in a humidified atmosphere containing 5% CO₂. The degree of collagen contraction was determined by scintillation counting of the ³H-inulin remaining in the contracted gel (means+SEM, N=3, FIG. 3).
This example shows that factors in cheese whey extract act on the human skin fibroblast to induce reorganization of collagen fibers and contraction of a collagen gel that is analogous to the contraction of a wound. [0174]

EXAMPLE 9

Whey-derived Growth Factor Extract Induces Closure of a Full Thickness Excisional Wound in Cultured Fetal Rat Skin

Cheese whey extract GFE-2 as produced in Example 2 was dissolved in Dulbecco's-Modified Eagle's Minimal Essential Medium (DMEM) and sterile filtered. [0175]
An in vitro organ culture model of wound repair was established as follows. A pregnant Sprague-Dawley rat was killed by CO[0176] ₂asphyxiation at 17 days gestation and fetal rats dissected free from the uterus. A 1 cm×1 cm piece of skin, including the epidermis and full thickness dermis, was dissected from the back of each fetus using fine scissors and forceps, and a 1 mm hole placed in the centre of the skin using a 19 G needle. The preparation was mounted on a cradle and placed in a culture dish containing DMEM done, dilutions of cheese whey extract or 10% fetal bovine serum (FBS). All preparations were photographed using a standard focal length jig prior to culture. Cultures were then maintained at 37° C. in a humidified atmosphere of 5% CO₂for 72 hours, and photographed at 24, 48 and 72 hours. The percentage of the wound remaining open was determined by planimetry. FIG. 4 shows that cheese whey extract at a concentration of 2.5 mg/ml is able to induce healing of the excisional defect after 72 h culture (mean+SEM, N=3). This experiment shows that cheese whey extract contains factors that promote healing of a full thickness excisional wound in organotypic fetal skin culture.

EXAMPLE 10

Cheese Whey Extract Enhances Wound Strength in Normal Rats

Cheese whey extract GFE-2 prepared as in Example 2 was formulated in a 1 mg/ml collagen solution to a final concentration of 2.5 mg/ml. [0177]
An in vivo model of incisional wound repair was established as follows. Male rats between 250 and 300 g were anaesthetized by inhalation of isofluorane and paired 6 cm full thickness incisions were placed through the skin 1.5 cm either side of the dorsal midline. Cheese whey extract (2.5 mg/ml) was then applied to one wound and vehicle (rat tail collagen, 1 mg/ml) applied to the contralateral wound, each rat therefore serving as its own control. Each wound received 100 μl of either the vehicle or the cheese whey extract preparation; experimental wounds therefore received 250 μg of cheese whey extract. The margins of both wounds were then apposed using sutures and animals housed individually after surgery. Rats were killed by CO[0178] ₂asphyxiation at days 3, 5 and 7, and the dorsal pelts removed. The maximum load (breaking strength) tolerated by the wounds was measured by placing 4 mm strips taken at 90° to the wound in a tensiometer. Wound strength (mean+SEM, N=16 wounds per time point) is expressed as breaking strength vs time (FIG. 5).
This example shows that the single application of cheese whey extract enhances healing of an incisional wound in normal rats. This enhancement is particularly evident between 3 and 5 days after wounding, and is maintained until [0179] day 7.

EXAMPLE 11

Cheese Whey Extract Enhances Wound Contraction and Epithelialization in Steroid-treated Rats

Cheese whey extract was prepared as in Example 2 and formulated as in Example 10. [0180]
An in vivo model of wound contraction and epithelialization was established as follows. Sprague Dawley rats, weighing between 250 and 300 grams were administered methyl-prednisolone (30 mg/kg). The rats were then anaesthetized using isofluorane/N[0181] ₂O/O₂, their backs clipped of hair and four square symmetrical wounds created down the midline of the back. A template containing four square holes, each measuring 1.5 by 1.5 cm, was used to create a line of wounds through the skin and panniculus carnosis muscle to the deep fascia of the back. The size and shape of the wounds are then recorded by taking analogue tracings onto acetate film. All rats were housed individually after surgery. Animals were reanaesthetized and wound areas measured on days 1, 3, 5, 7, 9, 12, 14, 16, 19, 21, 23, 26 and 28. Care was taken to delineate between the full thickness wound margin and the advancing epithelium which were recorded separately. Wounds were treated with the vehicle preparation (rat tail collagen at 1 mg/ml) or vehicle plus whey extract, daily for the first 10 days of the study. A further control group received no treatment and the wounds were left exposed. Data (means±SEM, N=28 wounds) are expressed as area inside full thickness wound margin vs time (FIG. 6(a); wound contraction) or area inside the epithelial margin of the wound vs time (ie incorporating wound epithelialization; FIG. 6(b)).
This experiment shows that cheese extract incorporated into a collagen vehicle enhances wound contraction and epithelialization in steroid-compromised animals. [0182]

EXAMPLE 12

Cheese Whey Extract Contains Factors That Promote Cell Division by Cultured Intestinal Epithelial Cells

GFE-2 was prepared as in Example 2. The GFE-2 was dissolved in Dulbecco's Modified Eagle's Medium (DMEM), passed through a 0.22 micron filter, and serially diluted in the same medium. [0183]
This example utilizes rat intestinal epithelial cells as a primary cell of intestinal wound repair. IEC-6 cells were maintained and passaged in DMEM containing 10% FBS. For each experiment, IEC-6 cells were sub-cultured onto 96-place tissue culture plates in DMEM containing 10% FBS and left in a 5% CO[0184] ₂, 37° C. humidified incubator overnight to ensure attachment. Sterile techniques were used throughout. The plates were then thoroughly washed in DMEM to remove any residual serum and the whey extract added at the indicated concentrations either alone or in the presence of 1% FBS or 5% FBS. The total volume in each well was 0.1 ml. After a 48 h incubation period the cell number was assessed using a dye-binding assay (Oliver et al., J. Cell. Sci. 92, 513, 1989).
This example shows that the whey extract contains factors that stimulate growth of intestinal epithelial cells, and therefore whey extract may be useful in promoting repair of intestinal ulceration or damage, or to enhance intestinal growth. [0185]

EXAMPLE 13

Oral Administration of a Milk Product Extract From Bovine Cheese Whey (GFE-2) Partially Prevents Loss of Small Intestinal Crypts and Villi in Rats With Methotrexate Induced Small Bowel Damage

In this Example 13, rats were injected with high doses of the chemotherapy agent, methotrexate, as an experimental model of damage to the lining of the alimentary tract. Oral administration to methotrexate-injected rats of a milk product extract purified from bovine cheese whey (GFE-2 as described in Example 2) provides evidence that the milk product extract can ameliorate chemotherapy damage to the small bowel. [0186]
Male Sprague Dawley rats, weighing on average 140 g and maintained in metabolism cages were fed a high-carbohydrate diet. Control rats received no GFE-2 whereas experimental rats were treated for 5 days with GFE-2. GFE-2 treated rats were fed a modified diet containing 31.2 g GFE-2/kg diet in place of the equivalent amount of casein. In addition, the GFE-2 fed rats were given GFE by stomach gavage on [0187] days 3, 4 and 5 of the experimental period so that the total dose of GFE-2 per day averaged 514 mg/day GFE-2. Control rats were fed the unmodified diet and gavaged by an identical protocol on days 3, 4 and 5 with an equivalent amount of bovine serum albumin to ensure an isonitrogenous diet.
One group of control rats and the GFE-2 treated rats (8 rats per group) were injected subcutaneously with 2.5 mg/kg methotrexate at the start of [0188] days 1, 2 and 3. An additional control group (“pair-fed”) received sham methotrexate injections, and was pair-fed to the methotrexate-injected control group.
Rats were maintained in the metabolism cages for 5 days, at which time they were killed for collection of the gastrointestinal tract. Tissue samples were collected from the proximal small bowel (duodenum and jejunum) as well as the distal small bowel (ileum). Tissue samples were fixed in methacarn, embedded in paraffin, sectioned and stained with haematoxylin-eosin for histological analysis. [0189]
Compared with the pair-fed controls, the methotrexate-injected control group showed loss of mucosal crypts in the jejunum, and to a lesser extend in the ileum. This is illustrated in FIG. 8([0190] a) as the area of intact crypts per unit area of total mucosa, and demonstrates that methotrexate causes loss of mucosal crypts (which contain the dividing cells of the epithelium) characteristic of chemotherapy damage. Also characteristic of chemotherapy damage in the small bowel, methotrexate injection caused stunting and loss of intestinal villi, being the functional compartment of the small bowel mucosa. This is illustrated in FIG. 8 (b) by a reduction in the surface length of the finger-like viiii per unit length of intestinal circumference in methotrexate-treated controls compared with the pair-fed group receiving no methotrexate.
Oral administration of GFE-2 for 5 days starting at the time of the first methotrexate injection partially prevented the loss of mucosal crypts and villi in both regions of the small bowel (FIG. 8) The effects of GFE-2 were statistically significant (P<0.05 by ANOVA) in the jejunum, where methotrexate-induced damage was more severe, and in the ileum for villus surface length. [0191]
The example demonstrates that oral administration of GFE-2 is able to partially prevent or accelerate repair of chemotherapy damage in the small bowel. [0192]

EXAMPLE 14

Sucrase Activity is Increased in the Damaged Mucosa of Rats Treated With Cheese Whey Extract

From the same experiment as described in Example 13, 4 cm lengths of small bowel were frozen for measurement of the activity of mucosal sucrase, an enzyme located on the surface of epithelial cells of the villus. Because sucrase is essential for digestion of dietary sucrose, the sucrase activity per unit length of intestine provides a measure of the functional capacity of the small bowel. [0193]
Five day's, oral administration of GFE-2 to methotrexate-injected rats significantly improved (P<0.05) the sucrase activity per unit length of ileum compared with the methotrexate-injected control group, or the pair-fed control group (FIG. 9). [0194]
This example demonstrates that GFE-2 improves the functional capacity of the chemotherapy-damaged small bowel. [0195]

EXAMPLE 15

Oral Administration of Cheese Whey Extract to Rats for 5 to 12 Days Reduces Bacterial Translocation Across the Gut

The ability of the gut epithelium to provide a barrier against bacterial invasion provides another measure of gut function that is improved by cheese whey extract. [0196]
140 g male Sprague Dawley rats were injected with methotrexate for three consecutive days as described in Example 13. Methotrexate-injected rats were administered oral GFE-2 by an identical protocol to that described in Example 13. One group of rats was killed on [0197] day 5 after is the start of methotrexate injections (as in Example 13), while in other groups, GFE-2 treatment was continued for a total of 8 or 12 days (8 rats per group). Control methotrexate treated rats and pair-fed control rats identical to those in Example 13 were killed on days 5, 8 and 12 (8 rats per group).
Rats were maintained in metabolism cages as in Example 13 until exsanguination on [0198] day 5, 8 or 12. The abdominal skin was soaked in 70% ethanol before the intestine was removed under aseptic conditions. All visible mesenteric lymph nodes were placed into a sterile pre-weighed container. Samples were then weighed and infusion solution was added to a final concentration of 100 mg/ml. Tissues were homogenized in this solution with sterile glass-reinforced grinders. For measurement of translocation of gram negative bacteria into mesenteric lymph nodes, 40 or 60 mg of each tissue homogenate was placed onto MacConkey agar II or blood agar plates and incubated aerobically at 35° C. for 48 hours. Enteric gram negative bacterial colonies were identified using API 20E strips, then counted. The incidence (proportion of animals exhibiting detectable bacterial translocation) and mean number of bacterial colonies per gram of tissue were calculated for each treatment group.
Pair fed control animals receiving no methotrexate showed no incidence of bacterial translocation across the gut. Methotrexate injection impaired the intestinal barrier so that all rats in the methotrexate-injected control group (FIG. 10; “No GFE-2”) had positive bacterial cultures from mesenteric lymph nodes on [0199] day 5. The incidence in this group diminished over the next 7 days, but remained at 60% on day 12 (FIG. 10 (a)). The number of colonies per gram of mesenteric lymph node was maximal on day 5, and then diminished thereafter in parallel with the incidence (FIG. 10(b)).
Oral administration of GFE-2 resulted in a lower incidence of translocation on [0200] days 8 and 12, with the difference between GFE-2 treated and control 2 methotrexate-injected rats reaching statistical significance by X²test (P<0.05) on day 12. The number of colonies per gram of mesenteric lymph node was also significantly lower in the GFE-2 treated group on both day 5 and 8.
The example demonstrates that oral administration of the milk product extract partially prevents chemotherapy-induced loss of barrier function in the gut. This could be expected to decrease the incidence of infection and sepsis following chemotherapy. [0201]
Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein. [0202]

EXAMPLE 16

Continuous topical application of GFE-2 to the hamster cheek pouch reduces the severity of 5-fluorouracil (5-FU)-induced chemotherapy-induced mucositis. [0203]
This experiment investigated the effects of GFE-2 administered topically on chemotherapy-induced oral mucositis in male Golden Syrian hamsters. The trial included continuous treatment of GFE-2 to the cheek pouch of 10 hamsters treated with 5-fluorouracil. [0204]
Hamsters were divided into two groups of five animals. The initial mean body weight of each group was similar. All hamsters were given intraperitoneal injections of 90 mg/kg of 5-FU on [0205] day 1, and 60 mg/kg on day 3. The cheek pouch was scratched on days 1, 2 and 3 with six strokes of a wire brush in one direction and six strokes in the other perpendicular direction to achieve a uniform wound.
Groups were treated with either a commercial mouthwash as vehicle, or 0.3 ml of GFE-2 at 40/mg/ml protein concentration. The cheek pouch liquid treatments were applied daily for one minute, during which time the hamsters were anaesthetized using isoflurane anesthesia. [0206]
The cheek pouch was assess on [0207] days 5, 7, 8, 11, 13 and 15. Monitoring was based on a visual assessment of the cheek pouch (graded on a 1-10 scale) taking into account the overall severity of the lesion, degree of bruising, swelling and scarring. Body weight was recorded as a percentage of the day 0 value.
Animals given a topical treatment of GFE-2 showed reduced mucositis compared to the vehicle treated group, measured as overall visual score (FIG. 5), total ulcer area and body weight loss (FIG. 6). Each of these effects was statistically significant by paired t-test favoring GFE-2 treatment. [0208]
This example suggested that topical administration of GFE-2 may reduce the severity of oral mucositis and related symptoms such as body weight loss. [0209]

Claims

We claim:

1. A pharmaceutical or veterinary composition for the treatment of surface wounds which composition comprises:

(a) a milk product extract that contains a mixture of cell growth factors with basic to approximately neutral isoelectric points obtained from a milk product of an ungulate mammal by first subjecting that product to a cation exchange matrix under conditions whereby casein, alpha lactalbumin, and beta lactoglobulin present in the milk product are not absorbed to the matrix, after which the absorbed growth factor mixture is eluted and then concentrated; and

2. A composition according to claim 1 further including an effective amount of at least one active ingredient selected from antibiotics, antiseptics, other growth promotants, anaesthetics, and mixtures thereof.

3. A composition according to claim 1 wherein the cell growth factors have isoelectric points between about 6.0 and about 10.5.

4. A pharmaceutical or veterinary composition for the treatment of gastrointestinal injuries, diseases or ulcers which composition comprises:

(a) a milk product extract that contains a mixture of cell growth factors with basic to aproximately neutral isoelectric points that are obtained from a milk product of an ungulate mammal by first subjecting that product to a cation exchange matrix under conditions whereby casein, alpha lactalbumin, and beta lactoglobulin present in the milk product are not absorbed to the matrix, after which the absorbed growth factor mixture is eluted and then concentrated; and

5. A composition according to claim 4 further including an effective amount of at least one active ingredient selected from antibiotics, antiseptics, other growth promotants, anaesthetics, and mixtures thereof.

6. A composition according to claim 4 wherein the cell growth factors have isoelectric points between about 6.0 and about 10.5.

7. A method of treating surface wounds in animals, including humans, which method comprises administering to a subject to be treated an effective amount of a pharmaceutical or veterinary composition, which composition comprises:

(a) a milk product extract that contains a mixture of cell growth factors with basic to approximately neutral isoelectric points that are obtained from a milk product of an ungulate mammal by first subjecting that product to a cation exchange matrix under conditions whereby casein, alpha lactalbumin, and beta lactoglobulin present in the milk product are not absorbed to the matrix, after which the absorbed growth factor mixture is eluted and then concentrated; and

8. The method according to claim 7 wherein the surface wounds are selected from the group consisting of ulcers, conditions that results from surgery, therapeutically induced wounds, wounds associated with disorders of the central nervous system, any exfoliative disease of the skin, wounds associated with local or systemic infection, congenital wounds, pathological wounds, traumatic and accidental wounds, and burns.

9. A method according to claim 7 wherein the cell growth factors have isoelectric points between about 6.0 and about 10.5.

10. A method for the treatment of gastrointestinal injuries, diseases or ulcers, which method comprises administering to a subject to be treated an effective amount of a pharmaceutical or veterinary composition, which composition comprises:

11. The method according to claim 10 wherein the gastrointestinal injuries, diseases or ulcers are selected from the group consisting of dental and oral wounds, peptic ulcers, inflammatory bowel diseases, ulcers associated with stress conditions, damage caused by radiotherapy and/or chemotherapy, inadequate gut function or damage associated with prematurity, diarrheal conditions, damage caused by food intolerance, cancer of the gastrointestinal tract, surgically induced damage, damage caused by esophageal reflux, conditions associated with loss of gut barrier function, congenital conditions resulting in inadequate gastrointestinal function or damage, and autoimmune diseases that affect the gut.

12. A method according to claim 10 wherein the cell growth factors have isoelectric points between about 6.0 and about 10.5.